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Third-party punishment (TPP) is an altruistic behavior or sense willing to punish transgressors as a third party not directly involved in the transgression. TPP is observed worldwide, regardless of tradition and culture, and is essential for morality in human society. Moreover, even preverbal infants display TPP-like judgement, suggesting that TPP is evolutionarily conserved and innate. Thus, it is possible that non-human animals display TPP-like behavior, although TPP has been said to be human-specific. We investigated whether or not male mature Wistar rats displayed TPP-like behaviors when they witnessed deadly aggression by an unknown aggressive mouse toward another unknown victim mouse. Normally reared rats did not display TPP-like behaviors, but rats reared with extensive affectionate handling by human caretakers as beloved pets contacted the unknown aggressive mice in a gentle manner leading to reduced aggression toward the unknown victim mice, even when the aggressive mice fought back. Furthermore, the handled rats touched unknown rat pups that were drowning in water and anesthesia-induced comatose rats more frequently than control rats. These findings suggest a possibility that TPP is not in fact human-specific and innate but rather an acquired behavior that flourishes in affectionate circumstances., (© 2024. The Author(s).)

Objective: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na + / H + exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells., Methods: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line., Results: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells., Conclusion: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Japanese Society of Otorhinolaryngology-Head and Neck Surgery, Inc. Published by Elsevier B.V. All rights reserved.)

In this study, a murine sepsis model was developed using the cecum ligation and puncture (CLP) technique. The expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in the brain increased 6 h after CLP but decreased 24 h later when elevated endogenous dopamine levels in the brain were sustained. Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride reduced dopamine levels in the striatum and increased mortality in septic mice. Dopamine D1-like receptors were significantly expressed in the brain, but not in the lungs. Intraperitoneally administered SKF-81297 (SKF), a blood-brain barrier-permeable D1-like receptor agonist, prevented CLP-induced death of septic mice with ameliorated acute lung injury and cognitive dysfunction and suppressed TNF-α and IL-1β expression. The D1-like receptor antagonist SCH-23390 abolished the anti-inflammatory effects of SKF. These data suggest that D1-like receptor-mediated signals in the brain prevent CLP-induced inflammation in both the brain and the periphery., Competing Interests: The authors have no competing interests., (© 2024 The Authors.)

Objective: The prognosis of glioblastoma (GBM) correlates with residual tumor volume after surgery. In fluorescence-guided surgery, 5-aminolevulinic acid (ALA) has been used to maximize resection while avoiding neurological morbidity. However, not all tumor cells, particularly glioma stem cells (GSCs), display 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA fluorescence). The authors searched for repositioned drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA fluorescence. In this study, they investigated whether BBR can enhance 5-ALA fluorescence in GSCs and whether BBR can be applied to clinical practice as a 5-ALA fluorescence enhancer., Methods: The effects of BBR on 5-ALA fluorescence in glioma and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. As 5-ALA is metabolized for heme synthesis, the effects of BBR on mRNA expressions of 7 enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed., Results: Fluorescence microscopy revealed much more intense 5-ALA fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS showed that BBR greatly enhanced 5-ALA fluorescence compared with 5-ALA alone, and enhancement was much higher for GSCs than for glioma cells. Among the 7 enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. An in vivo study showed that 5-ALA fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue surrounding the tumor mass., Conclusions: BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to significantly augment fluorescence-guided surgery and photodynamic therapy.

Sleep serves a multitude of roles in brain maturation and function. Although the neural networks involved in sleep regulation have been extensively characterized, it is increasingly recognized that neurons are not the sole conductor orchestrating the rhythmic cycle of sleep and wakefulness. In the central nervous system, microglia have emerged as an important player in sleep regulation. Within the last two decades, microglia have gained substantial attention for carrying out numerous nonimmune tasks that are crucial for brain development and function by co-opting similar mechanisms used in their conventional immune functions. Here, we highlight the importance of microglia in sleep regulation with recent findings reporting an arrhythmic sleep/wake cycle in the absence of microglia. Although the underlying mechanisms for such regulation are still being uncovered, it is likely that microglial contributions to the rhythmic control of the sleep/wake cycle come from their influence on synaptic strength and neuronal activity. We review the current literature to provide speculative signaling pathways and suggest key questions for future research. Advancing our knowledge of the mechanistic contribution of microglia to sleep regulation will not only further our insight into this critical biological process but also be instrumental in providing novel therapeutic strategies for sleep disorders., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

High invasiveness is a characteristic of glioblastoma (GBM), making radical resection almost impossible, and thus, resulting in a tumor with inevitable recurrence. GBM recurrence may be caused by glioma stem-like cells (GSCs) that survive many kinds of therapy. GSCs with high expression levels of CD44 are highly invasive and resistant to radio-chemotherapy. CD44 is a multifunctional molecule that promotes the invasion and proliferation of tumor cells via various signaling pathways. Among these, paired pathways reciprocally activate invasion and proliferation under different hypoxic conditions. Severe hypoxia (0.5-2.5% O 2 ) upregulates hypoxia-inducible factor (HIF)-1α, which then activates target genes, including CD44, TGF-β, and cMET, all of which are related to tumor migration and invasion. In contrast, moderate hypoxia (2.5-5% O 2 ) upregulates HIF-2α, which activates target genes, such as vascular endothelial growth factor (VEGF)/VEGFR2, cMYC, and cyclin D1. All these genes are related to tumor proliferation. Oxygen environments around GBM can change before and after tumor resection. Before resection, the oxygen concentration at the tumor periphery is severely hypoxic. In the reparative stage after resection, the resection cavity shows moderate hypoxia. These observations suggest that upregulated CD44 under severe hypoxia may promote the migration and invasion of tumor cells. Conversely, when tumor resection leads to moderate hypoxia, upregulated HIF-2α activates HIF-2α target genes. The phenotypic transition regulated by CD44, leading to a dichotomy between invasion and proliferation according to hypoxic conditions, may play a crucial role in GBM recurrence.

