Publications by authors named "Makoto Sawada"

99 Publications

Size and surface modification of silica nanoparticles affect the severity of lung toxicity by modulating endosomal ROS generation in macrophages.

Part Fibre Toxicol 2021 06 17;18(1):21. Epub 2021 Jun 17.

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.

Background: As the application of silica nanomaterials continues to expand, increasing chances of its exposure to the human body and potential harm are anticipated. Although the toxicity of silica nanomaterials is assumed to be affected by their physio-chemical properties, including size and surface functionalization, its molecular mechanisms remain unclear. We hypothesized that analysis of intracellular localization of the particles and subsequent intracellular signaling could reveal a novel determinant of inflammatory response against silica particles with different physico-chemical properties.

Results: We employed a murine intratracheal instillation model of amorphous silica nanoparticles (NPs) exposure to compare their in vivo toxicities in the respiratory system. Pristine silica-NPs of 50 nm diameters (50 nm-plain) induced airway-centered lung injury with marked neutrophilic infiltration. By contrast, instillation of pristine silica particles of a larger diameter (3 μm; 3 μm-plain) significantly reduced the severity of lung injury and neutrophilic infiltration, possibly through attenuated induction of neutrophil chemotactic chemokines including MIP2. Ex vivo analysis of alveolar macrophages as well as in vitro assessment using RAW264.7 cells revealed a remarkably lower cellular uptake of 3 μm-plain particles compared with 50 nm-plain, which is assumed to be the underlying mechanism of attenuated immune response. The severity of lung injury and neutrophilic infiltration was also significantly reduced after intratracheal instillation of silica NPs with an amine surface modification (50 nm-NH) when compared with 50 nm-plain. Despite unchanged efficacy in cellular uptake, treatment with 50 nm-NH induced a significantly attenuated immune response in RAW264.7 cells. Assessment of intracellular redox signaling revealed increased reactive oxygen species (ROS) in endosomal compartments of RAW264.7 cells treated with 50 nm-plain when compared with vehicle-treated control. In contrast, augmentation of endosomal ROS signals in cells treated with 50 nm-NH was significantly lower. Moreover, selective inhibition of NADPH oxidase 2 (NOX2) was sufficient to inhibit endosomal ROS bursts and induction of chemokine expressions in cells treated with silica NPs, suggesting the central role of endosomal ROS generated by NOX2 in the regulation of the inflammatory response in macrophages that endocytosed silica NPs.

Conclusions: Our murine model suggested that the pulmonary toxicity of silica NPs depended on their physico-chemical properties through distinct mechanisms. Cellular uptake of larger particles by macrophages decreased, while surface amine modification modulated endosomal ROS signaling via NOX2, both of which are assumed to be involved in mitigating immune response in macrophages and resulting lung injury.
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http://dx.doi.org/10.1186/s12989-021-00415-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210371PMC
June 2021

The sodium-glucose cotransporter-2 inhibitor Tofogliflozin prevents the progression of nonalcoholic steatohepatitis-associated liver tumors in a novel murine model.

Biomed Pharmacother 2021 Aug 21;140:111738. Epub 2021 May 21.

Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Department of Immunometabolism, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address:

Background: Diabetes and obesity contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). However, how diabetes and obesity accelerate liver tumorigenesis remains to be fully understood. Moreover, to verify the therapeutic potential of anti-diabetic drugs, there exists a strong need for appropriate animal models that recapitulate human pathophysiology of NASH and HCC.

Methods: We established a novel murine model of NASH-associated liver tumors using genetically obese melanocortin 4 receptor-deficient mice fed on Western diet in combination with a chemical procarcinogen, and verified the validity of our model in evaluating drug efficacy.

Findings: Our model developed multiple liver tumors together with obesity, diabetes, and NASH within a relatively short period (approximately 3 months). In this model, sodium glucose cotransporter 2 inhibitor Tofogliflozin prevented the development of NASH-like liver phenotypes and the progression of liver tumors. Tofogliflozin attenuated p21 expression of hepatocytes in non-tumorous lesions in the liver.

Interpretation: Tofogliflozin treatment attenuates cellular senescence of hepatocytes under obese and diabetic conditions. This study provides a unique animal model of NASH-associated liver tumors, which is applicable for assessing drug efficacy to prevent or treat NASH-associated HCC.
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http://dx.doi.org/10.1016/j.biopha.2021.111738DOI Listing
August 2021

Secretion of signal peptides via extracellular vesicles.

Biochem Biophys Res Commun 2021 Jun 5;560:21-26. Epub 2021 May 5.

Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, 464-8601, Japan; Department of Molecular Pharmacokinetics, Nagoya University Graduate School of Medicine, Nagoya, Aichi, 464-8601, Japan.

Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the proteins to the endoplasmic reticulum (ER). It was thought that the SPs are intracellularly degraded after translocation to the ER; however, recent studies showed cleaved SPs have diverse roles for controlling cell functions in auto- and/or intercellular manners. In addition, it still remains obscure how SP fragments translocate away from the site where they are produced. Extracellular vesicles (EV) are important for intercellular communication and can transport functional molecules to specific cells. In this study, we show that SPs are involved in EV from T-REx AspALP cells that were transfected with a human APP SP-inducible expression vector. There was no difference in the average particle size or particle concentration of EV collected from T-REx AspALP cells and T-REx Mock cells. When the SP content in the EV was examined by mass spectrometry, the C-terminal fragment of APP SP was identified in the exosomes (SEV) of T-REx AspALP cells. In our preparation of SEV fractions, no ER-specific proteins were detected; therefore, SPs may be included in SEV but not in the debris of degraded ER. This is the first indication that SPs are secreted from cells via EV.
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http://dx.doi.org/10.1016/j.bbrc.2021.04.073DOI Listing
June 2021

Detection of Alzheimer's disease-related neuroinflammation by a PET ligand selective for glial versus vascular translocator protein.

