全面了解啮齿动物不活动引起的步态改变
Summary
本协议描述了三维运动跟踪/评估，以描绘暴露于模拟废弃环境后大鼠的步态运动改变。
Abstract
众所周知，废弃会影响神经系统，关节运动会发生变化;然而，哪些结局正确表现出这些特征尚不清楚。本研究描述了一种运动分析方法，该方法利用视频捕获的三维（3D）重建。使用这项技术，在暴露于模拟微重力环境的啮齿动物中，通过尾巴卸载后肢，观察到了废弃引起的行走性能的改变。卸货2周后，大鼠在跑步机上行走，用四个电荷耦合器件（CCD）相机捕捉它们的步态运动。使用图像处理软件重建3D运动曲线并与对照受试者的运动曲线进行比较。重建的结局测量成功地描绘了畸形步态运动的不同方面：膝关节和踝关节的过度伸展以及站立阶段髋关节的较高位置。运动分析很有用，原因有几个。首先，它支持定量行为评估而不是主观观察（例如，在某些任务中通过/失败）。其次，一旦获得基本数据集，就可以提取多个参数以满足特定需求。尽管存在更广泛应用的障碍，但通过确定综合测量和实验程序，可以减轻该方法的缺点，包括劳动强度和成本。
Introduction
缺乏体力活动或废用会导致运动效应器的恶化，例如肌肉萎缩和骨质流失1和全身失调2。此外，最近人们注意到，不活动不仅影响肌肉骨骼组件的结构方面，还影响运动的定性方面。例如，即使在干预结束后1个月，暴露于模拟微重力环境的大鼠的肢体位置也与完整动物的肢体位置不同3，4。然而，关于不活动造成的运动缺陷的报告很少。此外，劣化的综合运动特性尚未完全确定。
目前的协议演示并讨论了运动学评估的应用，通过参考在承受后肢卸载的大鼠中因废弃而引起的步态运动缺陷来可视化运动改变。
已经表明，在模拟微重力环境后行走时四肢的过度伸展在人类5和动物4，6，7，8中都观察到。因此，为了普遍性，我们在本研究中重点关注一般参数：膝关节和踝关节的角度以及跖趾关节与髋关节之间的垂直距离（大致相当于髋关节的高度）在站立阶段（中间）。此外，讨论了视频运动学评估的潜在应用。
一系列运动学分析可能是评估神经控制功能方面的有效措施。然而，尽管运动分析已经从足迹观察或对捕获的视频9，10的简单测量发展到多个相机系统11，12，但通用方法和参数尚未建立。本研究旨在为该关节运动分析提供全面的参数。
在之前的工作13中，我们尝试使用综合视频分析来说明神经病变模型大鼠的步态改变。然而，一般来说，运动分析的潜在结果通常仅限于分析框架中提供的预定变量。出于这个原因，本研究进一步详细说明了如何纳入广泛适用的用户定义的参数。如果实施适当的参数，使用视频分析的运动学评估可能会进一步有用。
Protocol
本研究已获得京都大学动物实验委员会（Med Kyo 14033）的批准，并符合国立卫生研究院的指导方针（实验动物护理和使用指南，第8版）。7周龄的雄性Wistar大鼠用于本研究。 补充文件1中提供了代表程序顺序的示意图。 1. 让老鼠熟悉跑步机行走 注意：有关该程序的详细信息，请参阅先前发布的报告13 。 将?…
Representative Results
12只动物被随机分配到两组之一：卸载组（UL，n = 6）或对照组（Ctrl，n = 6）。对于UL组，动物的后肢被尾巴卸载2周（UL期），而Ctrl组动物则保持自由。卸载 2 周后，与 Ctrl 组相比，UL 组表现出明显的步态模式。 图1 显示了代表性受试者的归一化关节轨迹。在站立阶段，UL组在膝盖和脚踝的伸展（即脚踝的跖屈）比Ctrl组更突出，称为“脚趾行走”3，<…
Discussion
环境的改变导致运动系统的功能方面和肌肉骨骼成分波动26，27。收缩结构或环境中的畸变可能会影响功能能力，即使在解决机械/环境变形后仍然存在19.客观的运动分析有助于定量测量这些功能能力。如上所示，视频分析是获取此类参数的强大方法。
为了跟踪联合地标进行视频分析，使用红外标记和摄像头很普遍?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了日本科学促进会（JSPS）KAKENHI（编号18H03129，21K19709，21H03302，15K10441）和日本医学研究与发展机构（AMED）（编号15bk0104037h0002）的部分支持。
Materials
| Adhesive Tape | NICHIBAN CO.,LTD. | SEHA25F | Adhesive tape to secure thread on tails of rats for hindlimb unloading |
| Anesthetic Apparatus for Small Animals | SHINANO MFG CO.,LTD. | SN-487-0T | |
| Auto clicker | N.A. | N.A. | free software available to download to PC (https://www.google.com/search?client=firefox-b-1-d&q=auto+clicker) |
| CCD Camera | Teledyne FLIR LLC | GRAS-03K2C-C | CCD (Charge-Coupled Device) cameras for video capture |
| Cotton Thread | N.A. | N.A. | Thread to hang tails of rats from the ceiling of cage |
| ISOFLURANE Inhalation Solution | Pfizer Japan Inc. | (01)14987114133400 | |
| Joint marker | TOKYO MARUI Co., Ltd | 0.12g BB | 6 mm airsoft pellets that were used as semispherical markers with modification |
| Kine Analyzer | KISSEI COMTEC CO.,LTD. | N.A. | Software for analysis |
| Konishi Aron Alpha | TOAGOSEI CO.,LTD. | #31204 | Super glue to attach spherical markers on randmarks of rats |
| Motion Recorder | KISSEI COMTEC CO.,LTD. | N.A. | Software for video recording |
| Paint Marker | MITSUBISHI PENCIL CO., LTD | PX-21.13 | Oil based paint marker to mark toes of animals |
| Three-dimensional motion capture apparatus (KinemaTracer for small animals) | KISSEI COMTEC CO.,LTD. | N.A. | 3D motion analysis system that consists of four cameras (https://www.kicnet.co.jp/solutions/biosignal/animals/kinematracer-for-animal/ or https://micekc.com/en/) |
| Three-dimensional(3D) Calculator | KISSEI COMTEC CO.,LTD. | N.A. | Software fo marker tracking |
| Treadmill | MUROMACHI KIKAI CO.,LTD | MK-685 | Treadmill equipped with transparent housing, electrical shocker, and speed control unit |
| Wistar Rats (male, 7-week old) | N.A. | N.A. | Commercially available at experimental animal sources |
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Citer Cet Article
Tajino, J., Aoyama, T., Kuroki, H., Ito, A. Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents. J. Vis. Exp. (185), e63865, doi:10.3791/63865 (2022).