神経保護、神経毒性と神経炎症 Neuroprotection, Neurotoxicity and Neuroinflammation
P1-2-187 Retinol palmitateはNotch1シグナル経路を介してマウス脳虚血性細胞変化を予防する Retinol palmitate prevents ischemia-induced cell changes in hippocampal neurons through the Notch1 signaling pathway in mice ○谷口順子1, 島田潤一郎1, 森雅裕1, 田桑弘之2, 伊藤浩2, 桑原聡1 ○Junko Taniguchi1, Jun-Ichiro Shimada1, Masahiro Mori1, Hiroyuki Takuwa2, Hiroshi Ito2, Satoshi Kuwabara1 千葉大学大学院　医学研究院　神経内科学1, 放射線医学総合研究所　分子イメージングセンター2 Dept of Neurology, Graduate School of Medicine, Chiba Univ, Chiba1, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba2 Retinol palmitate, an analogue of vitamin A, plays multiple roles in the nervous system, including neural differentiation, axon outgrowth and neural patterning, and also is an antioxidative agent and thereby potential neuroprotectant for brain ischemia. The aim of this study is to investigate the protective effects of retinol palmitate against ischemia-induced brain injury in a bilateral common carotid arteries occlusion (BCCAO) model in mice. Ischemia induced by 20 min BCCAO resulted in significant neuronal morphological changes, and reactive astrocyte proliferation in the hippocampus, particularly in CA1 region, and these changes were accompanied by increased Notch1 expression. Intraperitoneal retinol palmitate administration before ischemia significantly reduced ischemic neurons with Notch1 expression; the effects were obvious by the treatment with retinol palmitate at 12mg/kg, but not at 1.2mg/kg. These results show that retinol palmitate prevents brain ischemia-induced neuronal injury with Notch1 expression, and the Notch1 signaling could be involved in the neuroprotective mechanisms. Retinol palmitate could be a treatment option for human brain infarction.	P1-2-188 虚血再灌流による視神経損傷モデルに対する水素分子の神経保護効果 Molecular hydrogen protects against ischemic injury in optic nerves ○山藤芽実1, 藤田慶大1秋元望1, 城戸瑞穂3, 田中喜典4, 中別府雄作5野田百美1 ○Megumi Yamafuji1, Kyota Fujita1, Margaret A Hamner2, Nozomi Akimoto1, Mizuho Kido3, Yoshinori Tanaka4, Yuusaku Nakabeppu5, Bruce R Ransom2, Mami Noda1 九州大院・薬・病態生理1, ワシントン大・医・神経2, 九州大院・歯・口腔常態制御3, （株）パナソニック・アプライアンス社・技術本部4, 九州大・生医研・脳機能制御5 Lab Pathophysiol, Sch of Pharm, Kyushu Univ, Fukuoka1, Dept Neurol, Sch of Med, Univ of Washington, Seattle2, Dept Oral Anat & Cell Biol, Sch of Dental Science, Kyushu Univ, Fukuoka3, Cooporate Enginnering Division, Appiances Company, Panasonic Corp, Japan4, Div Neurofunctional Genomics, Med Inst of Bioregulation, Kyushu Univ, Fukuoka5 Background: Molecular hydrogen selectively reduces hydroxyl radicals, the most cytotoxic of reactive oxygen species (ROS). Hydrogen (H2) in drinking water reduced dopaminergic neuronal loss by buffering ROS in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice (Fujita et al., 2009). Central nervous system (CNS) white matter (WM) ischemia is an important clinical problem and may produce injury, in part, by ROS-induced mitochondrial dysfunction. Using the mouse optic nerve (MON) WM model, we tested whether hydrogen in drinking water reduced functional WM ischemic injury. Results: A 60 min period of oxygen and glucose deprivation (OGD) caused prompt loss of the compound action potential (CAP) followed by an average 20% recovery. After mice received H2-containing drinking water for 7-10 days, the CAP area did not disappear during ischemia and recovered to a significantly great extent during reperfusion with oxygen and glucose. Immunostaining of axonal neurofilament by SMI-31 labeling also showed significant protection by previous drinking of H2-water. Accumulation of nuclear 8-oxoguanine, a marker of oxidative DNA damage, was observed mainly in oligodendrocytes after OGD. The level of 8-oxoguanine after OGD were significantly reduced in optic nerves from H2-water drinking mice.Conclusions: Our results show that several days of H2 exposure reduced the extent of CNS WM irreversible injury associated with OGD. The importance of these observations is that ischemic protection of myelinated CNS WM took several days to develop, lasted several days and provided partial protection in a novel manner from what has been previously described. These observations raise intriguing therapeutic options.