J Cereb Blood Flow Metab 2021 Aug 8;41(8):2076-2089. Epub 2021 Feb 8.

Department of Functional Brain Imaging Research, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan.

A substantial and constitutive expression of translocator protein (TSPO) in cerebral blood vessels hampers the sensitive detection of neuroinflammation characterized by greatly induced TSPO expression in activated glia. Here, we conducted in vivo positron emission tomography (PET) and in vitro autoradiographic imaging of normal and TSPO-deficient mouse brains to compare the binding properties of F-FEBMP, a relatively novel TSPO radioligand developed for human studies based on its insensitivity to a common polymorphism, with C-PK11195, as well as other commonly used TSPO radioligands including C-PBR28, C-Ac5216 and F-FEDAA1106. TSPO in cerebral vessels of normal mice was found to provide a major binding site for C-PK11195, C-PBR28 and F-FEDAA1106, in contrast to no overt specific binding of F-FEBMP and C-Ac5216 to this vascular component. In addition, F-FEBMP yielded PET images of microglial TSPO with a higher contrast than C-PK11195 in a tau transgenic mouse modeling Alzheimer's disease (AD) and allied neurodegenerative tauopathies. Moreover, TSPO expression examined by immunoblotting was significantly increased in AD brains compared with healthy controls, and was well correlated with the autoradiographic binding of F-FEBMP but not C-PK11195. Our findings support the potential advantage of comparatively glial TSPO-selective radioligands such as F-FEBMP for PET imaging of inflammatory glial cells.
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http://dx.doi.org/10.1177/0271678X21992457DOI Listing
August 2021

Personality traits associated with freezing of gait in Parkinson's disease patients.

Parkinsonism Relat Disord 2020 12 10;81:67-68. Epub 2020 Oct 10.

National Hospital Organization Matsue Medical Center, 5-8-31 Agenogi, Matsue-shi, Shimane, 690-8556, Japan.

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http://dx.doi.org/10.1016/j.parkreldis.2020.10.005DOI Listing
December 2020

Peripheral benzodiazepine receptor/18 kDa translocator protein positron emission tomography imaging in a rat model of acute brain injury.

Ann Nucl Med 2021 Jan 28;35(1):8-16. Epub 2020 Sep 28.

Department of Brain Function, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.

Objective: The activation of microglia in various brain pathologies is accompanied by an increase in the expression of peripheral benzodiazepine receptor/18 kDa translocator protein (PBR/TSPO). However, whether activated microglia have a neuroprotective or neurotoxic effect on neurons in the brain is yet to be determined. In this study, we investigated the ability of the novel PBR/TSPO ligand FEPPA to detect activated microglia in an animal model of primary neurotoxic microglia activation.

Methods: [F] FEPPA positron emission tomography (PET) imaging was performed before and after intraperitoneal administration of lipopolysaccharide (LPS) (LPS group) or saline (control group) in a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease. Images were compared between these groups. After imaging, the brains were collected, and the activated microglia at the disease sites were analyzed by the expression of inflammatory cytokines and immunohistochemistry staining. These results were then comparatively examined with those obtained by PET imaging.

Results: In the unilateral 6-OHDA lesion rat model, the PBR/TSPO PET signal was significantly increased in the LPS group compared with the saline group. As the increased signal was observed 4 h after the injection, we considered it an acute response to brain injury. In the post-imaging pathological examination, activated microglia were found to be abundant at the site where strong signals were detected, and the expression of the inflammatory cytokines TNF-α and IL-1β was increased. Intraperitoneal LPS administration further increased the expression of inflammatory cytokines, and the PBR/TSPO PET signal increased concurrently. The increase in inflammatory cytokine expression correlated with enhanced signal intensity.

Conclusions: PET signal enhancement by PBR/TSPO at the site of brain injury correlated with the activation of microglia and production of inflammatory cytokines. Furthermore, because FEPPA enables the detection of neurotoxic microglia on PET images, we successfully constructed a novel PET detection system that can monitor neurodegenerative diseases.
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http://dx.doi.org/10.1007/s12149-020-01530-2DOI Listing
January 2021

Inhibition of cellular inflammatory mediator production and amelioration of learning deficit in flies by deep sea Aspergillus-derived cyclopenin.

J Antibiot (Tokyo) 2020 09 24;73(9):622-629. Epub 2020 Mar 24.

Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 480-1195, Nagakute, Japan.

