脳血管障害と虚血 Cerebrovascular Disease and Ischemia
P2-1-215 脳梗塞急性期における運動による認知機能回復と海馬BDNF濃度の上昇との関係 The effect of the elevation of BDNF in hippocampus on recovery of cognitive function in acute-phase of cerebral infarction model rats ○氷見直之1, 高橋尚2, 岡部直彦1, 古我知成2, 中村恵美1, 成田和彦1, 宮本修1 ○Naoyuki Himi1, Hisashi Takahashi2, Naohiko Okabe1, Tomoshige Koga2, Emi Nakamura1, Kazuhiko Narita1, Osamu Miyamoto1 川崎医大・生理21, 川崎医療福祉大・リハビリテーション2 Dept Physiol 2, Kawasaki Med Sch, Kurashiki, Japan1, Dept Rehabilitation, Kawasaki Univ Med Welfare, Kurashiki, Japan2 We investigated the effectiveness of exercise in acute-phase on recovery of cognitive function after stroke. The injection of 3,000 particles of microsphere (MS, φ45μm) through the right internal carotid artery induced small infarctions in cortex, striatum and hippocampus in rats. The operated animals presented mild motor paralysis at 24 hours after MS injection. Mild exercise was started at the 24 hours after MS injection by a treadmill (15m/min × 30min/day for 7days) in the acute-phase exercise group. A late-phase exercise group started exercise at 8 days after MS injection, and non-exercise group and sham operated group were also examined as control groups. The cognitive function was evaluated by Morris water-maze test and significantly improved in the acute-phase exercise group compared with the other groups. On the other hand, both acute-phase and late-phase exercise groups showed an elevation of brain-derived neurotrophic factor (BDNF) in the hippocampus. To investigate the relationship between the elevation of BDNF in hippocampus and recovery of cognitive function, rats were infused BDNF into their hippocampus continuously by osmotic pump in acute-phase or late-phase after stroke. The rats that were infused BDNF in acute-phase showed significant recovery in cognitive function, however, rats with the BDNF infusion in late-phase showed no recovery. These results suggest that exercise in acute-phase after onset of cerebral infarction and the resultant elevation of BDNF level is critical to improve cognitive function.	P2-1-216 脳アミロイドアンギオパチーの副腎皮質ステロイド治療による脳血管アミロイド退縮機序のプロテオミクス解析 Proteomic analysis of AΒ amyloid and its related proteins from cerebral amyloid angiopathy with or without corticosteroid treatment ○亀谷富由樹1, 池田修一2 ○Fuyuki Kametani1, Shu-ichi Ikeda2 公益財団法人東京都医学総合研究所　認知症・高次脳機能研究分野1, 信州大学医学部脳神経内科、リウマチ・膠原病内科2 Department of Dementia and Higher Brain Function Research, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan1, Department of Medicine (Neurology and Rheumatology), Shinshu University School of Medicine, Matsumoto, Japan2 Recently, we reported the histopathological changes in cerebrovascular Aβ amyloid deposition between biopsied and autopsied findings in a cerebral amyloid angiopathy (CAA) patient actively treated with corticosteroid, showing possible post-treatment regression of cerebrovascular amyloid deposits. To clarify this mechanism, we analyzed amyloid and its related proteins from CAA with or without corticosteroid treatment using the proteomic techniques and compared their proteins' profiles. We found novel oxidized-amino acid residues, fragmentations and truncations of N-terminal and/or C-terminal peptides of Aβ and its derivatives from CAA extracts without treatment, while only intact Aβ was detectable from those with corticosteroid treatment. Furthermore, amyloid-associated proteins were significantly reduced in the CAA extracts with corticosteroid treatment, where the reduction of S100A9 was most remarkable. Accumulation of Aβ amyloid fibrils on cerebral vessels may secondarily induce local inflammation and promote vascular wall degeneration, leading to cerebral hemorrhages. S100A9 seems to be a key protein in the biological responses to vascular injury and also may accelerate the formation of Aβ amyloid fibrils. These suggest that corticosteroid treatment subsides local inflammation and activation of S100A9, resulting in suppression of Aβ amyloid fibril and its-associated protein formations in cerebrovascular walls. Corticosteroid treatment is, therefore, considered to be a potentially effective treatment for CAA.