P1-2-189 少量のメタンフェタミンを前投与したラット中脳においてはATF6を介した小胞体ストレス応答が活性化される The neuroprotective effect of low-dose methamphetamine pretreatment against high-dose methamphetamine neurotoxicity in the rat midbrain is mediated by an unfolded protein response involving ATF6 ○武市敏明1, 王路1, 北村修1 ○Toshiaki Takeichi1, Elaine Lu Wang1, Osamu Kitamura1 金沢医科大学　医学部　法医学講座1 Dept Legal Med, Kanazawa Med Univ, Kanazawa, Japan1 We have previously reported that low-dose methamphetamine (METH) pretreatment inhibits apoptosis induced by high-dose METH administration in the rat midbrain. In this study, we examined an unfolded protein response (UPR) and the expression of an apoptosis-related protein and its mRNA after the administration of high-dose METH to the midbrain of rats pretreated with low-dose METH.The expression of ATF6, which is one of the key proteins involved in the UPR, increased significantly; however, the mRNA levels of spliced XBP1, which is another key genes, remained unchanged. In addition, the expression of ATF4, which is located downstream of PERK-a downstream effectors in another UPR, increased significantly upon pretreatment with both low-dose METH and saline.Furthermore, the phosphorylation of Ser2481 in mTOR, which is an element in the mTOR complex 2, increased significantly.The expression of phospho-AKT1, which inhibited ASK1 activity via the dephosphorylation of ASK1, increased significantly, and that of phospho-ASK1 involved in an apoptotic pathway was inhibited.These findings suggest that the inhibition of apoptosis involves the inhibition of activated ASK1 via the mTORC2-AKT1 pathway through the ATF6.	P1-2-190 視神経損傷後のニューログロビンの発現－マウスとゼブラフィッシュの比較検討 Comparative study of neuroglobin in mouse and zebrafish retina after optic nerve injury ○世良真悠子1, 杉谷加代2, 藤川千恵子3, 大貝和裕2, 若杉桂輔4, 荒井國三1, 加藤聖3 ○Mayuko Sera1, Kayo Sugitani2, Chieko Fujikawa3, Kazuhiro Ogai2, Keisuke Wakasugi4, Kunizo Arai1, Satoru Kato3 金沢大・医薬保・薬1, 金沢大院・医・保2, 金沢大院・医・脳情報分子3, 東京大院・総合・生4 Dept Clin Drug Info, Faculty of Pharm, Inst Med Pharm Health Sci, Kanazawa Univ, Ishikawa1, Div Health Sci, Grad Sch Med Sci, Kanazawa Univ, Ishikawa2, Dept Mol Neurobiol, Grad Sch Med Sci, Kanazawa Univ, Ishikawa3, Dept Life Sci, Grad Sch Arts Sci, Univ of Tokyo, Tokyo4 Neuroglobin (Ngb), a new member of the globin family, is specifically expressed in neurons. Previous studies have shown that mammalian Ngb has a neuroprotective function, and overexpression of Ngb protects retinal ganglion cells (RGCs) from apoptotic cell death caused by glaucomatous, hypoxic and ischemic insults. In this study we investigated the changes of Ngb expression in mouse retina following optic nerve injury (ONI) where RGCs undergo apoptosis. Further, we performed a comparative study in zebrafish retina where RGCs can survive and regrow new axons following ONI. Mouse Ngb (Ngb-m) mRNA was faintly detected in control retina and increased by 1.7-fold at 3 days post-optic nerve injury (dpi). Immunohistochemical staining revealed that Ngb-m protein was mainly detected in RGCs. On the other hand, zebrafish Ngb (Ngb-z) mRNA was significantly increased by 2.5-fold at 3 dpi in the amacrine cells. In addition, Ngb-z expressing amacrine cells merged with NADPH diaphorase and neuronal nitric oxide synthase (nNOS)-positive cells. Taken together, the cellular localization of Ngb was different in mouse and zebrafish retinas after ONI, although the level of Ngb was upregulated in both animals. To investigate the functional role of upregulation of Ngb in optic nerve regeneration, we prepared retinal explant cultures in the presence of recombinant Ngb-z protein. The addition of recombinant Ngb-z induced significant neurite sprouting on fish retinal explant cultures. Therefore, Ngb-z might have the function of neurite sprouting. These results suggested that Ngb-m and Ngb-z have different function after ONI. To elucidate the function of Ngb-m, we are now in progress to study the effect of Ngb-m overexpression against oxidative stress.