In the course of screening lipopolysaccharide (LPS)-induced nitric oxide (NO) production inhibitors, two related benzodiazepine derivatives, cyclopenol and cyclopenin, were isolated from the extract of a deep marine-derived fungal strain, Aspergillus sp. SCSIOW2. Cyclopenol and cyclopenin inhibited the LPS-induced formation of NO and secretion of IL-6 in RAW264.7 cells at nontoxic concentrations. In terms of the mechanism underlying these effects, cyclopenol and cyclopenin were found to inhibit the upstream signal of NF-κB activation. These compounds also inhibited the expression of IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) in mouse microglia cells, macrophages in the brain. In relation to the cause of Alzheimer's disease, amyloid-β-peptide is known to induce inflammation in the brain. Therefore, the present study investigated the ameliorative effects of these inhibitors on an in vivo Alzheimer's model using flies. Learning deficits were induced by the overexpression of amyloid-β42 in flies, and cyclopenin but not cyclopenol was found to rescue learning impairment. Therefore, novel anti-inflammatory activities of cyclopenin were identified, which may be useful as a candidate of anti-inflammatory agents for neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41429-020-0302-9DOI Listing
September 2020

Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis-associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects.

Sci Rep 2020 01 22;10(1):983. Epub 2020 Jan 22.

Department of Molecular Medicine and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development.
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http://dx.doi.org/10.1038/s41598-020-57935-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976646PMC
January 2020

Novel Oxindole-Curcumin Hybrid Compound for Antioxidative Stress and Neuroprotection.

ACS Chem Neurosci 2020 01 18;11(1):76-85. Epub 2019 Dec 18.

NAGARAGAWA Research Center, API Co., Ltd. , Gifu 502-0071 , Japan.

Oxidative stress plays an important role in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. The oxindole compound GIF-2165X-G1 is a hybrid molecule composed of the oxindole skeleton of the neuroprotective compound GIF-0726-r and the polyphenolic skeleton of the antioxidant curcumin. We previously reported that novel oxindole derivatives such as GIF-0726-r and GIF-2165X-G1 prevent endogenous oxidative stress-induced cell death in mouse hippocampal HT22 cells. In this study, we present a detailed investigation of the effect of GIF-2165X-G1 on endogenous oxidative stress in HT22 cells in comparison with GIF-0726-r and curcumin. GIF-2165X-G1 exhibited more potent neuroprotective activity than GIF-0726-r or curcumin and had less cytotoxicity than that observed with curcumin. Both GIF-0726-r and GIF-2165X-G1 were found to have ferrous ion chelating activity similar to that exhibited by curcumin. GIF-2165 X-G1 and curcumin induced comparable antioxidant response element transcriptional activity. Although the induction of heme oxygenase-1, an antioxidant response element-regulated gene product, was much stronger in curcumin-treated cells than in GIF-2165X-G1-treated cells, it turned out that the induction of heme oxygenase-1 is dispensable for neuroprotection. These results demonstrate that the introduction of the polyphenol skeleton of curcumin to the oxindole GIF-0726-r improves neuroprotective features. Furthermore, intrastriatal injection of GIF-2165X-G1 alleviated apomorphine-induced rotation and prevented dopaminergic neuronal loss in a 6-hydroxydopamine mouse model of Parkinson's diseases. Collectively, our novel findings indicate that the novel oxindole compound GIF-2165X-G1 serves to delay the progression of Parkinson's disease by suppressing oxidative stress.
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http://dx.doi.org/10.1021/acschemneuro.9b00619DOI Listing
January 2020

Clinical features of freezing of gait in Parkinson's disease patients.

Brain Behav 2019 04 9;9(4):e01244. Epub 2019 Mar 9.

National Hospital Organization Matsue Medical Center, Matue, Japan.

Objective: To clarify the clinical features of freezing of gait (FOG) in Parkinson's disease (PD) patients by classification into two groups: Clinically observed FOG (CFOG) and self-reported FOG (SFOG).

Methods: Two hundred twenty-nine PD patients were medically examined in an examination room as well as subjected to a New Freezing of Gait Questionnaire (NFOG-Q) and analysis of nonmotor symptoms including sleep, cognition, depression, and fatigue.

Results: The prevalence of CFOG was 17.9%, while 53.7% of the patients without CFOG reported the presence of FOG via the NFOG-Q. Univariate analysis revealed that CFOG was associated with longer disease duration, motor dysfunction, sleepiness, fatigue, and cognitive dysfunction. These symptoms, excluding akinesia, apathy, rapid eye movement (REM) sleep Behavior Disorder, and cognitive dysfunction, were also associated with SFOG. Multivariate analysis revealed that long PD duration, postural instability, and gait difficulty (PIGD), along with fatigue, were independent factors for SFOG.

Conclusions: SFOG and CFOG have many common clinical features. Although the clinical relevance of SFOG remains unclear, careful attention should be paid to related features in clinical practice.
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http://dx.doi.org/10.1002/brb3.1244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456785PMC
April 2019

Anti-Claudin Antibodies as a Concept for Development of Claudin-Directed Drugs.

J Pharmacol Exp Ther 2019 02 7;368(2):179-186. Epub 2018 Dec 7.

Research Institute of Environmental Medicine, Nagoya University, Aichi, Japan (Y.H., M.S.) and Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., K.S., K.T., K.Y., T.D., M.K.)

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.
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http://dx.doi.org/10.1124/jpet.118.252361DOI Listing
February 2019

Hitomi X-ray studies of Giant Radio Pulses from the Crab pulsar.

Publ Astron Soc Jpn Nihon Tenmon Gakkai 2018 Apr;70(2)

Institute of Astronomy, University of Cambridge, Madingley Road, Cambridge, CB3 0HA, UK.