P2-1-217 内包領域での軸索傷害を焦点とした新規脳内出血病態進行メカニズム Novel mechanisms of pathological progression of intracerebral hemorrhage focused on axonal injury in internal capsule ○肱岡雅宣1, 松下英明1, 石橋勇人1, 久恒昭哲1, 礒濱洋一郎1, 香月博志1 ○Masanori Hijioka1, Hideaki Matsushita1, Hayato Ishibashi1, Akinori Hisatsune1, Yoichiro Isohama1, Hiroshi Katsuki1 熊本大学大学院　生命科学研究部　薬物活性学1 Dept. Chemico-Pharmacol. Sci., Grad. Sch. Pharm. Sci., Kumamoto Univ., Kumamoto1 Intracerebral hemorrhage (ICH) results from formation of blood clot in brain parenchyma by rupture of blood vessels. Putamen and thalamus are well known as focus sites of hemorrhage. Hematoma expansion to the internal capsule (IC), the neighboring site of putamen and thalamus, leads to poor prognosis. Moreover, our previous study showed that hematoma formation in IC aggravated behavioral dysfunction in mouse model of ICH. Anatomically, IC is constituted by axonal fibers connecting cerebral cortex and spinal cord. These axonal fibers may govern behavioral functions. Accordingly, we investigated the pathological progression mechanisms of in mice, focusing on axonal injury. Behavioral dysfunction was apparent from 3 h after induction of ICH by collagenase injection near IC. Whereas, axonal truncation revealed by neurofilament H immunoreactivity was detectable from 6 h after ICH. Amyloid precursor protein (APP), transported along microtubules by axonal transport, accumulated in axon tracts from 3 h after ICH. Administration of colchicine, an inhibitor of axonal transport, into IC caused behavioral dysfunction and axonal truncation. These data suggest that inhibition of axonal transport by hemorrhagic stroke leads to behavioral dysfunction and axonal truncation. Administration of thrombin, a coagulation factor contained in the blood, also caused APP accumulation and axonal truncation with the same time course as ICH. In addition to APP accumulation, thrombin cleaved APP to low-molecular-weight fragments from 3 h after administration. In a previous report (Nicolaev et al., Nature, 2009), N-terminal fragment of APP was shown to bind to death receptor-6 and produce axonal truncation. Therefore, APP fragments may lead to pathological progression by truncating axon fibers in IC. Overall, sequential events involving thrombin-mediated inhibition of axonal transport, cleavage of APP, and axon truncation by APP fragments may have crucial roles in ICH-associated prognosis.	P2-1-218 脳梗塞後急性期のハロペリドールの投与は機能回復を遅延させる Aplication of haloperidole for acute-phase stroke mouse retarded the functional recovery by intact hemisphere ○高鶴裕介1, 天野出月1, 鯉淵典之1 ○Yusuke Takatsuru1, Izuki Amano1, Noriyuki Koibuchi1 群馬大学大学院・医学系研究科・応用生理学分野1 Dept. Integrative Physiol., Gunma Univ. Grad. Sch. Med., Maebashi1 Appropriate functional recovery after stroke is most important for improvement of quality of life. Contralateral hemisphere of the infarction site plays an important role for the recovery. However, the underlying processes in contralateral hemisphere during the recovery have not yet been elucidated. We have previously reported that the increase in synaptic turnover in contralateral somatosensory cortex during 1st week after infarction of unilateral somatosensory cortex. The neuronal circuit was remodeled after this period to process bilateral information to remaining hemisphere, and functional compensation was achieved. In the present study, we find that the concentration of dopamine in unilateral somatosensory cortex was increased at 1st week after the stroke under free-moving condition using in vivo microdialysis. Application of haloperidol, a typical D2 receptor antagonist, during this time period retarded the functional recovery. Those results indicate that the role of dopamine is important for functional recovery after focal stroke.
