Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.neuint.2023.105479

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.3390/brainsci13010055

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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 β2 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 β2 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.

DOI： 10.1080/1547691X.2022.2029630

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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.

DOI： 10.5535/arm.22105

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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.

DOI： 10.1016/j.bbrc.2022.11.062

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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.

DOI： 10.3390/cells11223649

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

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.

DOI： 10.3389/fnins.2022.941363

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Background: Levodopa-carbidopa intestinal gel (LCIG) therapy is used in advanced Parkinson's disease (PD) and consists of continuous administration of levodopa directly into the jejunum through a percutaneous endoscopic gastro-jejunal (PEG-J) tube. Recently, the metabolism of levodopa by Enterococcus faecalis (E. faecalis) has been reported. Intestinal bacteria can also affect this therapy. Objectives: To investigate intestinal bacteria and examine its impact on levodopa blood concentration in patients with PD receiving LCIG therapy. Methods: We enrolled 6 patients receiving LCIG therapy in our department. After PEG-J tube replacement, intestinal bacteria were collected from the tip of the tube and were identified using culture and polymerase chain reaction (PCR) tests. Moreover, the presence of tyrosine decarboxylase, which metabolizes levodopa, was also confirmed by PCR test. The ability of these bacteria to metabolize levodopa was confirmed in vitro. Levodopa blood concentrations were also examined before PEG-J tube replacement. Results: Bacteria were detected in all 6 patients. E. faecalis was present in 4 patients. Moreover, tyrosine decarboxylase was detected in 2 patients. The identified bacteria displayed in vitro metabolization to dopamine in the 4 E. faecalis positive samples. The addition of carbidopa did not inhibit the metabolism of levodopa. However, there was no difference in the mean blood concentration of levodopa, regardless of the presence of E. faecalis. Conclusions: We found bacteria, including E. faecalis in the PEG-J tube. We observed levodopa metabolism in vitro, but there was no association with levodopa blood concentration. The effect of intestinal bacteria may be limited in patients receiving LCIG therapy.

DOI： 10.1002/mdc3.13417

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BACKGROUND: Amyotrophic lateral sclerosis (ALS) causes deterioration of respiratory function. Muscle weakness of the orbicularis oris interferes with the accurate assessment of respiratory function using spirometry. Reduced forced vital capacity (FVC) is an indicator that helps determine the appropriate timing to provide noninvasive ventilation (NIV) for the survival of ALS patients. We employed ultrasonography to evaluate changes in respiratory function by measuring the thickness of the rectus abdominis (RA) muscle as a possible alternative to spirometry. METHODS: Sixteen subjects with ALS were included in this study. The thickness of RA muscles was measured using ultrasonography, and respiratory fluctuations, such as vital capacity (VC), FVC, FEV1, percentage of predicted VC (%VC), percentage of predicted FVC (%FVC), percentage of predicted FEV1 (%FEV1), and FEV1/FVC, were evaluated using spirometry. RESULTS: Sixteen subjects underwent assessment by ultrasonography. A positive correlation was observed between the percent change in RA muscle thickness evaluated from maximal expiration to maximal inspiration and %VC (P = .001), %FVC (P = .001), FEV1 (P = .009), and %FEV1 (P = .02). CONCLUSIONS: RA ultrasonography was useful for predicting a reduction in VC in subjects with ALS and may help determine the best timing for introducing NIV.

DOI： 10.4187/respcare.09317

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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.

DOI： 10.3390/brainsci12020268

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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.

DOI： 10.1016/j.jneuroim.2021.577794

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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.

DOI： 10.1016/j.neo.2021.06.001

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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.

DOI： 10.3390/ijms22157824

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Authorship：Lead author   Publisher：MDPI AG  

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.

DOI： 10.3390/ijms22083907

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Language：Japanese   Publisher：(一社)日本生理学会  

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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.

DOI： 10.1016/j.neuint.2020.104857

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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.

DOI： 10.1016/j.neuint.2020.104672

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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.

DOI： 10.1016/j.bbrc.2019.12.041

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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.

DOI： 10.1002/glia.23698

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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.

DOI： 10.1016/j.ibror.2019.10.002

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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.

DOI： 10.1016/j.neuint.2018.11.005

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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.

DOI： 10.1080/21688370.2019.1593775

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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.

DOI： 10.1002/glia.23469

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.jphs.2018.03.005

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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.

DOI： 10.1016/j.bbadis.2017.12.022

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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.

DOI： 10.1016/j.bbrc.2018.01.065

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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.