P1-2-191 [18F]FDGを用いたイメージングによる脳組織グルコース代謝への非ステロイド性抗炎症薬セレコキシブの作用 Effect of non-steroidal anti-inflammatory drug celecoxib on cerebral glucose metabolism in rat brain slices measured with dynamic positron autoradiography ○浅井竜哉1,2,3, 福本真也2岡沢秀彦1,3 ○Tatsuya Asai1,2,3, Shinya Fukumoto2, Wei Wang1, Hidehiko Okazawa1,3 福井大・ 高エネ1, 福井大院・工2, 福井大・生命センター3 Biomed Imaging Res Ctr, Univ of Fukui, Fukui1, Grad School Eng, Univ of Fukui, Fukui2, Res & Ed Prog for Life Sci, Univ of Fukui, Fukui3 Background: Celecoxib (CXB) is a non-steroidal anti-inflammatory drug (NSAID) that specifically inhibits cyclooxygenase-2 and thus is widely used as a drug with less gastric side effects. Recent studies indicate that NSAIDs including CXB increase intracellular Ca2+ concentration and cause apoptosis in various tumor cells. We have examined whether CXB has any effect on the cerebral metabolic rate of glucose in rat brain slices by measuring [18F]FDG uptake with dynamic positron autoradiography. Methods: Sagittal brain slices (300-μm thickness) were prepared from Wistar rats (6-7 weeks old). The slices were incubated in a chamber filled with oxygenated Krebs-Ringer solution kept at 36°C and containing 100 kBq/mL of [18F]FDG. Sequential images of [18]FDG uptake were obtained every 15 min by replacing imaging plates after being scanned with an image analyzer to measure radioactivity signals from the frontal cortical region of the slices. Results: Application of CXB (10-100 μM) increased [18F]FDG uptake rate gradually with time over 2 h and the degree of the CXB-induced increase was concentration dependent. Addition of membrane-permeable Ca2+ chelator BABTA-AM (50 μM) caused a slight decrease in the basal [18F]FDG uptake rate and largely inhibited the effect of CXB. On the other hand, the removal of Ca2+ from the external solution had no effect on the basal and the CXB-induced [18F]FDG uptake rate. Conclusion: CXB caused an increase in the brain metabolism by elevating the intracellular Ca2+ concentration that were most likely released from the internal Ca2+ stores such as endoplasmic reticulum and/or mitochondria.	P1-2-192 発達期ジフェニルアルシン酸曝露による行動異常と小脳における酸化ストレス Developmental exposure to diphenylarsinic acid induced impaired learning and locomotor behavior and oxidative stress in rat cerebellum ○根岸隆之1, 松永友貴1, 小林弥生2, 平野靖史郎2, 田代朋子1 ○Takayuki Negishi1, Yuki Matsunaga1, Yayoi Kobayashi2, Seishiro Hirano2, Tomoko Tashiro1 青山学院大学　理工学部　化学・生命科学科1, 国立環境研究所2 Aoyamagakuin University, Kanagawa1, National Institute for Environmental Studies, Ibaraki2 Diphenlyarsinic acid (DPAA) was detected in the well water in Kamisu, Ibaraki, Japan in 2003, which was released accidentally from a wasted concrete lump in underground, and some individuals drinking DPAA-containing water in this area complained of cerebellar symptom. In addition, a few children suspected to be exposed to DPAA developmentally showed mental retardation. Here, we investigated neurodevelopmental effects of exposure to DPAA in rats. Neonatal Wistar rats were exposed to DPAA via milk of their mother drinking water containing DPAA (20 mg/L) which was relevant to the dose detected in the well water. After weaning, male rats were exposed to DPAA directly via drinking water containing DPAA (20 mg/L). Behavioral tests, measurement of cerebellar glutathione, and immunohistochemistry and Western blotting were performed at 6 and 12 weeks of age. Both at 6 and at 12 weeks of age, male rats developmentally exposed to DPAA showed