パーキンソン病モデル動物での視床下核刺激による認知機能変化の検討

帕金森病模型动物丘脑底核刺激所致认知功能变化的检测

• 批准号: 22590964
• 负责人: 滝山 容子
• 金额: $ 2万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2010
• 资助国家: 日本
• 起止时间: 2010 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22590964/
• 关键词: パーキンソン病; 視床下核脳深部刺激療法; 認知機能; 中脳腹側被蓋野; pre-pulse inhibition test

パーキンソン病(PD)で、運動機能を改善する視床下核脳深部刺激療法(STN-DBS)の、認知機能への直接的影響を検討する目的で、認知機能障害に特化したPDモデル動物を作成し、運動機能に影響されない行動実験でSTN-DBS施行後の認知機能変化を分析する実験系を確立することが、H22年度の目標である。PDの主病態は中脳黒質緻密部ドーパミン細胞の変性だが、中脳腹側被蓋野(VTA)の変性も認知機能に関わるためVTAを選択的に破壊し、認知機能障害に特化したPDモデル動物を作成した。全体の実験系は以下の通りで、現在まで1,2,4,5の行程が確立された。1 両側VTA障害モデルの作成 8週齢以降のWister ratにステレオ装置を用いて、神経毒である6-OHDA2μg/μlを両側VTA(前後:内耳道点より+3.8mm、左右:正中線より左右に0.7mm、深さ:硬膜表面より7.6mm)に1μl、0.125μl/minの速度で注入.対照(非障害)群は生理食塩水を注入。VTA障害群では動作緩慢、反応性低下が観察され認知機能変化が示唆された。2 3週間後STN-DBS刺激電極の装着 ステレオ装置を用いてDBS刺激電極をSTN(前後:内耳道点より+5.4mm、左右:正中線より左右に2.5mm、深さ:硬膜表面より7.7mm)に埋入。3 1週間後、刺激装置にて単発刺激(125Hz,60μsec,0.2mA 連続60分間)を1日1回3日間施行4 行動実験(pre-pulse inhibition test:注意力分析、他)施行5 免疫染色分析 行動実験後灌流固定し脳凍結切片を作成、1次抗体にAnti-Tyrosine hydroxylase Rabbit Ab、2次抗体にBiotinylated anti-rabbit IgGを用い、DAB発色、cresyl violet染色を行った。VTAへの刺入可否確認とVTAドーパミン細胞の脱落程度を細胞数で比較。VTA障害モデルでは40～50個/VTAと細胞破壊が確認されたが、黒質緻密部も一部破壊された。しかしこの方が認知機能障害を伴う本来のPDモデルに近い為、今後VTA+黒質緻密部破壊モデルでも行う。6 行動実験にて、VTA生理食塩水注入(非障害)群との比較(認知精神機能の変化の有無)、DBS非刺激(非治療)群との比較(DBS刺激影響の有無)を準備中である。

PD disease (PD), improvement of motor function, subbed-bed nuclear deep stimulation therapy (STN-DBS), direct impact on cognitive function, purpose of study, specialization of cognitive dysfunction STN-DBS treatment of PD animals and movements affecting motor functions After the operation, the cognitive function improvement analysis system was analyzed and the target for H22 was established. The main pathology of PD is the recognition of the abnormality of the substantia nigra pars compacta cells and the abnormality of the ventral tegmental field (VTA). Cognitive dysfunction is a specialization of VTA's selected VTA, and cognitive dysfunction is a specialization of PD and animals. The overall system is the following and the current itinerary of 1,2,4,5 is established. 1 The side VTA obstacle is created by Wister from 8 weeks old rat にステレオ device をいて, 神経toxic である6-OHDA2μg/μl を両lateral VTA (anterior and posterior: inner auditory canal point より+3.8mm, left and right: midline よりLeft and right 0.7mm, depth: dural surface (7.6mm), injection speed of 1μl, 0.125μl/min. Injection of physiological food and water for irradiation (non-barrier) group. VTA disorders include slow movements, low reflexes, and changes in cognitive function. After 2 to 3 weeks, the STN-DBS stimulation electrode was installed. The STN-DBS stimulation electrode was embedded in the STN device using the STN (anterior and posterior: inner auditory canal point +5.4mm, left and right: midline, left and right +2.5mm, depth: surface of the dura mater +7.7mm). 3 After 1 week, stimulation using a stimulation device (125Hz, 60μsec, 0.2mA for 60 minutes) was performed once a day for 3 days. 4 Pre-pulse inhibition test (pre-pulse inhibition test: attention analysis, etc.) was performed. 5 Immunostaining analysis After the operation, frozen sections were prepared by perfusion and fixation, and the primary antibody was Anti-Tyrosine hydroxylase Rabbit Ab. The secondary antibody was Biotinylated anti-rabbit IgG. DAB staining and cresyl violet staining were performed. It is possible to confirm whether the VTA has been punctured and to compare the degree of cell detachment and the number of cells in the VTA. 40 to 50 VTA barrier cells/VTA cells are broken and confirmed, and the black dense part is broken. This is a cognitive dysfunction person who is originally a PD person, but from now on the VTA + Black Density Department is going to be broken. 6. Comparison of action, VTA physiological food and water injection (non-impairment) group (presence or absence of changes in cognitive and mental functions), DBS non-stimulation (non-treatment) group comparison (presence or absence of DBS stimulation effect) are currently under preparation.