P2-1-219 新生児対酸素性虚血性脳症モデルおける臍帯血由来細胞移植の治療効果：個体差低減法を用いた評価 The therapeutic effects of the umbilical cord blood derived cells administration for neonatal hypoxic-ischemic encephalopathy: The novel method for reducing animal variability ○大嶌麻妃子1, 辻雅弘1, 山原研一1, 笠原由紀子2, 田口明彦2, 松山知弘3, 池田智明4 ○Makiko Ohshima1, Masahiro Tsuji1, Kenichi Yamahara1, Yukiko Kasahara2, Akihiko Taguchi2, Tomohiro Matsuyama3, Tomoaki Ikeda4 国立循環器病研究センター　再生医療部1, 先端医療振興財団　先端医療センター　再生医療研究部2, 兵庫医科大学　先端医学研究所　神経再生研究部門3, 三重大学大学院医学部　産科婦人科学教室4 Dept. Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, Osaka1, Department of Regenerative Medicine Research, Biomedical Research Innovation, Hyogo2, Institute for Advanced Medical Science, Hyogo College of Medicine, Hyogo3, Department of Obstetrics and Gynecology, Mie University School of Medicine, Mie4 Background&Objectives: Although the rodent model of neonatal hypoxic-ischemic encephalopathy (HIE) have been extensively used to establish therapeutic strategy, the variation on extent of cerebral injury make it difficult to evaluate the therapeutic effects in detail. Therefore we established a technique to analyze precisely the therapeutic effects despite inherent animal variability. The therapeutic effects of umbilical cord blood derived CD34+ cells administration were estimated using by this novel methods. Methods: Experiment 1) Postnatal-day-eight CB17 mice underwent left carotid artery ligation, followed by 30min of hypoxic exposure. Cortical surface cerebral blood flow (CBF) was measured by laser speckle imaging during 24h after the hypoxia. Experiment 2) HIE mice were divided into PBS-treated or cell-transplantated group so that the CBF level at 24h after the hypoxia became equal in the two groups. The CD34+ cells (1×105 cells/animal) were intravenously administrated at 48h. The cerebral morphological analysis and behavioral tests were performed at 7 weeks. Results&Conclusions: 1) The ratio of CBF at 24h was best correlated with the ratio of hemispheric volume at 7 days after HI (r2=0.86). These results demonstrated the level of CBF during reperfusion is available to predict the later brain injury whereby reduce the animal variability in this model. 2) HIE mice administrated CD34+ cells showed no difference in the cerebral injury and the rotarod test compared with PBS-treated mice. In the open field test, the cell-transplantation moderately improved hyperactivity observed in PBS-treated HIE mice but it did not reach statistical significance.	P2-1-220 ラット脳梗塞後のS-ニトロソグルタチオンと運動療法による神経修復と機能回復について Neurorepair and functional recovery by S-nitrosoglutathione and motor exercise in rats after ischemia reperfusion ○榊間春利1, , 吉田義弘1, 松田史代1 ○Harutoshi Sakakima1, Mushfiquddin Khan2, Yoshihiro Yoshida1, Fumiyo Matsuda1, Inderjit Singh2 鹿児島大学医学部保健学科　理学療法1, サウスカロライナ医科大学2 Sch Health Sci, Kagoshima Univ, Kagoshima, Japan1, Medical University of South Carolina, USA2 Stroke disability stems from insufficient neurorepair mechanisms. Limited improvement of neurobehavioral functions has been achieved through rehabilitation or neurovascular protective agents. Therefore, we combined motor exercise with a neurovascular protective agent S-nitrosoglutathione (GSNO) to enhance overall functional recovery. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) for 60 min followed by reperfusion in adult male rats. Injured animals were either treated with vehicle (IR group), GSNO (0.25 mg/kg, GSNO group), rotarod exercise (EX group) and GSNO plus exercise (GSNO+EX group). The groups were compared for 14 days in terms of neurorepair mechanisms and functional recovery. Treated groups (GSNO, EX and GSNO+EX) showed reduced infarction, decreased apoptotic neuronal cell death, enhanced expression of neurotrophic factors, improved tissue histology, and improved motor and neurological functions compared to the IR group. However, the GSNO+EX group showed greater functional recovery than either the GSNO or the EX group. A GSNO sub group, treated 24 hours after IR, still showed functional recovery. The protective effect of GSNO or exercise was blocked by an inhibition of AKT activity using the PI3 kinase inhibitor LY294002 compound. GSNO, like exercise, aids functional recovery in a 14-day treatment by stimulating neurorepair mechanisms. A combination of exercise and GSNO shows greater neurobehavioral improvement. Improved recovery with GSNO even administered 24 hours post-IR demonstrates its clinical relevance. Mechanistically, the improvements by GSNO and exercise may depend on AKT activity.