significantly increased locomotor activity in the open-field test and impaired learning ability in the step-through passive avoidance test. Immunohistochemistry and Western blotting for hemeoxygenase-1 (HO-1) and GFAP revealed that developmental exposure to DPAA increased expression of HO-1 and GFAP protein and induced deposition oxidative adducts both at 6 and at 12 weeks of age in the cerebellum. Concentration of total glutathione in the cerebellum was reduced by exposure to DPAA both at 6 and 12 weeks of age. These results indicated DPAA-induced oxidative stress in the cerebellum. Further, protein expression of NMDAR1 subunit and PSD95 was suppressed by developmental exposure to DPAA at 12 weeks of age, while that of GABAergic protein examined was not affected. These findings suggest that developmental exposure to DPAA induced oxidative stress in the cerebellum, which leads to abnormal behavioral development. In addition, developmental exposure to DPAA might disturb cerebellar glutamatergic neurotransmission in adulthood.
P1-2-193 1-ブロモプロパンの直接投与はラット海馬CA1切片の長期増強を抑制する Direct application of 1-bromopropane suppresses long-term potentiation in the rat CA1 hippocampal slices ○粟生修司1, 金光雅成1, 笛田由紀子2, 石田尾徹2, 保利一2 ○Shuji Aou1, Masanari Kanemitsu1, Yukiko Fueta2, Toru Ishidao2, Hajime Hori2 九州工業大学院・生命体工学・脳情報1, 産業医科大・産業保健・環境マネジメント2 Dept Brain Sci & Engineer, Kyushu Inst Technol, Kitakyushu1, Dept Environm Management, Sch Health Sci, Univ Occup & Environm Health2 Although many volatile organic compounds (VOCs) used in work places are neurotoxic, cellular mechanisms induced by their direct exposure to neurons are not well studied because of methodological difficulty concerning their high volatility. Occupational exposure of 1-bromopropane (1-BP) has been shown to elicit the neurotoxicity including impairment of memory function in human. Several studies revealed direct actions of 1-BP on NMDA, nACh and GABAA receptors, feedback inhibition in the hippocampus, and intracellular signals such as BDNF and Bcl-xL (Fueta et al, 2007). However the direct effects of 1-BP on learning and memory have not been well characterized. We developed a new exposure system of volatile organic compounds by dissolving high concentration of oxygen to the perfused medium applying gas-liquid equilibrium, and by using materials less adsorptive inside the tubing. In the present study, we examined the effects of direct application of 1-BP on long-term potentiation (LTP), considered as the cellular basis of learning and memory, in the CA1 of hippocampal slices in rats. The slices were made from young adult male rats (5-7 weeks). The field excitatory postsynaptic potentials (fEPSP) were recorded every 30 s from CA1 or DG in both groups. The slices of the 1-BP group were perfused with 1-BP at the concentration of 0.1, 1.0, 3.3, and 10 mM for 5 min just before tetanic stimulation, whereas those of the control group were free from 1-BP. The LTP was induced by two trains of 100 Hz stimulations for 1 s. The LTP of fEPSP were significantly and dose dependently suppressed immediately after tetanic stimulation in the CA1 and DG in the 1-BP group. These results indicate that neurotoxic effects of 1-BP on learning and memory functions are mediated by its direct effects on the hippocampal CA1 at least in part.	