To search for giant X-ray pulses correlated with the giant radio pulses (GRPs) from the Crab pulsar, we performed a simultaneous observation of the Crab pulsar with the X-ray satellite Hitomi in the 2 - 300 keV band and the Kashima NICT radio observatory in the 1.4 - 1.7 GHz band with a net exposure of about 2 ks on 25 March 2016, just before the loss of the Hitomi mission. The timing performance of the Hitomi instruments was confirmed to meet the timing requirement and about 1,000 and 100 GRPs were simultaneously observed at the main and inter-pulse phases, respectively, and we found no apparent correlation between the giant radio pulses and the X-ray emission in either the main or inter-pulse phases. All variations are within the 2 sigma fluctuations of the X-ray fluxes at the pulse peaks, and the 3 sigma upper limits of variations of main- or inter-pulse GRPs are 22% or 80% of the peak flux in a 0.20 phase width, respectively, in the 2 - 300 keV band. The values become 25% or 110% for main or inter-pulse GRPs, respectively, when the phase width is restricted into the 0.03 phase. Among the upper limits from the Hitomi satellite, those in the 4.5-10 keV and the 70-300 keV are obtained for the first time, and those in other bands are consistent with previous reports. Numerically, the upper limits of main- and inter-pulse GRPs in the 0.20 phase width are about (2.4 and 9.3) ×10 erg cm, respectively. No significant variability in pulse profiles implies that the GRPs originated from a local place within the magnetosphere and the number of photon-emitting particles temporally increases. However, the results do not statistically rule out variations correlated with the GRPs, because the possible X-ray enhancement may appear due to a > 0.02% brightening of the pulse-peak flux under such conditions.
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http://dx.doi.org/10.1093/pasj/psx083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999749PMC
April 2018

Effectiveness of Home-Based Exercises Without Supervision by Physical Therapists for Patients With Early-Stage Amyotrophic Lateral Sclerosis: A Pilot Study.

Arch Phys Med Rehabil 2018 10 31;99(10):2114-2117. Epub 2018 Mar 31.

Department of Neurology, National Hakone Hospital, Kanagawa, Japan.

Objective: To verify the effects of structured home-based exercises without supervision by a physical therapist in patients with early-stage amyotrophic lateral sclerosis (ALS).

Design: A historical controlled study that is part of a multicenter collaborative study.

Setting: Rehabilitation departments at general hospitals and outpatient clinics with a neurology department.

Participants: Patients (N=21) with ALS were enrolled and designated as the home-based exercise (Home-EX) group, and they performed unsupervised home-based exercises. As a control group, 84 patients with ALS who underwent supervised exercise with a physical therapist for 6 months were extracted from a database of patients with ALS and matched with the Home-EX group in terms of their basic attributes and clinical features.

Intervention: The Home-EX group was instructed to perform structured home-based exercises without supervision by a physical therapist that consisted of muscle stretching, muscle training, and functional training for 6 months.

Main Outcome Measures: The primary outcome was the score on the ALS Functional Rating Scale-Revised (ALSFRS-R), which is composed of 3 domains: bulbar function, limb function, and respiratory function. The score ranges from 0 to 48 points, with a higher score indicating better function.

Results: In the Home-EX group, 15 patients completed the home-based exercises for 6 months, and 6 patients dropped out because of medical reasons or disease progression. No adverse events were reported. The Home-EX group was found to have a significantly higher respiratory function subscore and total score on the ALSFRS-R than the control group at follow-up (P<.001 and P<.05, respectively).

Conclusions: Structured home-based exercises without supervision by a physical therapist could be used to alleviate functional deterioration in patients with early-stage ALS.
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http://dx.doi.org/10.1016/j.apmr.2018.02.015DOI Listing
October 2018

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

Neuropharmacology 2018 06 15;135:242-252. Epub 2018 Mar 15.

Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu, Japan.

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells.
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http://dx.doi.org/10.1016/j.neuropharm.2018.03.015DOI Listing
June 2018

Inducible nitric oxide synthase during the late phase of sepsis is associated with hypothermia and immune cell migration.

Lab Invest 2018 05 14;98(5):629-639. Epub 2018 Feb 14.

Department of Emergency and Critical Care Medicine, Nagoya University Graduate School of Medicine, Aichi, 466-8560, Japan.

Hypothermia is a significant sign of sepsis, which is associated with poor prognosis, but few mechanisms underlying the regulation of hypothermia are known. Inducible nitric oxide synthase (iNOS) is a key inflammatory mediator of sepsis. However, the therapeutic benefit of iNOS inhibition in sepsis is still controversial, and requires elucidation in an accurate model system. In this study, wild-type (WT) mice showed temperature drops in a biphasic manner at the early and late phase of sepsis, and all mice died within 48 h of sepsis. In contrast, iNOS-knockout (KO) mice never showed the second temperature drop and exhibited improved mortality. Plasma nitric oxide (NO) levels of WT mice increased in the late phase of sepsis and correlated to hypothermia. The results indicate that iNOS-derived NO during the late phase of sepsis caused vasodilation-induced hypothermia and a lethal hypodynamic state. The expression of the iNOS mRNA was high in the lung of WT mice with sepsis, which reflects the pathology of acute respiratory distress syndrome (ARDS). We obtained the results in a modified keyhole-type cecal ligation and puncture model of septic shock induced by minimally invasive surgery. In this accurate and reproducible model system, we transplanted the bone marrow cells of GFP transgenic mice into WT and iNOS-KO mice, and evaluated the role of increased pulmonary iNOS expression in cell migration during the late phase of sepsis. We also investigated the quantity and type of bone marrow-derived cells (BMDCs) in the lung. The number of BMDCs in the lung of iNOS-KO mice was less than that in the lung of WT mice. The major BMDCs populations were CD11b-positive, iNOS-negative cells in WT mice, and Gr-1-positive cells in iNOS-KO mice that expressed iNOS. These results suggest that sustained hypothermia may be a beneficial guide for future iNOS-targeted therapy of sepsis, and that iNOS modulated the migratory efficiency and cell type of BMDCs in septic ARDS.
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http://dx.doi.org/10.1038/s41374-018-0021-zDOI Listing
May 2018

Visualization of Arc promoter-driven neuronal activity by magnetic resonance imaging.