Recurrent glioblastoma multiforme (GBM) is largely attributed to peritumoral infiltration of tumor cells. As higher CD44 expression in the tumor periphery correlates with higher risk of GBM invasion, the present study analyzed the relationship between CD44 expression and magnetic resonance imaging (MRI)-based invasiveness of GBM on a large scale. We also quantitatively evaluated GBM invasion using 5-aminolevulinic acid (5-ALA) spectroscopy to investigate the relationship between CD44 expression and tumor invasiveness as evaluated by intraoperative 5-ALA intensity. Based on MRI, GBM was classified as high-invasive type in 28 patients and low-invasive type in 22 patients. High-invasive type expressed CD44 at a significantly higher level than low-invasive type and was associated with worse survival. To quantitatively analyze GBM invasiveness, the relationship between tumor density in the peritumoral area and the spectroscopic intensity of 5-ALA was investigated. Spectroscopy showed that the 5-ALA intensity of infiltrating tumor cells correlated with tumor density as represented by the Ki-67 staining index. No significant correlation between CD44 and degree of 5-ALA-based invasiveness of GBM was found, but invasiveness of GBM as evaluated by 5-ALA matched the classification from MRI in all except one case, indicating that CD44 expression at the GBM periphery could provide a reliable biomarker for invasiveness in GBM.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Microglia play a central role in neuroinflammatory processes by releasing proinflammatory mediators. This process is tightly regulated along with neuronal activities, and neurotransmitters may link neuronal activities to the microglia. In this study, we showed that primary cultured rat microglia express the dopamine (DA) D1 receptor (D1R) and D4R, but not D2R, D3R, or D5R. In response to a D1R-specific agonist SKF-81297 (SKF), the cultured microglia exhibited increased intracellular cAMP levels. DA and SKF suppressed lipopolysaccharide (LPS)-induced expression of interleukin-1β (IL-1β) and tumor necrosis α (TNFα) in cultured microglia. Microglia in the normal mature rat prefrontal cortex (PFC) were sorted and significant expression of D1R, D2R, and D4R was observed. A delirium model was established by administering LPS intraperitoneally to mature male Wistar rats. The model also displayed sleep-wake disturbances as revealed by electroencephalogram and electromyogram recordings as well as increased expression of IL-1β and TNFα in the PFC. DA levels were increased in the PFC 21 h after LPS administration. Increased cytokine expression was observed in sorted microglia from the PFC of the delirium model; however, TNFα, but not IL-1β expression, was abruptly decreased 21 h after LPS administration in the delirium model, whereas DA levels were increased. A D1R antagonist SCH23390 partially abolished the TNFα expression change. This suggests that endogenous DA may play a role in suppressing neuroinflammation. Administration of the DA precursor L-DOPA or SKF to the delirium model rats inhibited the expression of IL-1β and TNFα. The simultaneous administration of clozapine, a D4R antagonist, strengthened the suppressive effects of L-DOPA. These results suggest that D1R mediates the suppressive effects of LPS-induced neuroinflammation, in which microglia may play an important role. Agonists for D1R may be effective for treating delirium., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 (GABA A R) for hypnotic/sedative activity. To investigate whether BU could potentiate GABA A ergic neurotransmission, we conducted a whole-cell patch-clamp recording from pyramidal neurons in rat cortical slices to detect spontaneous GABA A R-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 GABA A R-mediated Cl - flux may be a possible mechanism that contributes to its hypnotic/sedative activity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Activated microglia potentially cause neurodegeneration in Parkinson's disease (PD). Matrix metalloproteinase (MMP)-9 plays a crucial role in the pathogenesis of PD, but the modulator of microglial release of MMP-9 remains obscure. Given the modulatory effect of chloride intracellular channel protein 2 (CLIC2) on MMPs, we aimed to determine the role of CLIC2 in regulating microglial MMP expression and activation. We found that CLIC2 is expressed in microglia and neurons in rat brain tissue and focused on the function of CLIC2 in primary cultured microglia. Exposure to recombinant CLIC2 protein enhanced microglial invasion activity, and its knockdown abolished this activity. Moreover, increased activation of MMP-9 was confirmed by the addition of the CLIC2 protein, and CLIC2 knockdown eliminated this activation. Additionally, increased expression of CLIC2 was observed in PD-modeled tissue. In conclusion, CLIC2 increases MMP-9 activity in the microglia, which are involved in PD pathogenesis.