P1-2-194 Effects of inhalation exposure to secondary organic aerosol generated from diesel exhaust particles on hippocampal function in BALB/c mice ○Tin Tin Win Shwe1, Yuji Fujitani1, Hideko Sone1, Akiko Furuyama1, Hiroshi Nitta1, Seishiro Hirano1 National Institute for Environmental Studies1 Currently, the importance of secondary organic aerosol (SOA) formation in urban atmosphere is well recognized. SOA is produced in atmosphere through oxidation of volatile organic compounds by light, ozone, and NOx. Recently, we have reported that exposure to primary particles from diesel exhaust affect learning ability in mice. In vivo and in vitro studies have shown that SOA induced adverse effects in the lung and heart. However, the effect of SOA on the brain is still unclear. In the present study, we investigated the effect of one month inhalation exposure to SOA on learning ability and memory function-related gene expressions in the hippocampus of mice. We generated the SOA by oxidation of diesel exhaust particle (DEP) adding with ozone (O3). Five-week-old female BALB/c mice were exposed to clean air, DEP or DEP+O3 (SOA) 5 h per day, 5 days a week for four weeks. Twenty-four hours after last exposure to SOA, we investigated the neurotoxic effect of SOA exposure on learning ability and N-methyl-D-aspartate (NMDA) receptor and its signal transduction pathway-related genes and inflammatory markers using a novel object recognition test and a real-time RT-PCR analysis, respectively. Mice exposed to SOA could not discriminate between familiar and novel objects. Although no significant changes in the expression levels of the inflammatory markers in the hippocampus were observed, the expression of NMDA receptor subunit NR1 was significantly increased in the hippocampus of SOA-exposed mice compared with the control mice. We also found that plasma 8-hydroxy-2'-deoxyguanosine (8OHdG), an oxidative stress marker, was slightly increased in SOA-exposed mice. These findings indicated that exposure to SOA for one month impaired the novel object recognition ability by modulation of NMDA receptor subunit expression in the hippocampus. The present study suggests that oxidative stress may be involved in the SOA-induced memory impairment observed in the mouse model.
P1-2-195 Potential role of fibronectin in microglia/macrophage activation following cryoinjury in the rat brain ○Taekyun Shin1, Heechul Kim1,2, Meejung Ahn1,3, Sungyoung Choi1, Minsoo Kim1, Ki-Bum Sim4, Juhwan Kim5, Changjong Moon5 Dept Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju, Republic of Korea1, Paik Institute for Clinical Research, Inje University, Pusan, Republic of Korea2, Department of Anatomy, School of Medicine, Jeju National University, Jeju, Republic of Korea3, Department of Neurosurgery, School of Medicine, Jeju National University, Jeju, Republic of Korea4, Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea5 To investigate whether fibronectin, an extracellular matrix (ECM) and plasma protein, plays a role in the activation of microglia/macrophages after brain injury, we examined the changes in fibronectin and arginase-1, a marker for alternatively activated macrophages, in a rat cryoinjury model using Western blot analysis, real-time reverse transcription PCR and immunohistochemistry. The protein and mRNA level of fibronectin and arginase-1 significantly increased in the injury site of the ipsilateral cerebral cortex at days 4 and 7 after cryoinjury but was decreased at day 14. The immunohistochemical analysis revealed fibronectin expression in ED1-positive microglia/macrophages and reactive astrocytes, in the lesion core and in the lesion periphery, respectively. Fibronectin immunoreactivity in the lesion was similar to arginase-1 except that fibronectin was detected in the ECM after cryoinjury. The present results suggest that fibronectin was extravasated into injured brain lesions via an impaired blood-brain barrier and stimulated glial cells including microglia and infiltrating macrophages in the lesion core and periphery to become alternatively activated microglia/macrophages, which modulated CNS inflammation after brain injury.	