Neurosci Lett 2018 02 21;666:92-97. Epub 2017 Dec 21.

Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, 464-8601, Japan.

Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.
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http://dx.doi.org/10.1016/j.neulet.2017.12.041DOI Listing
February 2018

Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis.

EBioMedicine 2018 Jan 7;27:71-85. Epub 2017 Dec 7.

Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.

Angiogenesis, a prominent feature of pathology, is known to be guided by factors secreted by living cells around a lesion. Although many cells are disrupted in a response to injury, the relevance of degenerating cells in pathological angiogenesis is unclear. Here, we show that the release of lactate dehydrogenase A (LDHA) from degenerating neurons drives central nervous system (CNS) angiogenesis. Silencing neuronal LDHA expression suppressed angiogenesis around experimental autoimmune encephalomyelitis (EAE)- and controlled cortical impact-induced lesions. Extracellular LDHA-mediated angiogenesis was dependent on surface vimentin expression and vascular endothelial growth factor receptor (VEGFR) phosphorylation in vascular endothelial cells. Silencing vimentin expression in vascular endothelial cells prevented angiogenesis around EAE lesions and improved survival in a mouse model of glioblastoma. These results elucidate novel mechanisms that may mediate pathologic angiogenesis and identify a potential molecular target for the treatment of CNS diseases involving angiogenesis.
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http://dx.doi.org/10.1016/j.ebiom.2017.10.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828296PMC
January 2018

LC-MS/MS imaging with thermal film-based laser microdissection.

Anal Bioanal Chem 2018 Jan 28;410(2):491-499. Epub 2017 Nov 28.

Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan.

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.
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http://dx.doi.org/10.1007/s00216-017-0739-2DOI Listing
January 2018

Optogenetic control of cell differentiation in channelrhodopsin-2-expressing OS3, a bipotential glial progenitor cell line.

Neurochem Int 2017 Mar 6;104:49-63. Epub 2017 Jan 6.

Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.

Alterations in the intracellular ion environment have been identified as one of the signals playing a critical role in the control of cellular proliferation and differentiation; however, the mechanisms responsible for signal transduction remain unclear. Recent studies have reported that channelrhodopsin-2 (ChR2) is a rapidly gated blue light (BL)-sensitive cation channel suitable for the non-invasive control of ion influx. We herein examined the expression of differentiation-associated markers by photo-activation and its signal transduction in ChR2-expressing OS3 (OS3ChR2) cells, which are clonal bipotential glial progenitor cells. Increases were observed in intracellular Na and Ca concentrations in OS3ChR2 cells with BL exposure. Alterations in the intracellular ion environment, particularly in Ca, led to increases in the expression of oligodendrocyte markers including galactocerebrosides (GalC) and decreases in that of astrocyte markers such as glial fibrillary acidic protein (GFAP). These alterations also triggered activation of the ERK1/2 signaling pathway, which is involved in cell survival, and PI3K/Akt/mTOR signaling pathway, which is involved in oligodendrocyte differentiation, characterized by GalC expression. Moreover, when photo-activated OS3ChR2 cells were injected into mice with lysophosphatidyl choline (LPC)-induced demyelination, deficits in motor function were reduced. Our results demonstrated that signal transduction by ChR2-expressing glial progenitor cells may be controlled through alterations induced in the intracellular ion environment by photo-activation and results in oligodendrocyte differentiation from glial progenitor cells. Our results also suggest that ChR2-expressing glial progenitor cells have potential as a useful tool for therapeutic approaches to brain and spinal cord disorders associated with oligodendrocyte dysfunctions.
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http://dx.doi.org/10.1016/j.neuint.2016.12.022DOI Listing
March 2017

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.

Glia 2016 11 27;64(11):1938-61. Epub 2016 Jul 27.

Department of Molecular Cell Function, Faculty of Life Sciences, Kumamoto University, 5-1 Ohe-Honmachi, Kumamoto, 862-0973, Japan.

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961.
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http://dx.doi.org/10.1002/glia.23034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129557PMC
November 2016

Soluble Siglec-9 suppresses arthritis in a collagen-induced arthritis mouse model and inhibits M1 activation of RAW264.7 macrophages.

Arthritis Res Ther 2016 06 7;18(1):133. Epub 2016 Jun 7.

Department of Oral and Maxillofacial Surgery/Protective Care for Masticatory Disorders, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.

Background: The aim of this study was to assess the effects of soluble sialic acid-binding immunoglobulin-type lectin (sSiglec)-9 on joint inflammation and destruction in a murine collagen-induced arthritis (CIA) model and in monolayer cultures of murine macrophages (RAW264.7 cells and peritoneal macrophages) and fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis.