Objective: To determine the appropriate time points to start regular exercise which could reduce age-related anxiety and impaired social behavior., Methods: For this study, 8-week-old male Wistar rats were divided into three groups: no exercise (NoEX), short-term exercise (S-Ex), and long-term exercise (L-Ex) groups. S-Ex-group rats started treadmill exercise at 12 months of age, while L-Ex rats started from at 2 months of age. Exercise rats were forced to walk on the treadmill three times per week, with 1- to 2-day intervals for 10 minutes during the first 2 weeks, at 10 m/min until 17 months of age, and at 8 m/min thereafter. At 19 months of age, behavioral tests were performed to assess the effects of exercise on age-induced behavioral change as well as quantitative polymerase chain reaction were done to uncover the mechanism behind the behavioral changes., Results: Anxiety-like behavior was improved by long-term exercise. Additionally, rats belonging to the S-Ex and L-Ex groups showed improved social behavior and increased curiosity about interesting objects. The qPCR data showed that treadmill exercise suppressed the expression of immediate-early genes in the prefrontal cortex of the aged rats., Conclusion: This study suggests that long-term exercise represses early response genes, and in this way, it increases resistance to stress, diminishes anxiety-related behavior, and improves social behavior. These findings underscore the need to consider appropriate time to start exercise to prevent stress induced anxiety related behavior.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Sepsis is a pathology accompanied by increases in myeloid cells and decreases in lymphoid cells in circulation. In a murine sepsis model induced by cecum ligation and puncture (CLP), increasing numbers of neutrophils and decreasing levels of B-cells in circulation are among the earliest changes in the immune system. However, to date, the mechanisms for these changes remain to be elucidated. The study here sought to elucidate mechanisms underlying the changes in the leukocyte levels after CLP and also to determine what, if any, role for an involvement of the sympathetic nervous system (SNS). Here, male C57/BL6 mice were subjected to CLP or sham-CLP (abdominal wall incised, but cecum was not punctured). The changes in the number of circulating leukocytes over time were then investigated using flow cytometry. The results showed that a sham-CLP led to increased polymorphonuclear cells (PMN; most of which are neutrophils) and decreased B-cells in the circulation to an extent similar to that induced by CLP. Effects of adrenergic agonists and antagonists, as well as of adrenalectomy, were also examined in mice that underwent CLP or sham-CLP. Administering adrenaline or a β 2 adrenergic receptor agonist (clenbuterol) to mice 3 h before sacrifice produced almost identical changes to as what was seen 2 h after performing a sham-CLP. In contrast, giving a β 2 adrenergic receptor antagonist ICI118,551 1 h before a CLP or sham-CLP suppressed the expected changes 2 h after the operations. Noradrenaline and an α1 adrenergic receptor agonist phenylephrine did not exert significant effects. Adrenalectomy 24 h before a sham-CLP significantly abolished the expected sham-CLP-induced changes seen earlier. Clenbuterol increased splenocyte expression of Cxcr4 (a chemokine receptor gene); adrenalectomy abolished sham-CLP-induced Cxcr4 expression. A CXCR4 antagonist AMD3100 repressed the sham-CLP-induced changes. From these results, it may be concluded that sepsis-induced activation of the SNS may be one cause for immune dysfunction in sepsis - regardless of the pathogenetic processes.

In addition to genetic factors, environmental factors play a role in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). This study used Lister hooded rats (LHRs) as ADHD model animals to evaluate the effects of environmental factors. Male LHR pups were kept in four rearing conditions from postnatal day 23 (4 rats in a standard cage; 12 rats in a large flat cage; and 4 or 12 rats in an enriched environment [EE]) until 9 weeks of age. EE rearing but not rearing in a large flat cage decreased the activity of LHRs in the open field test that was conducted for 7 consecutive days. In the drop test, most rats reared in an EE remained on a disk at a height, whereas most rats reared in a standard cage fell off. RNA sequencing revealed that the immediate-early gene expression in the medial prefrontal cortex of LHRs reared in an EE was reduced. cFos-expressing neurons were reduced in number in LHRs reared in an EE. These results suggest that growing in an EE improves ADHD-like behaviors and that said improvement is due to the suppression of neuronal activity in the mPFC.

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 &lt; .05 and q &lt; 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of CINP.)

CLICs are the dimorphic protein present in both soluble and membrane fractions. As an integral membrane protein, CLICs potentially possess ion channel activity. However, it is not fully clarified what kinds of roles CLICs play in physiological and pathological conditions. In vertebrates, CLICs are classified into six classes: CLIC1, 2, 3, 4, 5, and 6. Recently, in silico analyses have revealed that the expression level of CLICs may have prognostic significance in cancer. In this review, we focus on CLIC2, which has received less attention than other CLICs, and discuss its role in the metastasis and invasion of malignant tumor cells. CLIC2 is expressed at higher levels in benign tumors than in malignant ones, most likely preventing tumor cell invasion into surrounding tissues. CLIC2 is also expressed in the vascular endothelial cells of normal tissues and maintains their intercellular adhesive junctions, presumably suppressing the hematogenous metastasis of malignant tumor cells. Surprisingly, CLIC2 is localized in secretory granules and secreted into the extracellular milieu. Secreted CLIC2 binds to MMP14 and inhibits its activity, leading to suppressed MMP2 activity. CLIC4, on the other hand, promotes MMP14 activity. These findings challenge the assumption that CLICs are ion channels, implying that they could be potential new targets for the treatment of malignant tumors.

Ischemic stroke is a leading cause of mortality and permanent disability. Chronic stroke lesions increase gradually due to the secondary neuroinflammation that occurs following acute ischemic neuronal degeneration. In this study, the ameliorating effect of a cytokine mixture consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 was evaluated on ischemic brain injury using a rat stroke model prepared by transient middle cerebral artery occlusion (tMCAO). The mixture reduced infarct volume and ameliorated ischemia-induced motor and cognitive dysfunctions. Sorted microglia cells from the ischemic hemisphere of rats administered the mixture showed reduced mRNA expression of tumor necrosis factor (TNF)-α and IL-1β at 3 days post-reperfusion. On flow cytometric analysis, the expression of CD86, a marker of pro-inflammatory type microglia, was suppressed, and the expression of CD163, a marker of tissue-repairing type microglia, was increased by the cytokine treatment. Immunoblotting and immunohistochemistry data showed that the cytokines increased the expression of the anti-apoptotic protein Bcl-xL in neurons in the ischemic lesion. Thus, the present study demonstrated that cytokine treatment markedly suppressed neurodegeneration during the chronic phase in the rat stroke model. The neuroprotective effects may be mediated by phenotypic changes of microglia that presumably lead to increased expression of Bcl-xL in ischemic lesions, while enhancing neuronal survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Matsumoto, Choudhury, Takeda, Sato, Kihara, Mikami, Inoue, Yano, Watanabe, Kumon, Kunieda and Tanaka.)