P2-1-221 Withdrawn	P2-1-222 アストロサイト依存型虚血耐性のメカニズム Mechanism of astrocytes-dependent ischemic tolerance ○平山友里1, 松尾由理2, 小泉修一1 ○Yuri Hirayama1, Yuri Ikeda-Matsuo2, Schuichi Koizumi1 山梨大院・医・薬理1, 北里大・薬・薬理2 Dept. Neuropharmacol., Interdisciplinary Grad. Sch. Med. and Engineering, Univ. Yamanashi1, Dept. Pharmacol., Sch. Pharm. Sci., Univ. Kitasato2 A brief episode of ischemia (preconditioning; PC) makes the brain resistant against subsequent lethal ischemia. This phenomenon is known as PC-induced neural ischemic tolerance. The ischemic tolerance has been shown in numerous experimental models of cerebral ischemia. We previously developed an ischemic tolerance model using a middle cerebral artery occlusion (MCAO) in mice, and found that activation of astrocytes by PC was essential for the ischemic tolerance. However, a mechanism underlying the astrocytes-dependent ischemic tolerance remains largely unknown. Here, we show that P2X7 receptors (ATP-gated cation channels) are the responsible molecule that causes the astrocytes-dependent ischemic tolerance. MCA was occluded transiently for 15 min for PC and 1 h for subsequent lethal MCAO, and then, infarct area was assessed by 2,3,5-triphenyl tetrazolium chloride staining 3 days after. P2X7 receptors were up-regulated in astrocytes by PC. Fluorocitrate, a metabolic inhibitor of astrocytes, inhibited the PC-induced activation of astrocytes, which also inhibited the up-regulation of P2X7 receptors and ischemic tolerance. Although P2X7 receptors were also expressed in microglia by PC, minocycline, an inhibitor of microglia, had no effect on ischemic tolerance. In P2X7 receptor deficient mice (P2X7-KO), the PC-induced ischemic tolerance disappeared, although astrocytic activation by PC in P2X7-KO was similar to that in wild-type mice. Taken together, these results suggest that PC-activated astrocytes should induce ischemic tolerance by up-regulating their P2X7 receptors.
P2-1-223 脳虚血/再灌流負荷後のスナネズミの海馬組織におけるアストロサイトとミクログリアの変動におよぼすリコピン摂取の影響 Influence of lycopene intake to changes of astrocyte and microglia on hippocampal tissue of Mongolian gerbil after cerebral ischemia/reperfusion ○藤田公和1, 芳本信子2, 今田英己1, 松本岳3, 矢賀部隆史3, 稲熊隆博3, 永田豊1, 宮地栄一1 ○Kimikazu Fujita1, Nobuko Yoshimoto2, Hideki Imada1, Gaku Matsumoto3, Takafumi Yakabe3, Takahiro Inakuma3, Yutaka Nagata1, Eiichi Miyachi1 藤田保健衛生大学医学部生理学1, 名古屋文理大学短期大学部2, カゴメ株式会社　総合研究所3 Dept.Physiol.Sch.Med.fujita Health Univ. Toyoake,Aichi,Japan.1, Dept.Nutrition and Food Sciences. Nagoya Bunri Univ.Col.2, Research Institute KAGOME Co.Ltd.3 Several days following transient ischemia/reperfusion treatment, apoptotic degenerative changes in hippocampal neurons in gerbil brain are observed. Cerebral ischemia increases the activity of astrocyte and microglia, and this cellular response may play a key role in brain injury development. In the present study, changes in astrocyte (GFAP) and microglia (Iba-1) in lycopene-induced neurodegenerative depression were investigated using Mongolian gerbil brains. Animals were fed either a normal or a lycopene-containing diet (5 mg/100 g feed) freely. After attaining a body weight of 60-65 g, animals were subjected to transient cerebral ischemia for 10 min or a sham operation. Animals were decapitated under flothane anesthesia, and the brains were removed at intervals of 3h, 1, 3 and 7 d post-treatment. GFAP and Iba-1 expression in the hippocampus were analyzed using Western blotting. Expression of GFAP protein in the hippocampus gradually increased in control (ischemia and non-lycopene) group after ischemia/reperfusion. In contrast, GFAP protein level was significantly lower in lycopene-treated ischemia group than the control group at 3 and 7d after ischemia. The results indicate that astrocyte activation decreased in the gerbil hippocampus due to lycopene and suggest that the oral administration of lycopene could protect neurons from ischemic damage.	