应用被动头部运动在啮齿动物头部产生定义的加速度
1Department of Rehabilitation for Motor Functions,National Rehabilitation Center for Persons with Disabilities, 2Department of Assistive Technology,National Rehabilitation Center for Persons with Disabilities, 3Department of Metabolism and Endocrinology, Graduate School of Medicine,Juntendo University, 4Department of Rehabilitation Medicine,Graduate School of Medicine, The University of Tokyo, 5Department of Clinical Research,National Rehabilitation Center for Persons with Disabilities
Summary
本协议描述了一种定制设计的“被动头部运动”系统，该系统再现了啮齿动物头部在以中等速度跑步期间产生的机械加速度。它允许从体育锻炼的有益影响中剖析机械因素/元素。
Abstract
运动被广泛认为对各种疾病和身体疾病有效，包括与脑功能障碍有关的疾病和疾病。然而，运动有益效果背后的分子机制知之甚少。许多体育锻炼，特别是那些被归类为有氧运动的锻炼，如慢跑和步行，在足部与地面接触时会产生冲动力。因此，推测机械冲击可能与运动如何导致机体稳态有关。为了在大脑上测试这一假设，开发了一种定制设计的“被动头部运动”（以下简称PHM）系统，该系统可以产生具有受控和定义大小和模式的垂直加速度，并再现机械刺激，这些刺激可能应用于啮齿动物的头部在跑步机以中等速度跑步时，这是一种典型的干预措施，用于测试运动对动物的影响。通过使用该系统，证明PHM概括了小鼠前额叶皮层（PFC）神经元中的血清素（5-羟色胺，以下简称5-HT）受体亚型2A（5-HT2A）信号传导。这项工作为应用PHM和测量其在啮齿动物头部产生的机械加速度提供了详细的协议。
Introduction
运动有益于治疗或预防几种身体疾病，包括糖尿病和原发性高血压等生活方式疾病1。与此相关的是，关于运动对大脑功能的积极影响的证据也已积累2。然而，运动对大脑益处的分子机制仍然主要未阐明。大多数体育活动和锻炼至少在某种程度上会在头部产生机械加速度。虽然各种生理现象是机械调节的，但在大多数情况下，机械负荷的重要性已在肌肉骨骼系统中得到记录3，4，5。虽然大脑在体育活动中也受到机械力的影响，特别是所谓的冲击运动，但很少研究生理大脑功能的机械调节。由于头部机械加速度的产生在体育锻炼中相对常见，因此推测机械调节可能与运动对大脑功能的益处有关。
5-HT2A 受体信号传导对于调节神经系统中起作用的各种生化信号中的情绪和行为至关重要.它涉及多种精神疾病6，7，8，运动已被证明具有治疗效果。5-HT2A受体是5-HT2受体的一个亚型，属于5-羟色胺家族，也是G蛋白偶联受体（GPCR）家族的成员，其信号传导通过其内化调节，配体依赖性或非依赖性9。头部抽搐是啮齿动物的特征行为，其数量（频率）明确表示其前额叶皮层（PFC）神经元中5-HT2A受体信号传导的强度10，11。利用这种致幻反应对施用5-HT（头部抽搐反应，以下简称HTR;见补充视频1）的严格特异性，测试了上述关于运动对大脑功能的机械影响的假设。因此，我们分析和比较了接受强制运动（跑步机跑步）或运动模拟机械干预（PHM）的小鼠的HTR。
Protocol
所有动物实验均获得国家残疾人康复中心机构动物护理和使用委员会的批准。8-9周龄雄性Sprague-Dawley大鼠用于测量跑步机跑步和PHM期间头部的加速度。9-10周龄雄性C57BL / 6小鼠用于PFC的行为测试和组织学分析。 这些动物是从商业来源获得的（见 材料表）。 1. 测量跑步机跑步过程中沿 x、y 和 z 轴的加速度大小 用吸入1.5%异氟醚麻醉大鼠。注意…
Representative Results
在跑步机以中等速度（20 m / min）跑步期间，大鼠头部垂直加速度的峰值幅度约为1.0× g （图1C）。PHM系统（图1D）的设置是为了在啮齿动物的头部产生1.0 × g 的垂直加速度峰值。 与对照小鼠（每天麻醉而不使用PHM30分钟/天，持续7天）相比，向小鼠施用PHM（2Hz，30分钟/天，持续7天）显着减弱了其HTR（图2<…
Discussion
使用开发的PHM应用系统，我们已经证明其PFC神经元中的5-HT信号传导是机械调节的。由于运动效果的复杂性，很难在健康促进的背景下准确剖析运动的后果。重点是机械方面，以排除可能与运动活动一起或随后发生的代谢事件的参与或贡献，例如能量消耗。这里描述的方法有望在生物医学研究中更广泛地用于探索运动对大脑功能影响的机制。
目前的系统需要麻醉才能使实验动?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了日本厚生劳动省校内研究基金的支持;日本学术振兴会科学研究补助金（KAKENHI 15H01820、15H04966、18H04088、20K21778、21H04866、21K11330、20K19367）;文部科学省支持的私立大学战略研究基金会项目，2015-2019年，日本文部科学省（S1511017）;内藤科学与工程基金会。这项研究还获得了再生康复研究与培训联盟（AR3T）的资助，该联盟得到了Eunice Kennedy Shriver国家儿童健康与人类发展研究所（NICHD），国家神经疾病和中风研究所（NINDS）和美国国立卫生研究院国家生物医学成像和生物工程研究所（NIBIB）的支持，奖励号为P2CHD086843。
Materials