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., (© 2022. The Author(s).)

Myeloid differentiation primary response gene 88 (MyD88) is essential for microglial activation. Despite the significant role of microglia in regulating sleep homeostasis, the contribution of MyD88 to sleep is yet to be determined. To address this, we performed electroencephalographic and electromyographic recordings on MyD88-KO mice and wild-type mice to investigate their sleep/wake cycles. In the daytime, MyD88-KO mice exhibited prolonged wakefulness and shorter non-rapid eye movement sleep duration. Tail suspension and sucrose preference tests revealed that MyD88-KO mice displayed a depressive-like phenotype. We determined monoamines in the prefrontal cortex (PFC) using high-performance liquid chromatography and observed a decreased content of serotonin in the PFC of MyD88-KO mice. Flow cytometry revealed that CD11b, CD45, and F4/80 expressions were elevated at Zeitgeber time (ZT) 1 compared to at ZT13 only in wild-type mice. Furthermore, MFG-E8 and C1qB-tagged synapses were enhanced at ZT1 in the PFC of wild-type mice but not in MyD88-KO mice. Primary cultured microglia from MyD88-KO mice revealed decreased phagocytic ability. These findings indicate that genetic deletion of MyD88 induces insomnia and depressive behavior, at least in part, by affecting microglial homeostasis functions and lowering the serotonergic neuronal output., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mitochondrial dysfunction and exacerbated neuroinflammation are critical factors in the pathogenesis of both familial and non-familial forms of Parkinson's disease (PD). This study aims to understand the possible ameliorative effects of zonisamide on microglial mitochondrial dysfunction in PD. We prepared 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and lipopolysaccharide (LPS) co-treated mouse models of PD to investigate the effects of zonisamide on mitochondrial reactive oxygen species generation in microglial cells. Consequently, we utilised a mouse BV2 cell line that is commonly used for microglial studies to determine whether zonisamide could ameliorate LPS-treated mitochondrial dysfunction in microglia. Flow cytometry assay indicated that zonisamide abolished microglial reactive oxygen species (ROS) generation in PD models. Extracellular flux assays showed that LPS exposure to BV2 cells at 1 μg/mL drastically reduced the mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Zonisamide overcame the inhibitory effects of LPS on mitochondrial OCR. Our present data provide novel evidence on the ameliorative effect of zonisamide against microglial mitochondrial dysfunction and support its clinical use as an antiparkinsonian drug.

The axon initial segment (AIS) is a structural neuronal compartment of the proximal axon that plays key roles in sodium channel clustering, action potential initiation, and signal propagation of neuronal outputs. Mutations in constitutive genes of the AIS, such as ANK3, have been identified in patients with neurodevelopmental disorders. Nevertheless, morphological changes in the AIS in neurodevelopmental disorders have not been characterized. In this study, we investigated the length of the AIS in rodent models of attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). We observed abnormalities in AIS length in both animal models. In ADHD model rodents, we observed shorter AIS length in layer 2/3 (L2/3) neurons of the medial prefrontal cortex (mPFC) and primary somatosensory barrel field (S1BF). Further, we observed shorter AIS length in S1BF L5 neurons. In ASD model mice, we observed shorter AIS length in L2/3 and L5 neurons of the S1BF. These results suggest that impairments in AIS length are common phenomena in neurodevelopmental disorders such as ADHD and ASD and may be conserved across species. Our findings provide novel insight into the potential contribution of the AIS to the pathophysiology and pathogenesis of neurodevelopmental disorders., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: We previously reported that glioma stemlike cells (GSCs) exist in the area of the tumor periphery showing no gadolinium enhancement on magnetic resonance imaging. In the present work, we analyzed glucose metabolism to investigate whether lactate could be predictive of tumor invasiveness and of use in detection of the tumor invasion area in glioblastoma multiforme (GBM)., Methods: The expression of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase (PDH) was investigated in 20 patients. In GSC lines, LDH-A and PDH expression also was examined in parallel to assessments of mitochondrial respiration. We then investigated the relationship between lactate/creatine ratios in the tumor periphery measured by magnetic resonance spectroscopy, using learning-compression-model algorithms and phenotypes of GBMs., Results: In 20 GBMs, high-invasive GBM expressed LDH-A at significantly higher expression than did low-invasive GBM, whereas low-invasive GBM showed significantly higher expression of PDH than did high-invasive GBM. The highly invasive GSC line showed higher expression of LDH-A and lower expression of PDH compared with low-invasive GSC lines. The highly invasive GSC line also showed the lowest consumption of oxygen and the lowest production of adenosine triphosphate. Lactate levels, as measured by magnetic resonance spectroscopy, showed a significant positive correlation with LDH-A transcript levels, permitting classification of the GBMs into high-invasive and low-invasive phenotypes based on a cutoff value of 0.66 in the lactate/creatine ratio., Conclusions: In the tumor periphery area of the highly invasive GBM, aerobic glycolysis was the predominant pathway for glucose metabolism, resulting in the accumulation of lactate. The level of lactate may facilitate prediction of the tumor-infiltrating area on GBM., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive glioma stem-like cells (GSCs) in tumors. Upon GBM recurrence, GSCs with highly invasive and highly migratory activities must assume a less-motile state and proliferate to regenerate tumor mass. Elucidating the molecular mechanism underlying this transition from a highly invasive phenotype to a less-invasive, proliferative tumor could facilitate the identification of effective molecular targets for treating GBM. Here, we demonstrate that severe hypoxia (1% O 2 ) upregulates CD44 expression via activation of hypoxia-inducible factor (HIF-1α), inducing GSCs to assume a highly invasive tumor. In contrast, moderate hypoxia (5% O 2 ) upregulates osteopontin expression via activation of HIF-2α. The upregulated osteopontin inhibits CD44-promoted GSC migration and invasion and stimulates GSC proliferation, inducing GSCs to assume a less-invasive, highly proliferative tumor. These data indicate that the GSC phenotype is determined by interaction between CD44 and osteopontin. The expression of both CD44 and osteopontin is regulated by differential hypoxia levels. We found that CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo. Mouse brain tumors generated from CD44-knockdown GSCs exhibited diminished invasiveness, and the mice survived significantly longer than control mice. In contrast, siRNA-mediated silencing of the osteopontin gene decreased GSC proliferation. These results suggest that interaction between CD44 and osteopontin plays a key role in tumor progression in GBM; inhibition of both CD44 and osteopontin may represent an effective therapeutic approach for suppressing tumor progression, thus resulting in a better prognosis for patients with GBM., (Copyright © 2021. Published by Elsevier Inc.)