P2-1-224 内包出血後の麻痺側前肢の集中使用は出血側運動野の体部位再現を変化させ運動機能改善を促進する Intensive use of impaired limb alters forelimb representation of affected motor cortex and facilitates functional recovery in rats with capsular hemorrhage ○石田章真1,2, 梅田達也2, 伊佐正2, 飛田秀樹1 ○Akimasa Ishida1,2, Tatsuya Umeda2, Tadashi Isa2, Hideki Hida1 名古屋市立大学大学院　医学研究科　脳神経生理学1, 生理学研究所・認知行動発達機構研究部門2 Dept. Neurophysiol. and Brain Sci., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya1, Dept. Dev. Physiol., Natl. Inst. Physiol. Sci., Okazaki2 Intensive use of affected upper limb is an effective method to promote functional recovery of the forelimb after hemiplegic stroke. Previously, we demonstrated the better recovery of motor function in forced impaired limb use (FLU)-treated rats than non-treated rats after internal capsule hemorrhage (ICH). In the present study, we investigated the FLU effect on reorganization of the motor cortex after ICH and assessed the functional role of the reorganized sites in the recovery. Wistar rats received collagenase injection (type IV, 15 Units/ml, 1.4 μl) into the internal capsule for induction of ICH, followed by FLU with a one-sleeve plaster cast for a week. The FLU-treated group rats were forced to perform all the daily movements only with their affected forelimb. We carried out serial investigation of motor map in the motor cortex on the ipsilesional side by intracortical microstimulation (ICMS) at 5 day before and 1, 10 and 26 day after ICH. Behavioral assessments of forelimb motor functions (single pellet reaching task, horizontal ladder step) were made at 12 and 28 day. Forelimb movements were not elicited at all by ICMS in the ipsilesional motor cortex on day 1 after ICH. On day 10, ICMS-induced movements were observed only from small area of the caudal forelimb area (CFA). In comparison to the non-treated control, forelimb movements were elicited from larger area in the CFA and rostral forelimb area (RFA) in the FLU-treated rats. The forelimb motor maps were further expanded in the FLU-group at day 26. Behavioral assessments showed better recovery in the FLU-treated ICH rats than in the non-treated ICH rats. Moreover, microinjection of muscimol (1 μM, 1 μl) into the newly-emerged "forelimb areas" of the CFA and RFA resulted in impairment of the recovered forelimb movements. These data suggested that the FLU promoted reorganization of the ipsilesional motor cortex which underlies the FLU-induced functional recovery.
P2-1-225 ミクログリア/マクロファージにおけるTRPM2を介したNO産生が脳虚血傷害の進展に関与する TRPM2-mediated NO production in microglia/macrophage contribute to the progression of cerebral ischemic injury in mice ○崎元伸哉1, 白川久志1, 宗像将也1, 中川貴之1, 金子周司1 ○Shinya Sakimoto1, Hisashi Shirakawa1, Masaya Munakata1, Takayuki Nakagawa1, Shuji Kaneko1 京都大院・薬・生体機能解析1 Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto1 Ischemic stroke is the third leading cause of death worldwide. Emerging evidence indicates that the activation of inflammatory cells in ischemic brain tissue can extend the brain infarction, but the activation mechanisms of inflammatory cells remain to be elucidated. Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable nonselective cation channel, is highly expressed in the brain and immune cells. We have previously demonstrated that TRPM2 plays a critical role in the progression of cerebral ischemic injury, which could be mediated through the activation of microglia/macrophage. However, the mechanisms of the protective effect in TRPM2-KO mice remain to be clearly elucidated. In this study, to examine whether resident microglia or peripheral macrophages are more responsible for cerebral ischemic injuries, we generated bone marrow chimeric mice by transplanting bone marrow from GFP-wild-type or GFP-TRPM2-KO mice into irradiated recipients of both genotypes. Analysis of chimeric mice revealed that both central and peripheral deficiency of TRPM2 improved neurological deficits, suggesting that both microglia and macrophages play important roles in brain ischemic injuries. Next, we focused on inducible nitric oxide synthase (iNOS) in microglia/macrophage and found that administration of a selective iNOS inhibitor 1400W prevented ischemic neuronal injuries in WT mice, but not TRPM2-KO mice. Moreover, the production of nitric oxide induced by toll-like receptor agonist, lipopolysaccharide and lipoteichoic acid, was markedly reduced in microglia as well as macrophages derived from TRPM2-KO mice. These results indicate that TRPM2 could mediate the cerebral ischemic injury through NO release from both microglia and macrophage.	