Methods: DBA/1J mice were immunized with type II collagen. Effects of sSiglec-9 were evaluated using a physiologic arthritis score, histological analysis, serum tumor necrosis factor (TNF)-α concentration, and the proportion of forkhead box P3 (Foxp3)-positive regulatory T (Treg) cells. In vivo biofluorescence imaging was used to assess the distribution of sSiglec-9. Levels of M1 (TNF-α, interleukin [IL]-6, and inducible nitric oxide synthase) and M2 (CD206, Arginase-1, and IL-10) macrophage markers and phosphorylation of intracellular signaling molecules were examined in macrophages, and levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were examined in FLS.

Results: sSiglec-9 significantly suppressed the clinical and histological incidence and severity of arthritis. The proportion of Foxp3-positive Treg cells significantly improved and serum TNF-α concentration decreased in vivo. Although sSiglec-9 reduced the expression of M1 markers in macrophages, it did not affect the expression of M2 markers and MMPs in FLS. Nuclear factor (NF)-kB p65 phosphorylation was attenuated by sSiglec-9, and chemical blockade of the NF-kB pathway reduced M1 marker expression in RAW264.7 cells.

Conclusions: In this study, we have demonstrated the therapeutic effects of sSiglec-9 in a murine CIA model. The mechanism underlying these effects involves the suppression of M1 proinflammatory macrophages by inhibiting the NF-kB pathway. sSiglec-9 may provide a novel therapeutic option for patients with rheumatoid arthritis refractory to currently available drugs.
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http://dx.doi.org/10.1186/s13075-016-1035-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4897938PMC
June 2016

Nanoparticles speckled by ready-to-conjugate lanthanide complexes for multimodal imaging.

Nanoscale 2015 Sep 24;7(36):14829-37. Epub 2015 Jul 24.

Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-Cho, Takamatsu, Kagawa 761-0395, Japan.

Multimodal and multifunctional contrast agents receive enormous attention in the biomedical imaging field. Such contrast agents are routinely prepared by the incorporation of organic molecules and inorganic nanoparticles (NPs) into host materials such as gold NPs, silica NPs, polymer NPs, and liposomes. Despite their non-cytotoxic nature, the large size of these NPs limits the in vivo distribution and clearance and inflames complex pharmacokinetics, which hinder the regulatory approval for clinical applications. Herein, we report a unique method that combines magnetic resonance imaging (MRI) and fluorescence imaging modalities together in nanoscale entities by the simple, direct and stable conjugation of novel biotinylated coordination complexes of gadolinium(III) to CdSe/ZnS quantum dots (QD) and terbium(III) to super paramagnetic iron oxide NPs (SPION) but without any host material. Subsequently, we evaluate the potentials of such lanthanide-speckled fluorescent-magnetic NPs for bioimaging at single-molecule, cell and in vivo levels. The simple preparation and small size make such fluorescent-magnetic NPs promising contrast agents for biomedical imaging.
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http://dx.doi.org/10.1039/c5nr00959fDOI Listing
September 2015

Insular neural system controls decision-making in healthy and methamphetamine-treated rats.

Proc Natl Acad Sci U S A 2015 Jul 6;112(29):E3930-9. Epub 2015 Jul 6.

Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Nagoya 466-8560, Japan;

Patients suffering from neuropsychiatric disorders such as substance-related and addictive disorders exhibit altered decision-making patterns, which may be associated with their behavioral abnormalities. However, the neuronal mechanisms underlying such impairments are largely unknown. Using a gambling test, we demonstrated that methamphetamine (METH)-treated rats chose a high-risk/high-reward option more frequently and assigned higher value to high returns than control rats, suggestive of changes in decision-making choice strategy. Immunohistochemical analysis following the gambling test revealed aberrant activation of the insular cortex (INS) and nucleus accumbens in METH-treated animals. Pharmacological studies, together with in vivo microdialysis, showed that the insular neural system played a crucial role in decision-making. Moreover, manipulation of INS activation using designer receptor exclusively activated by designer drug technology resulted in alterations to decision-making. Our findings suggest that the INS is a critical region involved in decision-making and that insular neural dysfunction results in risk-taking behaviors associated with altered decision-making.
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http://dx.doi.org/10.1073/pnas.1418014112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517258PMC
July 2015

Multi-Acupuncture Point Injections and Their Anatomical Study in Relation to Neck and Shoulder Pain Syndrome (So-Called Katakori) in Japan.

PLoS One 2015 5;10(6):e0129006. Epub 2015 Jun 5.

Department of Anatomy, Tokai University School of Medicine, Isehara-shi, Kanagawa, Japan.

Katakori is a symptom name that is unique to Japan, and refers to myofascial pain syndrome-like clinical signs in the shoulder girdle. Various methods of pain relief for katakori have been reported, but in the present study, we examined the clinical effects of multi-acupuncture point injections (MAPI) in the acupuncture points with which we empirically achieved an effect, as well as the anatomical sites affected by liquid medicine. The subjects were idiopathic katakori patients (n = 9), and three cadavers for anatomical investigation. BL-10, GB-21, LI-16, SI-14, and BL-38 as the WHO notation were selected as the acupuncture point. Injections of 1 mL of 1% w/v mepivacaine were introduced at the same time into each of these points in the patients. Assessment items were the Pain Relief Score and the therapeutic effect period. Dissections were centered at the puncture sites of cadavers. India ink was similarly injected into each point, and each site that was darkly-stained with India ink was evaluated. Katakori pain in the present study was significantly reduced by MAPI. Regardless of the presence or absence of trigger points, pain was significantly reduced in these cases. Dark staining with India ink at each of the points in the anatomical analysis was as follows: BL-10: over the rectus capitis posterior minor muscle and rectus capitis posterior major muscle fascia; GB-21: over the supraspinatus muscle fascia; LI-16: over the supraspinatus muscle fascia; SI-14: over the rhomboid muscle fascia; and BL-38: over the rhomboid muscle fascia. The anatomical study suggested that the drug effect was exerted on the muscles above and below the muscle fascia, as well as the peripheral nerves because the points of action in acupuncture were darkly-stained in the spaces between the muscle and the muscle fascia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457803PMC
March 2016