P1-2-196 Temporal profiles of inflammatory cytokines and inflammation-related genes in BV-2 microglia with trimethyltin ○Juhwan Kim1, Younghoon Son1, Sung-Ho Kim1, Jong-Choon Kim1, Taekyun Shin2, Changjong Moon1 Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea1, Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju, South Korea2 Trimethyltin (TMT) is a potent neurotoxic chemical, results in neuroinflammation in central nervous system. In the present study, the expressions of pro-inflammatory (interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha), anti-inflammatory cytokines (IL-4 and IL-10), and inflammation-related genes (inducible nitric oxide (iNOS), arginase (ARG)-1, CD-206, and CD-32) in BV-2 cells after TMT treatment (0-10 mM) were examined to investigate the TMT-induced change in expressions of inflammation-related signals in murine microglia. The mRNA levels of IL-1beta, IL-6, TNF-alpha, and iNOS were significantly increased, whereas the levels of ARG-1, CD-206, and CD-32 were significantly decreased in BV-2 cells after TMT treatment, within time- (1-12 h post-treatment) and/or dose-dependent manner (0-10 mM). Therefore, it is suggested that the dynamic changes of inflammatory cytokines and inflammation-related genes in TMT-treated microglia are associated with the pathological processing of TMT-induced neuroinflammation.
P1-2-197 Minocycline improve prepulse inhibition (PPI) deficits through inhibition of microglial activation in the dentate gyrus of Gunn rat: a neuroinflammatory animal model of schizophrenia ○Kristian Liaury1,3, Tsuyoshi Miyaoka1, Toshiko Tsumori2, Motohide Furuya1, Rei Wake1, Masa Ieda1, Keiko Tsuchie1, Michiyo Fukushima1, Andi Jayalangkara Tanra3, Jun Horiguchi1 Department of Psychiatry, Shimane University Faculty of Medicine1, Department of Anatomy and Morphological Neuroscience, Shimane University Faculty of Medicine2 Schizophrenia is a chronic and devastating illness with the etiology that remains unclear. Our previous study showed the evidence of microglial activation within the dentate gyrus of Gunn rat with hyperbilirubinemia. Consequently, we suggested that neuroinflammation may play an important role in the pathophysiology of schizophrenia and supports the possibility of using Gunn rat as an animal model of schizophrenia. (Liaury K et al. J Neuroinflammation, 2012).We recently reported that minocycline, a second generation tetracycline, improved the recognition memory impairment (NORT) test in Gunn rat through the inhibition of microglial activation in the dentate gyrus. On the other hand, we also found that Gunn rats clearly showed impaired in prepulse inhibition (PPI) for the acoustic startle response. PPI is a measure of sensory gating, which is clearly reduced in schizophrenia patients. Therefore, in the present study, we examined whether or not minocyline improve impaired PPI test in Gunn rats. After intraperitoneal administrations of either saline or minocycline (40 mg/kg) once a day for 14 consecutive days in Gunn rats and Wistar rats as a control, we performed the PPI test. Later, we measured the ratio of activated and resting microglial cells in the dentate gyrus using immunohistochemical technique. As a result, we found that Gunn rats treated with minocycline showed improvement in PPI test compared to Gunn rats treated with saline. Moreover, Gunn rats treated with minocycline showed less ratio of activated and resting microglial cells compared to saline and control group. These results suggest that minocycline improved cognitive dysfunction and also support the therapeutic strategy of schizophrenia through the inhibition of microglia activation.

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