DOI： 10.1002/glia.23199

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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.

DOI： 10.1016/j.jphs.2017.05.007

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Language：English   Publishing type：Research paper (scientific journal)  

The low molecular weight organic compound bromovalerylurea (BU) has long been used as a hypnotic/sedative. In the present study, we found that BU suppressed mRNA expression of proinflammatory factors and nitric oxide release in lipopolysaccharide (LPS)-treated rat primary microglial cell cultures. BU prevented neuronal degeneration in LPS-treated neuron-microglia cocultures. The anti-inflammatory effects of BU were as strong as those of a synthetic glucocorticoid, dexamethasone. A rat hemi-Parkinsonian model was prepared by injecting 6-hydroxydopamine into the right striatum. BU was orally administered to these rats for 7 days, which ameliorated the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and alleviated motor deficits. BU suppressed the expression of mRNAs for interferon regulatory factors (IRFs) 1, 7 and 8 in the right (lesioned) ventral midbrain as well as those for proinflammatory mediators. BU increased mRNA expression of various neuroprotective factors, including platelet-derived growth factor and hepatocyte growth factor, but it did not increase expression of alternative activation (M2) markers. In microglial culture, BU suppressed the LPS-induced increase in expression of IRFs 1 and 8, and it reduced LPS-induced phosphorylation of JAK1 and STATs 1 and 3. Knockdown of IRFs 1 and 8 suppressed LPS-induced NO release by microglial cells. These results suggest that suppression of microglial IRF expression by BU prevents neuronal cell death in the injured brain region, where microglial activation occurs. Because many Parkinsonian patients suffer from sleep disorders, BU administration before sleep may effectively ameliorate neurological symptoms and alleviate sleep dysfunction.

DOI： 10.1016/j.neuint.2016.06.013

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Language：English   Publishing type：Research paper (scientific journal)  

Exercise may be one of the most effective and sound therapies for stroke; however, the mechanisms underlying the curative effects remain unclear. In this study, the effects of forced treadmill exercise with electric shock on ischemic brain edema were investigated. Wistar rats were subjected to transient (90 min) middle cerebral artery occlusion (tMCAO). Eighty nine rats with substantially large ischemic lesions were evaluated using magnetic resonance imaging (MRI) and were randomly assigned to exercise and non-exercise groups. The rats were forced to run at 4-6m/s for 10 min/day on days 2, 3 and 4. Brain edema was measured on day 5 by MRI, histochemical staining of brain sections and tissue water content determination (n=7, each experiment). Motor function in some rats was examined on day 30 (n=6). Exercise reduced brain edema (P<0.05-0.001, varied by the methods) and ameliorated motor function (P<0.05). The anti-glucocorticoid mifepristone or the anti-mineralocorticoid spironolactone abolished these effects, but orally administered corticosterone mimicked the ameliorating effects of exercise. Exercise prevented the ischemia-induced expression of mRNA encoding aquaporin 4 (AQP4) and Na(+)/H(+) exchangers (NHEs) (n=5 or 7, P<0.01). Microglia and NG2 glia expressed NHE1 in the peri-ischemic region of rat brains and also in mixed glial cultures. Corticosterone at ~10nM reduced NHE1 and AQP4 expression in mixed glial and pure microglial cultures. Dexamethasone and aldosterone at 10nM did not significantly alter NHE1 and AQP4 expression. Exposure to a NHE inhibitor caused shrinkage of microglial cells. These results suggest that the stressful short-period and slow-paced treadmill exercise suppressed NHE1 and AQP4 expression resulting in the amelioration of brain edema at least partly via the moderate increase in plasma corticosterone levels.

DOI： 10.1016/j.expneurol.2015.12.016

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Noradrenaline (NA) has marked anti-inflammatory effects on activated microglial cells. The present study was conducted to elucidate the mechanisms underlying the NA effects using rat primary cultured microglial cells. NA, an α1 agonist, phenylephrine (Phe) and a β2 agonist, terbutaline (Ter) suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) release by microglia and prevented neuronal degeneration in LPS-treated neuron-microglia coculture. The agents suppressed expression of mRNA encoding proinflammatory mediators. Both an α1-selective blocker terazocine and a β2-selective blocker butoxamine overcame the suppressive effects of NA. cAMP-dependent kinase (PKA) inhibitors did not abolish the suppressive NA effects. LPS decreased IκB leading to NFκB translocation into nuclei, then induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). NA inhibited LPS-induced these changes. When NFκB expression was knocked down with siRNA, LPS-induced STAT1 phosphorylation and IRF1 expression was abolished. NA did not suppress IL-6 induced STAT1 phosphorylation and IRF1 expression. These results suggest that one of the critical mechanisms underlying the anti-inflammatory effects of NA is the inhibition of NFκB translocation. Although inhibitory effects of NA on STAT1 phosphorylation and IRF1 expression may contribute to the overall suppressive effects of NA, these may be the downstream events of inhibitory effects on NFκB. Since NA, Phe and Ter exerted almost the same effects and PKA inhibitors did not show significant antagonistic effects, the suppression by NA might not be dependent on specific adrenergic receptors and cAMP-dependent signaling pathway.