P2-1-226 脳梗塞後痙縮発症マウスの損傷反対側補足運動野から投射する神経線維は、赤核、脳幹網様核および脊髄で軸索発芽の増加が生じる Descending fibers from cortical neurons in contralesional supplemental motor area are spouted and increased in the red nucleus, reticular nucleus, and cervical spinal cord in spastic mice after the stroke ○李佐知子1, 加藤寛聡1 ○Sachiko Lee1, Hiroaki Kato1 名古屋大院・医・リハビリ1 Dept Health Sciences, Nagoya Univ, Nagoya1 Spasticity is characterized by a velocity-dependent increase in muscle tone with exaggerated tendon reflexes. Spasticity is a common disorder in patients with injuries of the brain and spinal cord, such as stroke, multiple sclerosis, spinal cord injury and other CNS lesions. Approximately 20% to 40% of stroke patients exhibit spasticity. The spasticity has been considered as increase of motoneuron excitability. However, the exact mechanisms of spasticity after the stroke are still unclear. One reason was that no rodent models for spasticity resulting from cortical injuries. In this study, we report about a novel spasticity model and the plasticitical changes of neuronal network after the stroke in mice. We lesioned the rostral and caudal forelimb motor areas in mice with photothrombotic injury. We used the rate dependent depression (RDD) of the Hoffman reflex as a reliable measurement of spasticity. To detect motoneuron excitability, we measured the mRNA expression levels of c-fos and choline acetyltransferase (ChAT) with a quantitative real-time reverse transcription polymerase chain reactions. Moreover, we counted a number of sprouting fibers, that projected from contralesional rostral forelimb area, using with anterograde tracer in the red nucleus, reticular nucleus, and cervical spinal cord after the stroke. The RDD in the affected muscles of infarcted animals were significantly weakened 3 days after stroke until 8 weeks after the stroke, except for 3 weeks after the stroke, compared to sham animals. The mRNA expression levels of c-fos and ChAT in affected motoneurons were significantly increased in stroke animals compared to those of sham animals. The newly sprouting fibers in supplementary motor area descending pathways were increased into the denervated side of the red nucleus, gigantocellular reticular nucleus, and cervical spinal cord in 2 week-injured group. Now we are investigating whether the newly sprouting fibers relate to spasticity.
P2-1-227 新生児脳障害モデルラットに対するラット臍帯血幹細胞療法 Rat umbilical cord blood-derived cell therapy for neonatal hypoxic-ischemic encephalopathy model of rats ○中西圭子1, 伊藤美春2, 佐藤義朗2, 東雄二郎1 ○Keiko Nakanishi1, Miharu Ito2, Yoshiaki Sato2, Yujiro Higashi1 愛知県コロニー研・周生期1, 名古屋大病院・総合周産母子・新生児2 Dept Perinatol, Inst Dev Res, Aichi Human Ser Ctr, Kasugai, Japan1, Div Neonatol, Ctr Maternal-Neonatal Care, Nagoya Univ Hosp,2 Despite recent advances in the treatment of neonatal hypoxic-ischemic encephalopathy (HIE) using therapeutic hypothermia, considerable number of infants still has moderate/severe neurological disability. Stem cell therapy using umbilical cord blood (UCB) cells, which are readily available at birth, could be a promising option for the treatment of neurological diseases. The UCB mononuclear cells of GFP transgenic rats were cultured with various growth factors such as SCFs. At postnatal day 7 (P7), rat pups underwent hypoxia-ischemia (HI) by right common carotid artery ligation followed by 2 hours exposure to 8 % oxygen (Rice-Vannucci model). The HI rat pups were subjected to intraperitoneal injection of UCB-derived cells (UCBDCs) propagated in vitro. At 3 weeks after the transplantation, the infarct area of UCBDC-treated rats reduced compared to that of control. Transplantation of UCBDC ameliorated motor function deterioration of HIE model rats. Our data indicate that UCBDC is a promising stem cell source, although the mechanisms by which transplanted cells mediate functional recovery need to be clarified.	