The abilities to invade surrounding tissues and metastasize to distant organs are the most outstanding features that distinguish malignant from benign tumors. However, the mechanisms preventing the invasion and metastasis of benign tumor cells remain unclear. By using our own rat distant metastasis model, gene expression of cells in primary tumors was compared with that in metastasized tumors. Among many distinct gene expressions, we have focused on chloride intracellular channel protein 2 (CLIC2), an ion channel protein of as-yet unknown function, which was predominantly expressed in the primary tumors. We created CLIC2 overexpressing rat glioma cell line and utilized benign human meningioma cells with naturally high CLIC2 expression. CLIC2 was expressed at higher levels in benign human brain tumors than in their malignant counterparts. Moreover, its high expression was associated with prolonged survival in the rat metastasis and brain tumor models as well as with progression-free survival in patients with brain tumors. CLIC2 was also correlated with the decreased blood vessel permeability likely by increased contents of cell adhesion molecules. We found that CLIC2 was secreted extracellularly, and bound to matrix metalloproteinase (MMP) 14. Furthermore, CLIC2 prevented the localization of MMP14 in the plasma membrane, and inhibited its enzymatic activity. Indeed, overexpressing CLIC2 and recombinant CLIC2 protein effectively suppressed malignant cell invasion, whereas CLIC2 knockdown reversed these effects. Thus, CLIC2 suppress invasion and metastasis of benign tumors at least partly by inhibiting MMP14 activity., (Copyright © 2021. Published by Elsevier Inc.)

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.

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.

Antiangiogenic therapy with bevacizumab (Bev), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), is a common treatment for recurrent glioblastoma (GBM), but its survival benefit is limited. Resistance to Bev is thought to be a major cause of ineffectiveness on Bev therapy. To optimize Bev therapy, identification of a predictive biomarker for responsiveness to Bev is required. Based on our previous study, we focused on the expression and functions of CD44 and VEGF in the Bev therapy. Here, we analyze a relationship between CD44 expression and responsiveness to Bev and elucidate the role of CD44 in anti-VEGF therapy. CD44 and VEGF expression in the tumor core and periphery of 22 GBMs was examined, and the relationship between expression of these molecules and progression-free time on Bev therapy was analyzed. The degree of CD44 expression in the tumor periphery was evaluated by the ratio of the mRNA expression in the tumor periphery to that in the tumor core (P/C ratio). VEGF expression was evaluated by the amount of the mRNA expression in the tumor periphery. To elucidate the roles of CD44 in the Bev therapy, in vitro and in vivo studies were performed using glioma stem-like cells (GSCs) and a GSC-transplanted mouse xenograft model, respectively. GBMs expressing high P/C ratio of CD44 were much more refractory to Bev than those expressing low P/C ratio of CD44, and the survival time of the former was much shorter than that of the latter. In vitro inhibition of VEGF with siRNA or Bev-activated CD44 expression and increased invasion of GSCs. Bev showed no antitumor effects in mice transplanted with CD44-overexpressing GSCs. The P/C ratio of CD44 expression may become a useful biomarker predicting responsiveness to Bev in GBM. CD44 reduces the antitumor effect of Bev, resulting in much more highly invasive tumors., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Microglia as a defensive arm of the nervous system emerged early in evolution. The surveilling microglia with motile and ramified processes are the main phenotype in the healthy CNS; the surveilling microglial patrol neuronal somata, dendrites, dendritic spines and axons. Increasing evidence suggests that microglia play fundamental roles in development, maturation and ageing of the brain, as well as contribute to a variety of physiological brain processes including sleep and circadian rhythm. Physiological state of microglia is tightly regulated by brain microenvironment and controlled by a sophisticated system of receptors and signalling cascades including ionotropic and metabotropic purinoceptors, pattern-recognition receptors, and receptors for chemokines and cytokines. Microglia also utilise ion channels and transporters in regulating ionic homeostasis and various aspects of microglial function. The major ion transporters expressed by microglia include Na + /H + exchanger 1 and Na + /Ca 2+ exchangers, which are involved in regulation of pH i and Ca 2+ homeostasis during microglial physiological responses. Microglial cells control development, maturation and plasticity of neuronal ensembles through controlled physiological phagocytosis of synapses or synaptic fragments - processes known as synaptic pruning and trogocytosis. This special issue on "Physiological roles of microglia" is an assembly of papers written by the leading experts in this research field. We start this special issue with this snapshot of microglial physiological functions as a prelude to the indepth discussion of microglia in physiological processes in the nervous system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Microglial cells in normal mature brains have long been considered to be cells that are resting until pathological events take place, activating the microglial cells. However, it is currently well known that the microglia that have resting ramified morphology in normal mature brains move actively in the brain parenchyma and phagocytose synapses, thus forming and maintaining neural circuits. This review summarizes recent findings on the roles of microglia in mature brains, with special reference to phagocytosis of synapses and higher brain functions. Phagocytic elimination of synapses by microglia may affect the balance between excitatory and inhibitory synaptic transmission, termed the E/I balance. When impaired synaptic elimination by microglia leads to disturbed E/I balance, various problems may follow in brain functions: in memory and cognitive functions, sleep, movement, social behaviors, and thinking. In addition to the roles of microglia in normal developing and mature brains, impaired microglial phagocytosis functions also correlate with disturbances to these higher brain functions that are caused by neurological, mental, and developmental disorders; Parkinson's and Alzheimer's diseases, autism spectrum disorder, attention deficit/hyperactivity disorder, and schizophrenia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Although del Río-Hortega originally reported that leptomeningeal cells are the source of ramified microglia in the developing brain, recent views do not seem to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1 + macrophages were peeled off the brain surface. Leptomeningeal macrophages strongly expressed CD68 and CD163, but microglia in the brain parenchyma did not. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF, resulting in the formation of large cell aggregates in which many proliferating macrophages were present. In contrast, colony-stimulating factor 1 (CSF1) did not enhance the generation of Iba1 + cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1 + cells in the presence of serum, which express CD68 and CD163 at much lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningeal-derived cells resembled parenchymal microglia better than primary microglia. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia-like cells that grow independently of CSF1.