Rapid Trimming of Cell Surface Polysialic Acid (PolySia) by Exovesicular Sialidase Triggers Release of Preexisting Surface Neurotrophin.

J Biol Chem 2015 May 6;290(21):13202-14. Epub 2015 Mar 6.

From the Bioscience and Biotechnology Center and School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan,

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation.
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http://dx.doi.org/10.1074/jbc.M115.638759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505574PMC
May 2015

Radiosynthesis and in vivo evaluation of two imidazopyridineacetamides, [(11)C]CB184 and [ (11)C]CB190, as a PET tracer for 18 kDa translocator protein: direct comparison with [ (11)C](R)-PK11195.

Ann Nucl Med 2015 May 24;29(4):325-35. Epub 2015 Jan 24.

Department of Clinical and Experimental Neuroimaging, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Obu, Aichi, 474-8522, Japan,

Objective: We report synthesis of two carbon-11 labeled imidazopyridines TSPO ligands, [(11)C]CB184 and [(11)C]CB190, for PET imaging of inflammatory process along with neurodegeneration, ischemia or brain tumor. Biodistribution of these compounds was compared with that of [(11)C]CB148 and [(11)C](R)-PK11195.

Methods: Both [(11)C]CB184 and [(11)C]CB190 having (11)C-methoxyl group on an aromatic ring were readily prepared using [(11)C]methyl triflate. Biodistribution and metabolism of the compounds were examined with normal mice. An animal PET study using 6-hydroxydopamine treated rats as a model of neurodegeneration was pursued for proper estimation of feasibility of the radioligands to determine neuroinflammation process.

Results: [(11)C]CB184 and [(11)C]CB190 were obtained via O-methylation of corresponding desmethyl precursor using [(11)C]methyl triflate in radiochemical yield of 73 % (decay-corrected). In vivo validation as a TSPO radioligand was carried out using normal mice and lesioned rats. In mice, [(11)C]CB184 showed more uptake and specific binding than [(11)C]CB190. Metabolism studies showed that 36 % and 25 % of radioactivity in plasma remained unchanged 30 min after intravenous injection of [(11)C]CB184 and [(11)C]CB190, respectively. In the PET study using rats, lesioned side of the brain showed significantly higher uptake than contralateral side after i.v. injection of either [(11)C]CB184 or [(11)C](R)-PK11195. Indirect Logan plot analysis revealed distribution volume ratio (DVR) between the two sides which might indicate lesion-related elevation of TSPO binding. The DVR was 1.15 ± 0.10 for [(11)C](R)-PK11195 and was 1.15 ± 0.09 for [(11)C]CB184.

Conclusion: The sensitivity to detect neuroinflammation activity was similar for [(11)C]CB184 and [(11)C](R)-PK11195.
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http://dx.doi.org/10.1007/s12149-015-0948-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835529PMC
May 2015

Novel positively charged nanoparticle labeling for in vivo imaging of adipose tissue-derived stem cells.

PLoS One 2014 3;9(11):e110142. Epub 2014 Nov 3.

Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan; Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho 2217-14, Takamatsu 761-0395, Japan.

Stem cell transplantation has been expected to have various applications for regenerative medicine. However, in order to detect and trace the transplanted stem cells in the body, non-invasive and widely clinically available cell imaging technologies are required. In this paper, we focused on magnetic resonance (MR) imaging technology, and investigated whether the trimethylamino dextran-coated magnetic iron oxide nanoparticle -03 (TMADM-03), which was newly developed by our group, could be used for labeling adipose tissue-derived stem cells (ASCs) as a contrast agent. No cytotoxicity was observed in ASCs transduced with less than 100 µg-Fe/mL of TMADM-03 after a one hour transduction time. The transduction efficiency of TMADM-03 into ASCs was about four-fold more efficient than that of the alkali-treated dextran-coated magnetic iron oxide nanoparticle (ATDM), which is a major component of commercially available contrast agents such as ferucarbotran (Resovist), and the level of labeling was maintained for at least two weeks. In addition, the differentiation ability of ASCs labeled with TMADM-03 and their ability to produce cytokines such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), were confirmed to be maintained. The ASCs labeled with TMADM-03 were transplanted into the left kidney capsule of a mouse. The labeled ASCs could be imaged with good contrast using a 1T MR imaging system. These data suggest that TMADM-03 can therefore be utilized as a contrast agent for the MR imaging of stem cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110142PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4217721PMC
August 2015

Protective effect of INI-0602, a gap junction inhibitor, on dopaminergic neurodegeneration of mice with unilateral 6-hydroxydopamine injection.

J Neural Transm (Vienna) 2014 Nov 18;121(11):1349-55. Epub 2014 Apr 18.

Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, 464-8601, Japan.

INI-0602, a novel gap junction hemichannel inhibitor, was administered to hemi-Parkinsonism mice generated by striatal 6-hydroxydopamine injection. INI-0602 prevented the toxic activation of microglia, such as the increased number of the activated form, enlargement of cell bodies and induction of proinflammatory cytokines, such as IL-1β and TNFα, in the ipsilateral striatum. On the other hand, INI-0602 induced the expression of neurotrophic factors, such as brain-derived neurotrophic factor and NT-4/5, in the 6-hydroxydopamine-treated striatum. INI-0602 treatment blocked not only dopaminergic loss in both the striatum and substantia nigra, but also apomorphine-induced rotational behavior.
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http://dx.doi.org/10.1007/s00702-014-1209-zDOI Listing
November 2014

The effect of lipid emulsion on intracellular bupivacaine as a mechanism of lipid resuscitation: an electrophysiological study using voltage-gated proton channels.

Anesth Analg 2013 Dec;117(6):1293-301

From the Departments of *Anesthesiology, and †Physiology, Osaka City University Graduate School of Medicine, Osaka; and ‡Department of Brain Life Science, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.

Background: Lipid resuscitation has become a standard treatment for local anesthetic (LA) systemic toxicity, but its mechanisms remain to be fully elucidated. Although the partitioning effect is one of the proposed mechanisms, it is difficult to evaluate its impact independently from several other mechanisms or to examine the intracellular concentration of a LA, which is primarily responsible for LA systemic toxicity. We recently reported that LAs as weak bases reduced voltage-gated proton currents by increasing intracellular pH, which could be estimated from the reversal potentials of the channels (Vrev). Using this characteristic, we examined the partitioning effect in detail and showed its impact on lipid resuscitation.

Methods: A whole-cell voltage clamp technique was used to record proton channel currents in a rat microglial cell line (GMI-R1). We used Intralipid® 20% as lipid emulsion. The effects of lipid emulsion on the intracellular concentrations of LAs were evaluated by measuring the current amplitude and the Vrev. The intracellular concentrations of LAs were calculated by the Henderson-Hasselbalch equation, using estimated intracellular pH. To confirm the importance of partitioning, we separated lipid by centrifugation. Data are means ± SD unless otherwise stated.

Results: Bupivacaine (1 mM) decreased proton currents to 43% ± 10% of the control and shifted the Vrev to positive voltages (from -88.0 ± 4.1 to -76.0 ± 5.5 mV, n = 5 each, P = 0.02). An addition of the lipid emulsion recovered the currents to 79% ± 2% of the control and returned the Vrev toward the control value (to -86.0 ± 7.1 mV, n = 5, P = 0.03). Both recoveries of the current and Vrev in the centrifuged aqueous extract were almost the same as in the 4% lipid solution (-85.6 ± 4.9 mV, n = 5, P = 0.9, 95% confidence interval for difference = -9.3 to 8.6). When 1 mM bupivacaine was applied extracellularly, the intracellular concentration of the charged form of bupivacaine was estimated to reach about 18.1 ± 3.9 mM but decreased to 5.4 ± 1.8 mM by the 4% lipid solution.

Conclusions: Here we quantitatively evaluated for the first time the partitioning effect of lipid emulsion therapy on the intracellular concentration of bupivacaine in real-time settings by analyzing behaviors of voltage-gated proton channels. Our results suggested that lipid emulsion markedly reduced the intracellular concentration of bupivacaine, which was mostly due to the partitioning effect. This could contribute to our understanding of the mechanisms underlying lipid resuscitation, especially the importance of the partitioning effect.
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http://dx.doi.org/10.1213/ANE.0000000000000011DOI Listing
December 2013

Glutamate release from astrocyte cell-line GL261 via alterations in the intracellular ion environment.

J Neural Transm (Vienna) 2014 8;121(3):245-57. Epub 2013 Oct 8.

Department of Brain Function, Division of Stress Adaptation and Protection, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, 464-8601, Japan.

Astrocytes modify and maintain neural activity and functions via gliotransmitter release such as, glutamate. They also change their properties and functions in response to alterations of ion environment resulting from neurotransmission; however, the direct evidence for whether intracellular ion alteration in astrocytes triggers gliotransmitter release is not indicated. Recent studies have reported that channelrhodopsin-2 (ChR2) is useful for alteration of intracellular ion environment in several types of cells with blue light exposure. Here, we show that ChR2-expressing GL261 (GLChR2) cells, clonal astrocytes, change their properties by photo-activation. Increased intracellular sodium and calcium ion concentrations and an altered membrane potential were observed in GLChR2 cells with blue light exposure. Alterations in the intracellular ion environment caused intracellular acidification and the inhibition of proliferation. In addition, it triggered glutamate release from GLChR2 cells. Glutamate from GLChR2 cells acted on N18 cells, clonal neuronal cells, as both a transmitter and neurotoxin depending on photo-activation. Our results show that the properties of ChR2-expressing astrocytes can be controlled by blue light exposure, and cation influx through photo-activated ChR2 might trigger functional cation influx via endogenous channels and result in the increase of glutamate release. Further, our results suggest that ChR2-expressing glial cells could become a useful tool in understanding the roles of glial cell activation and neural communication in the regulation of brain functions.
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http://dx.doi.org/10.1007/s00702-013-1096-8DOI Listing
October 2014
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