P2-1-228 脳虚血障害によって誘導されるオリゴデンドロサイトのアポトーシスに対するSema4Dの欠失の保護作用 Sema4D deficiency protects oligodendroctes from ischemia-induced apoptosis ○石口満津子1, 澤野俊憲2, 山口航3, 古山達雄3, 古郷幹彦1, 稲垣忍2 ○Mitsuko Ishiguchi1, Toshinori Sawano2, Wataru Yamaguchi3, Tatsuo Furuyama3, Mikihiko Kogo1, Shinobu Inagaki2 大阪大学大学院歯学研究科　顎口腔病因病態制御学講座　口腔外科学第一教室1, 大阪大学大学院医学系研究科　保健学専攻生体情報科学講座　神経生物学研究室2, 香川県立保健医療大学3 1st Department of oral and maxillofacial surgery Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan1, Group of Neurobiology, Division of Health Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan2, Kagawa Prefectural College of Health Science, Mure, Takamatsu, Kagawa, Japan3 Sema4D is a member semaphorin family of secreted and membrane-bound proteins, are known to function as repulsive axon guidance molecules.Ischemic injury can induce oligodendrogenesis, which involves the proliferation of oligodendrocyte progenitor cells (OPCs) (Tanaka et al.,2003).This suggest the presence of a regulatory mechanism that functions to maintain the appropriate number of mature oligodendrocytes and myelin sheaths during recovery from injury.We previously showed that Sema4D-deficiency increased the number of oligodendrocytes in the peri-infarct area after the occlusion of the middle cerebral artery.However, it is not known how Sema4D increased oligodendrocyte in number. In this study we examined whether Sema4D deficiency affects the proliferation and/or apoptosis of oligodendrocytes in the peri-infarct area after focal cerebral ischemia.Sema4D knockout (KO) mice and the wild type mice (WT) of 7-week-old were subjected to cerebral ischemia by ligation and disconnection of the distal portion of the left middle artery (MCA). Permanent focal ischemia was produced and subjected to immunohistochemical staining at 1, 3, 7, and 28 days after the injury. To label newly generated cells, mice were injected intraperitoneally with BrdU 4 hours before sacrifice. To detect apoptotic cells Tunel staining was used.BrdU and NG2 positive cells were found more in the KO mice at postoperated day (POD) 7, suggesting that the proliferation was enhanced in the KO mice.GSTπ positive oligodendrocytes decreased in number in the WT mice at POD7, but not in the KO mice, suggesting that Sema4D deficiency participates in the maintenance of the number of oligodendrocytes.The numbers of Tunel positive OPCs and oligodendrocytes were lower in the KO mice than in the WT mice.These results suggest that Sema4D deficiency protectively functions to apoptosis of oligodendrocyte after cerebral ischemia.Sema4D is most likely involved in neural restoration after stroke.
P2-1-229 出血性脳機能障害に対するTRPC3阻害薬の寛解作用 TRPC3 inhibitor Pyr3 improves neuronal dysfunction and attenuates astrogliosis after intracerebral hemorrhage in mice ○白川久志1, 宗像将也1, 永安一樹1, 宮之原遵1, 三宅崇仁1, 中川貴之1, 金子周司1 ○Hisashi Shirakawa1, Masaya Munakata1, Kazuki Nagayasu1, Jun Miyanohara1, Takahito Miyake1, Takayuki Nakagawa1, Shuji Kaneko1 京都大院・薬・生体機能解析1 Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto1 Intracerebral hemorrhage (ICH) stems from the rupture of blood vessels in the brain, with the subsequent accumulation of blood in the parenchyma. Increasing evidence suggests that blood-derived factors induce excessive inflammatory responses that are involved in the progression of ICH-induced brain injury. Thrombin, a major blood-derived factor, leaks into the brain parenchyma upon blood-brain barrier disruption and induces brain injury and astrogliosis. We have previously reported that thrombin dynamically upregulates transient receptor potential canonical 3 (TRPC3) channel, which contributes to pathological astrogliosis through a feed-forward upregulation of its own expression. Here, we investigated whether Pyr3, a specific TRPC3 inhibitor, can improve neuronal dysfunctions and attenuate astrogliosis after ICH in mice. In collagenase or autologous blood infusion-induced ICH model, no difference was observed in hematoma formation between control and ICH groups. Neurological deficits, motor dysfunction and neuronal injury were all significantly improved by intracerebroventricularly and intraperitoneally administration of Pyr3 in a dose-dependent manner. The number of S100-positive astrocytes in the perihematomal area was increased at 1 day after ICH and remained elevated until day 7, whereas Pyr3 markedly prevented the increase. Moreover, delayed administration of Pyr3 at 6 h or 1 day following blood or collagenase infusion, respectively, also improved the symptoms. Taken together, Pyr3, a specific inhibitor of TRPC3, reduced the perihematomal accumulation of astrocytes and ameliorated ICH-induced brain injury. Therefore, TRPC3 provides a new therapeutic target for the treatment of hemorrhagic brain injury.	P2-1-230 Withdrawn