| 5-hydroxytryptophan (5-HTP) | Sigma-Aldrich | H9772 | Serotonin (5-HT) precursor |
| Brushless motor driver | Oriental motor | BMUD30-A2 | Speed changer build-in motor driver |
| C57BL/6 mice | Oriental yeast company | C57BL/6J | Mice used in this study |
| Cryostat | Leica | CM33050S | Microtome to cut frozen samples |
| DC Motor | Oriental motor | BLM230-GFV2 | Motor |
| Donkey anti-goat Alexa Fluor 568 | Invitrogen | A-11057 | Secondary antibody used for immunohistochemical staining |
| Donkey anti-mouse Alexa Fluor 647 | Invitrogen | A-31571 | Secondary antibody used for immunohistochemical staining |
| Donkey anti-rabbit Alexa Fluor 488 | Invitrogen | A-21206 | Secondary antibody used for immunohistochemical staining |
| Donkey serum | Sigma-Aldrich | S30-100ML | Blocker of non-specific binding of antibodies in immunohistochemical staining |
| Fluorescence microscope | Keyence | BZ-9000 | Fluorescence microscope |
| Goat polyclonal anti-5-HT2A receptor | Santa Cruz Biotechnology | sc-15073 | Primary antibody used for immunohistochemical staining |
| Isoflurane | Pfizer | v002139 | Inhalation anesthetic |
| KimWipe | NIPPON PAPER CRECIA | S-200 | Paper cloth for cleaning surfaces, parts, instruments in labratory |
| Liquid Blocker | Daido Sangyo | PAP-S | Marker used to make the slide surface water-repellent |
| Mouse monoclonal anti-NeuN (clone A60) | EMD Millipore (Merck) | MAB377 | Primary antibody used for immunohistochemical staining |
| NinjaScan-Light | Switchscience | SSCI-023641 | Accelerometer to measure accelerations |
| OCT compound | Sakura Finetek | 45833 | Embedding agent for preparing frozen tissue sections |
| ProLong Gold Antifade Mountant | Invitrogen | P36934 | Mounting medium to prevent flourscence fading |
| Rabbit polyclonal anti-c-Fos | Santa Cruz Biotechnology | sc-52 | Primary antibody used for immunohistochemical staining |
| Slide box | AS ONE | 03-448-1 | Opaque box to store slides |
| Spike2 | Cambridge electronic design limited (CED) | N/A | Application software used to analyze acceleration |
| Sprague-Dawley rats | Japan SLC | Slc:SD | Rats used in this study |
| Treadmill machine | Muromachi | MK-680 | System used in experiments of forced running of rats and mice |
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Citar este artigo
Maekawa, T., Sakitani, N., Ryu, Y., Takashima, A., Murase, S., Fink, J., Nagao, M., Ogata, T., Shinohara, M., Sawada, Y. Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents. J. Vis. Exp. (185), e63100, doi:10.3791/63100 (2022).