DOI： 10.1016/j.neuint.2015.07.010

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Sepsis is a severe pathologic event, frequently causing death in critically ill patients. However, there are no approved drugs to treat sepsis, despite clinical trials of many agents that have distinct targets. Therefore, a novel effective treatment should be developed based on the pathogenesis of sepsis. We recently observed that an old hypnotic drug, bromvalerylurea (BU) suppressed expression of many kinds of pro- and anti-inflammatory mediators in LPS- or interferon-γ activated alveolar and peritoneal macrophages (AMs and PMs). Taken the anti-inflammatory effects of BU on macrophages, we challenged it to septic rats that had been subjected to cecum-ligation and puncture (CLP). BU was subcutaneously administered to septic rats twice per day. Seven days after CLP treatment, 85% of septic rats administrated vehicle had died, whereas administration of BU reduce the rate to 50%. Septic rats showed symptoms of multi-organ failure; respiratory, circulatory and renal system failures as revealed by histopathological analyses, blood gas test and others. BU ameliorated these symptoms. BU also prevented elevated serum-IL-6 level as well as IL-6 mRNA expression in septic rats. Collectively, BU might be a novel agent to ameliorate sepsis by preventing the onset of MOF.

DOI： 10.1016/j.bbrc.2015.02.111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Physical Fitness and Sports Medicine  

The variable effects of mechanical stimuli induced by exercise on the human body are becoming better understood. Additionally, the indispensable effect of constant gravity on the human body to construct and maintain living organisms is known from observed muscle and bone regression induced by long-term recumbency or visits to gravity-free environments, such as space. Based on reactions of the body, cells faced with various inputs largely depend on gene expression, biochemical processes, or both. Thus, it is easy to imagine that physical input can be converted into a chemical process. The conversion process that changes physical forces (mechanical stimuli) into chemical reactions in a cell is called mechanotransduction. A growing number of studies examining mechanotransduction have led to a new phase in the understanding of exercise-induced effects on organisms.

DOI： 10.7600/jpfsm.4.83

CiNii Books

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Authorship：Lead author   Language：English  

Zonisamide has been proven as an effective drug for the recovery of degenerating dopaminergic neurons in the animal models of Parkinson's disease. However, several lines of evidence have questioned the neuroprotective capacity of zonisamide in animal models of Parkinson's disease. Although it suppresses dopaminergic neurodegeneration in animal models, the cellular and molecular mechanisms underlying the effectiveness of zonisamide are not fully understood. The current study demonstrates the effects of zonisamide on astrocyte cultures and two 6-hydroxydopamine-induced models of Parkinson's disease. Using primary astrocyte cultures, we showed that zonisamide up-regulated the expression of mRNA encoding mesencephalic astrocyte-derived neurotrophic factor, vascular endothelial growth factor, proliferating cell nuclear antigen, metallothionein-2, copper/zinc superoxide dismutase, and manganese superoxide dismutase. Similar responses to zonisamide were found in substantia nigra where the rats were pre-treated with 6-hydroxydopamine. Notably, pharmacological inhibition of 6-hydroxydopamine-induced toxicity by zonisamide pre-treatment was also confirmed using rat mesencephalic organotypic slice cultures of substantia nigra. In addition to this, zonisamide post-treatment also attenuated the nigral tyrosine hydroxylase-positive neuronal loss induced by 6-hydroxydopamine. Taken together, these studies demonstrate that zonisamide protected dopamine neurons in two Parkinson's disease models through a novel mechanism, namely increasing the expression of some important astrocyte-mediated neurotrophic and anti-oxidative factors.