P2-1-231 Functional improvement of ischemic stroke by epidural application of sonic hedgehog in rats ○Ding-I Yang1, Shiang-Suo Huang2 Institute of Brain Science, National Yang-Ming University1, Department of Pharmacology and Institute of Medicine, College of Medicine, Chung Shan Medical University, Taichung, Taiwan2 Sonic hedgehog (SHH) is a morphogen important for neural development during embryogenesis. Recently, beneficial actions of SHH in ischemic injury have been noted. To test whether epidural application of the biolgically active N-terminal fragment of SHH (SHH-N) may reduce the extent of ischemic brain injury, male Long Evans rats were exposed to a 60-min episode of middle cerebral artery occlusion (MCAO) with topical application of SHH-N and/or its specific inhibitor, cyclopamine, through fibrin glue over the peri-infarct cortex. We found that epidural application of SHH-N substaintially reduced infarction volumes after 7 days of the stroke onset, which was reversed by cyclopamine; SHH-N also improved behavioral outcomes as assessed by global neurological functions, rotarod test, and grasping power test. Furthermore, SHH-N attenuated the extents of protein oxidation, lipid peroxidation, and apoptosis induced by focal ischemia/reperfusion. Immunohistochemical staining coupled with bromodeoxyuridine (BrdU) incorporation revealed that SHH-N enhanced post-ischemic angiogenesis, increased the number of nestin-positive neural progenitor cells, and suppressed astrocytosis. Our results thus revealed multifaceted protective mechanisms of SHH-N in focal cerebral ischemia/reperfusion.	P2-1-232 Focal brain cooling suppresses periodic epileptiform discharges and lessens brain injury after focal cerebral ischemia in rats ○Yeting He1, Masami Fujii1, Takao Inoue1, Sadahiro Nomura1, Hiroyuki Kida2, Yuichi Maruta1, Ichiro Kunitsugu3, Takeshi Yamakawa4, Michiyasu Suzuki1 Dept Neurosurg, Yamaguchi Univ, Japan1, Dept Systems Neurosc, Yamaguchi Univ Japan2, Dept Brain Sci & Eng, Kyushu Institute Technol Japan3 It is well known that periodic epileptiform discharges (EDs) was induced in acute cerebral ischemia model. On the other hand, we have reported that focal brain cooling (FBC) terminates EDs in drug-induced seizure model. Therefore, the aim of this study was to investigate whether FBC also has a favorable effect on focal cerebral ischemia in rats. Under general anesthesia, a small craniotomy was made and focal cerebral ischemia was induced with the photothrombosis technique (illumination after intravenous inject Rose Bengal at 1.3 mg/100 g of body weight in 0.9% sterile saline) in the primary sensorimotor area(SI-MI). An additional larger craniotomy was made in the SI-MI area and FBC was performed at a temperature of 15°C for 5 h. Electrocorticograms (ECoG) were recorded with needle-type electrodes placed on the cortex at the border zone of the ischemic focus for 5 h during cooling and for 1 h after cooling. After recording of the ECoG, rats were sacrificed and the infarct area was measured with triphenyltetrazolium chloride staining. Seven rats were used in the cooling and non-cooling groups. In addition, in a long-team study, neurological testing was performed, included neurobehavioral testing and grip strength testing every day for 5days after focal cerebral ischemia. FBC suppressed all the ECoG frequency bands (1-30 Hz) during and after cooling (p<0.05), except for the delta frequency band in the precooling versus rewarming periods. The injured areas (sections at the ischemic core level) in the cooling and non-cooling groups were 0.99 ± 0.30 and 1.71 ± 0.54 mm2, respectively. Lesion size was significantly reduced in the cooling group (p = 0.026). In the cooling group the neurological deficits were improved compare with the non-cooling group during 1 and 2 day after focal cerebral ischemia (p<0.05). FBC suppresses periodic EDs and has a protective effect on focal cerebral ischemia. These results suggest that FBC has potential for treatment of cerebral infarction.

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