Appropriate animal models are necessary to determine the molecular and cellular mechanisms underlying attention-deficit/hyperactivity disorder (ADHD). This study used a battery of behavioral tests to compare Lister hooded rats (LHRs), an old outbred strain frequently used for autistic epilepsy research, with Wistar rats and spontaneously hypertensive rats (SHRs), a commonly used ADHD model. The open field, elevated plus maze, light/dark box, and drop tests demonstrated that LHRs were the most hyperactive animals and displayed the most inattentive- and impulsive-like behaviors, which are characteristics of ADHD. The radial arm maze, social interaction, and Morris water maze tests showed that LHRs did not display deficits characteristic of autism or intellectual disability. Although LHRs did not show different monoamine contents, the mRNA expression levels of various genes linked to ADHD (Cdh13, Drd5, Foxp2, Maoa, Sema6d, Slc9a9, and St3gal3) and tyrosine hydroxylase protein expression levels were lower in the prefrontal cortex of LHRs compared with that of Wistar rats or SHRs. c-Fos, synapsin I, and tau protein expression levels in the prelimbic region of the medial prefrontal cortex were also increased in LHRs compared with Wistar rats. Atomoxetine and guanfacine, commonly used non-stimulant treatments for ADHD, ameliorated ADHD-like behaviors in LHRs. These results suggest that LHRs can serve as a better ADHD model to develop novel pharmacological interventions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3-7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Microglia, the immunocompetent cells in the central nervous system (CNS), have long been studied as pathologically deteriorating players in various CNS diseases. However, microglia exert ameliorating neuroprotective effects, which prompted us to reconsider their roles in CNS and peripheral nervous system (PNS) pathophysiology. Moreover, recent findings showed that microglia play critical roles even in the healthy CNS. The microglial functions that normally contribute to the maintenance of homeostasis in the CNS are modified by other cells, such as astrocytes and infiltrated myeloid cells; thus, the microglial actions on neurons are extremely complex. For a deeper understanding of the pathophysiology of various diseases, including those of the PNS, it is important to understand microglial functioning. In this review, we discuss both the favorable and unfavorable roles of microglia in neuronal survival in various CNS and PNS disorders. We also discuss the roles of blood-borne macrophages in the pathogenesis of CNS and PNS injuries because they cooperatively modify the pathological processes of resident microglia. Finally, metabolic changes in glycolysis and oxidative phosphorylation, with special reference to the pro-/anti-inflammatory activation of microglia, are intensively addressed, because they are profoundly correlated with the generation of reactive oxygen species and changes in pro-/anti-inflammatory phenotypes.

Objectives: The secretory enzyme lysyl oxidase like 2 (LOXL2) is speculated to contribute to tumor progression through its functions in the remodeling of extracellular matrix and epithelial-mesenchymal transition. We previously identified elevated expression of LOXL2 in metastatic human head and neck squamous cell carcinoma (HNSCC) cells in a mouse lymph node metastases model. Here we performed a case series study examining LOXL2 expression levels in human serum from HNSCC patients to evaluate whether LOXL2 is worth evaluation in a large cohort study., Methods: LOXL2 protein levels in three serum samples from HNSCC patients were assessed by immunoblotting and LOXL2 tissue expression was examined in one human tongue squamous cell carcinoma (SCC) tissue by immunohistochemistry as a representative of HNSCC tissue. Serum samples were further fractionated in exosomes and supernatants by ultracentrifugation, which were then subjected to immunoblot and in vitro LOX activity analyses. Exosomal LOXL2 levels of 36 serum samples from HNSCC patients and seven healthy volunteers were measured using polymer sedimentation exosome preparation followed by ELISA measurement and subjected to statistical analyses., Results: Immunoblot analyses revealed that LOXL2 was present in serum exosomal fractions from three HNSCC patients, and we observed approximately threefold higher levels of LOXL2 in HNSCC patients compared with three healthy volunteers. Immunohistochemical LOXL2 staining was detected in HNSCC cells in addition to non-cancerous lipid tissues and some muscles in human tongue HNSCC tissue. Further measurements of exosomal LOXL2 by ELISA showed over ninefold higher mean LOXL2 levels in patients compared with controls. Statistical analysis revealed a correlation between elevated serum exosomal LOXL2 levels and low-grade, but not high-grade, HNSCC., Conclusions: Our case series study that elevated serum exosomal LOXL2 levels exhibited a correlation with low-grade HNSCCs. A follow-up large cohort clinical study will be required to determine the potential clinical utility of LOXL2 as a new biomarker and/or therapy target for HNSCCs., Level of Evidence: 4 Laryngoscope, 130:E327-E334, 2020., (© 2019 The American Laryngological, Rhinological and Otological Society, Inc.)