DOI： 10.1016/j.ejphar.2012.05.012

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Randomized clinical trials have shown that pramipexole has an antidepressant effect in patients with Parkinson's disease. We investigated the comparative efficacy of pramipexole toward dopamine receptor D(2) and D(3) expression in rat brain. Groups of rats were treated subacutely with pramipexole (1 mg/kg), imipramine (10 mg/kg), or bromocriptine (5 mg/kg), with appropriate controls. Using real-time RT-PCR and immunoblotting, dopamine receptor D(2) and D(3) expression was up-regulated in the striatum following pramipexole treatment, while imipramine and bromocriptine had no significant effects. These findings support that pramipexole exerts additional therapeutic benefits such as decreasing depression by increasing dopamine receptor D(3) expression in the striatum.

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/brb3.11

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/brb3.11

Authorship：Lead author   Language：English  

Zonisamide is an antiepileptic drug that also improves the cardinal symptoms of Parkinson's disease. This study investigated the effects of zonisamide on dopaminergic neuronal degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Six groups of mice were treated as follows: 1) normal saline; 2) MPTP, 15 mg/kg×4 every 2h; 3) MPTP and zonisamide, 40 mg/kg×1, 1h after the last MPTP dose; 4) MPTP and zonisamide, 1 day after the last dose of MPTP; 5) MPTP and zonisamide, 1h before the first MPTP dose; and 6) zonisamide, 40 mg/kg. MPTP-treatment decreased the contents of dopamine as well as the number and area of tyrosine hydroxylase (TH)-positive neurons. Concurrent treatment of mice with zonisamide and MPTP did not show any inhibition of the toxic effect of MPTP towards dopamine contents at 1 week after treatment but it increased the number and area of TH-positive neurons compared to the MPTP-treated group. Surviving TH-positive neurons had recovery of dopamine production after several weeks. Moreover, zonisamide increased the number of S100β-positive and glial fibrillary acidic protein (GFAP)-positive astrocytes and dopamine turnover. These results suggest that zonisamide acts as a neuro-protectant against MPTP-induced dopaminergic neuronal degeneration as shown by an increase of TH-positive neurons and this may be mediated by increased S100β secretion.

DOI： 10.1016/j.brainres.2011.02.017

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MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces parkinsonism in humans and animals. The effects of zonisamide on dopamine neurons were studied in MPTP-treated common marmosets (Callithrix jacchus). Groups of animals (n = 3) were treated with MPTP (2.5 mg/kg, every 24 h × 3); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (a known MAO-B inhibitor) (2 mg/kg administered 1 h before each MPTP dose); and saline controls. An immunohistochemical study of the substantia nigra was performed 14 days after MPTP treatment in each group. MPTP reduced the mean number of tyrosine hydroxylase (TH)-positive neurons to 10% of the normal control group and mean cell size was significantly (P < 0.001) reduced from 424 to 159 μm2. In the group pre-treated with zonis amide, the mean number of TH-positive neurons was reduced to 26% of that in the normal control group and the mean neuron size was significantly (P < 0.05) increased from 159 to 273 μm2 compared with the group treated with MPTP alone. Moreover, in the group pre-treated with selegiline, the mean number of TH-positive neurons was 47% of that in the normal control group and the mean neuron size was increased significantly (P < 0.01) from159 to 319 μm2 compared to the group treated with MPTP alone. This observation suggests that zonisamide reduces MPTP toxicity. © 2010 The Japanese Pharmacological Society.

DOI： 10.1254/jphs.10120FP

Scopus

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The effect of zonisamide, an antiepileptic agent with anti-parkinsonian effects, was studied on dopamine neurons of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57 mice and common marmosets. Groups of mice (n = 8 - 9) were treated with: MPTP (15 mg/kg every 2 h x4); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (2 mg/kg administered 1 h before the first MPTP dose); zonisamide (40 mg/kg x4); and saline controls. Groups of common marmosets (n = 4 - 6) were treated with: MPTP (2.5 mg/kg every 24 h x3); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (2 mg/kg administered 1 h before the first MPTP dose); and saline controls. Brain dopamine and its metabolites were determined by HPLC. Dopamine content decreased in the striatum of MPTP-treated mice and monkeys. Co-administration of selegiline inhibited the effect of MPTP and dopamine contents were similar to those of the controls. Co-administration of zonisamide did not inhibit the effect of MPTP on dopamine content, but increased striatal dopamine turnover of animals treated with MPTP plus zonisamide more than in those treated with MPTP alone. MPTP treatment caused a compensatory increase of dopamine turnover in the striatum by remaining neurons. Zonisamide may help dopaminergic neurons by increasing striatal dopamine turnover following MPTP treatment.

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