Chronic constriction injury of the sciatic nerve is frequently considered as a cause of chronic neuropathic pain. Marked activation of microglia in the posterior horn (PH) has been well established with regard to this pain. However, microglial activation in the anterior horn (AH) is also strongly induced in this process. Therefore, in this study, we compared the differential activation modes of microglia in the AH and PH of the lumbar cord 7 days after chronic constriction injury of the left sciatic nerve in Wistar rats. Microglia in both the ipsilateral AH and PH demonstrated increased immunoreactivity of the microglial markers Iba1 and CD11b. Moreover, abundant CD68 + phagosomes were observed in the cytoplasm. Microglia in the AH displayed elongated somata with tightly surrounding motoneurons, whereas cells in the PH displayed a rather ameboid morphology and were attached to myelin sheaths rather than to neurons. Microglia in the AH strongly expressed NG2 chondroitin sulfate proteoglycan. Despite the tight attachment to neurons in the AH, a reduction in synaptic proteins was not evident, suggesting engagement of the activated microglia in synaptic stripping. Myelin basic protein immunoreactivity was observed in the phagosomes of activated microglia in the PH, suggesting the phagocytic removal of myelin. CCI caused both motor deficit and hyperalgesia that were evaluated by applying BBB locomotor rating scale and von Frey test, respectively. Motor defict was the most evident at postoperative day1, and that became less significant thereafter. By contrast, hyperalgesia was not severe at day 1 but it became worse at least by day 7. Collectively, the activation modes of microglia were different between the AH and PH, which may be associated with the difference in the course of motor and sensory symptoms., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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-1β, 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., Competing Interests: Declaration of competing interest Nothing to declare., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2019 Wiley Periodicals, Inc.)

Physical exercise is one of the best interventions for improving traumatic brain injury (TBI) outcomes. However, an argument has been raised regarding the timing at which physical exercise should be initiated. In this study, male Wistar rats were subjected to stab wounding of the right hemisphere to develop a TBI model and were forced to walk once on a treadmill at a 5-m/min pace at 24 h or 48 h after TBI for 10 min. Injured brain tissue was dissected after TBI to evaluate the effects of exercise. Behavioral abnormalities and motor impairment were assessed by various behavioral tests between 2 and 3 weeks after TBI. Exercise did not affect the circulating corticosterone levels and the weight of the adrenal glands. Exercise particularly that at 24 h, worsened the motor impairment of the left forelimbs. Quantitative reverse-transcription polymerase chain reaction showed that exercise at 24 h increased proinflammatory cytokines and chemokines on the third day while suppressing the proinflammatory reactions on the fourth day. Exercise at both time points decreased expression of transforming growth factor (TGF) β1 and its receptor TGFβR1. Exercise at 24 h increased phosphorylation of IκB kinase on the fourth day, which may be correlated with the decreased effects of TGFβ1. Even a low-intensity exercise activity could cause deleterious effects when it is initiated within 48 h after the onset of severe TBI, probably because of the resulting proinflammatory effects. Therefore, rehabilitation exercise programs should be initiated after 48 h of TBI onset., (© 2019 The Author(s).)

Carbon monoxide (CO) causes not only acute fatal poisoning but also may cause a delayed neurologic syndrome called delayed encephalopathy (DE), which occasionally occurs after an interval of several days to several weeks post-exposure. However, the mechanisms of DE have not been fully elucidated. This study aimed to clarify the pathophysiology of CO-induced DE and its distinctive features compared with hypoxemic hypoxia. Rats were randomly assigned to three groups; the air group, the CO group (exposed to CO), and the low O 2 group (exposed to low concentration of O 2 ). Impairment of memory function was observed only in the CO group. The hippocampus tissues were collected and analyzed for assessment of CO-induced changes and microglial reaction. Demyelination was observed only in the CO group and it was more severe and persisted longer than that observed in the low O 2 group. Moreover, in the CO group, decreased in microglial cell numbers were observed using flow cytometry, and microglia with detached branches were observed were observed using immunohistochemistry. Conversely, microglial cells with shortened branches and enlarged somata were observed in the low O 2 group. Furthermore, mRNAs encoding several neurotrophic factors expressed by microglia were decreased in the CO group but were increased in the low O 2 group. Thus, CO-induced DE displayed distinctive pathological features from those of simple hypoxic insults: prolonged demyelination accompanying a significant decrease in microglial cells. Decreased neurotrophic factor expression by microglial cells may be one of the causes of CO-induced DE., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Chloride intracellular channel protein 2 (CLIC2) belongs to the CLIC family of conserved metazoan proteins. Although CLICs have been identified as chloride channels, they are currently considered multifunctional proteins. CLIC2 is the least studied family member. We investigated CLIC2 expression and localization in human hepatocellular carcinoma, metastatic colorectal cancer in the liver, and colorectal cancer. Significant expression of mRNAs encoding CLIC1, 2, 4, and 5 were found in the human tissues, but only CLIC2 was predominantly expressed in non-cancer tissues surrounding cancer masses. Fibrotic or dysfunctional (aspartate aminotransferase ≥40) non-cancer liver tissues and advanced stage HCC tissues expressed low levels of CLIC2. Endothelial cells lining blood vessels but not lymphatic vessels in non-cancer tissues expressed CLIC2 as well as high levels of the tight junction proteins claudins 1 and 5, occludin, and ZO-1. Most endothelial cells in blood vessels in cancer tissues had very low expressions of CLIC2 and tight junction proteins. CD31 + /CD45 - endothelial cells isolated from non-cancer tissues expressed mRNAs encoding CLIC2, claudin 1, occludin and ZO-1, while similar cell fractions from cancer tissues had very low expressions of these molecules. Knockdown of CLIC2 expression in human umbilical vein endothelial cells (HUVECs) allowed human cancer cells to transmigrate through a HUVEC monolayer. These results suggest that CLIC2 may be involved in the formation and/or maintenance of tight junctions and that cancer tissue vasculature lacks CLIC2 and tight junctions, which allows the intravasation of cancer cells necessary for hematogenous metastasis.

Parkinson's disease (PD) is a frequent neurodegenerative disease causing bradykinesia, tremor, muscle rigidity and postural instability. Although its main pathology is progressive dopaminergic (DArgic) neuron loss in the substantia nigra, motor deficits are thought not to become apparent until most DArgic neurons are lost, probably due to compensatory mechanisms that overcome the decline of DA level in the striatum. Even in animal PD models, it is difficult to detect motor deficits when most DArgic neurons are functional. In this study, we performed various behavioral tests (apomorphine-induced rotation, cylinder, forepaw adjustment steps (FAS), beam walking, rota-rod, and open-field), using 6-hydroxydopamine (OHDA) and lipopolysaccharide (LPS)-induced hemi-PD model rats with various striatal DA levels, to find the best way to predict the DA level from earlier disease stages. Different from the 6-OHDA-induced model, reduction in the striatal DA levels in the LPS-model was less significant. Among the behavioral tests, data from cylinder and FAS tests, which evaluate forelimb movements, best correlated with decline of the DA level. They also correlated well with decreased body weight gain. The beam and apomorphine tests showed less significant correlation than the cylinder and FAS tests. Open-field and rota-rod tests were not useful. Expressional levels of mRNA encoding tyrosine hydroxylase (TH), a marker of DArgic neurons, correlated well with the DA level. Metabotropic glutamate receptor 4 mRNA expression correlated with the striatal DA level and may be related to compensatory mechanisms. These results suggest that motor impairments of PD should be evaluated by forelimb movements, or hands and forearms in clinical settings, rather than movement of the body or large joints. The combination of cylinder and FAS tests may be the best to evaluate the rat PD models, in which many DArgic neurons survive., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

OBJECTIVEHigh invasiveness of malignant gliomas frequently causes early local recurrence of the tumor, resulting in extremely poor outcome. To control such recurrence, novel therapies targeted toward infiltrating glioma cells around the tumor border are required. Here, the authors investigated the antitumor activity of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas to explore the possibility for clinical use of 5-ALA-mediated SDT (5-ALA-SDT).METHODSIn vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry and TUNEL staining. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined by analysis of the effect of pretreatment with the radical scavenger edaravone. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated.RESULTSThe 5-ALA-SDT inhibited cell growth and changed cell morphology, inducing cell shrinkage, vacuolization, and swelling. Flow cytometric analysis and TUNEL staining indicated that 5-ALA-SDT induced apoptotic cell death in all gliomas. The 5-ALA-SDT generated significantly higher ROS than in the control group, and inhibition of ROS generation by edaravone completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. The proliferative activity of the entire tumor was markedly decreased. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact.CONCLUSIONSThe 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field, whereas the surrounding brain tissue remained normal, resulting in longer survival of the HIFU-treated mice compared with that of untreated mice. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.

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-1β (IL-1β), and CD68 at higher levels than microglia. In contrast, microglia expressed transforming growth factor β1 (TGFβ1), 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 TGFβ1 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., (© 2018 Wiley Periodicals, Inc.)

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and a subpopulation of glioma stem-like cells (GSCs) is likely responsible for the invariable recurrence following maximum resection and chemoradiotherapy. As most GSCs that are located in the perivascular and perinecrotic niches should be removed during tumor resection, it is very important to know where surviving GSCs are localized. Here, we investigated the existence and functions of GSCs in the tumor periphery, which is considered to constitute the invasion niche for GSCs in GBM, by analyzing expression of stem cell markers and stem cell-related molecules and measuring particular activities of cultured GSCs. In addition, the relationship between GSCs expressing particular stem cell markers and pathological features on MRI and prognosis in GBM patients was analyzed. We showed that GSCs that express high levels of CD44 are present in the tumor periphery. We also found that vascular endothelial growth factor (VEGF) is characteristically expressed at a high level in the tumor periphery. Cultured GSCs obtained from the tumor periphery were highly invasive and have enhanced migration phenotype, both of which were markedly inhibited by CD44 knockdown. Higher expression of CD44 in the tumor periphery than in the core was correlated with a highly invasive feature on MRI and was associated with early tumor progression and worse survival, whereas lower expression of CD44 in the tumor periphery corresponded to low invasion and was associated with longer survival. The low invasion type on MRI tended to show high levels of VEGF expression in the tumor periphery, thus presenting the tumor with high proliferative activity. These results imply the significance of GSCs with high levels of CD44 expression in the tumor periphery compared to the core, not only in tumor invasion but also rapid tumor progression and short survival in patients with GBM.

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., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

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-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 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-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 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-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 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-β1. 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-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation., (Copyright © 2017 Elsevier B.V. All rights reserved.)