使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍
Summary
提供了一个实验管道，用于使用MouseWalker（MW）工具箱定量描述自由行走的小鼠的运动模式，范围从初始视频记录和跟踪到定量后分析。使用小鼠脊髓挫伤损伤模型来证明MW系统的有用性。
Abstract
复杂且高度协调的运动程序（例如步行和跑步）的执行取决于脊柱和脊髓上回路的节律激活。胸椎脊髓损伤后，与上游回路的通信受损。这反过来又导致协调的丧失，恢复潜力有限。因此，为了更好地评估给予药物或治疗后的恢复程度，有必要使用新的、更详细和准确的工具来量化脊髓损伤动物模型中步态、肢体协调和其他运动行为的精细方面。多年来，已经开发了几种测定方法来定量评估啮齿动物的自由行走行为;然而，它们通常缺乏与步进步态策略、足迹模式和协调相关的直接测量。为了解决这些缺点，提供了MouseWalker的更新版本，它将受挫的全内反射（fTIR）走道与跟踪和量化软件相结合。这个开源系统已经适应于提取几个图形输出和运动学参数，并且一组后量化工具可以分析提供的输出数据。这份手稿还展示了这种方法如何与已经建立的行为测试相结合，定量描述脊髓损伤后的运动缺陷。
Introduction
四肢的有效协调并非四足动物所独有。人类的前肢-后肢协调对于完成多项任务仍然很重要，例如游泳和行走时的速度改变1.各种肢体运动学2 和运动程序1，3，4 以及本体感觉反馈回路5 在人类和其他哺乳动物之间是保守的，在分析运动障碍（例如脊髓损伤 （SCI）6，7，8）的治疗方案时应考虑这些选择。
为了行走，来自前肢和后肢的几个脊柱连接需要正确连接和有节奏地激活，这需要来自大脑的输入和来自体感系统的反馈2，9，10。这些连接在中央模式发生器（CPG）中达到顶峰，它们分别位于前肢和后肢的颈椎和腰椎水平1，9，10。通常，在 SCI 之后，神经元连接的破坏和抑制性神经胶质瘢痕12 的形成限制了运动功能的恢复，结果从完全瘫痪到一组肢体功能受限不等，具体取决于损伤的严重程度。精确量化 SCI 后运动功能的工具对于监测恢复和评估治疗或其他临床干预措施的效果至关重要6.
SCI 小鼠挫伤模型的标准度量测定是巴索小鼠量表 （BMS）13，14，这是一种非参数评分，考虑了空旷场地的躯干稳定性、尾部位置、足底步和前肢-后肢协调。尽管BMS在大多数情况下非常可靠，但它需要至少两名经验丰富的评分员来观察动物运动的所有角度，以解释自然变异性并减少偏差。
还开发了其他测定方法来定量评估SCI后的运动性能。其中包括旋转测试，它测量在旋转圆柱体上花费的时间15;水平梯子，测量错过栏杆和正梯子抓斗的数量16，17;以及横梁行走测试，该测试测量动物在穿越窄梁时所花费的时间和失败次数18.尽管反映了运动缺陷的组合，但这些测试都没有产生关于前肢 – 后肢协调的直接运动信息。
为了具体和更彻底地分析步行行为，已经开发了其他测定方法来重建步数循环和步态策略。一个例子是足迹测试，其中动物的墨水爪子在一张白纸上画一个图案19.虽然执行简单，但提取步幅等运动学参数既麻烦又不准确。此外，缺乏动态参数，例如步进周期的持续时间或腿定时协调，限制了其应用;事实上，这些动态参数只能通过分析啮齿动物穿过透明表面的逐帧视频来获取。对于SCI研究，研究人员使用跑步机从侧面分析了步行行为，包括重建步数循环和测量每个腿关节的角度变化4，20，21。尽管这种方法可以提供非常丰富的信息6，但它仍然专注于一组特定的肢体，并且缺乏额外的步态特征，例如协调性。
为了填补这些空白，Hamers及其同事开发了一种基于光学触摸传感器的定量测试，使用受挫全内反射（fTIR）22。在这种方法中，光线通过内部反射 穿过 玻璃传播，在按压爪子时散射，最后被高速相机捕获。最近，这种方法的开源版本，称为MouseWalker，已经可用，这种方法将fTIR走道与跟踪和量化软件包23相结合。使用这种方法，用户可以提取大量定量参数，包括步数、空间和步态模式、脚印定位和前肢-后肢协调，以及视觉输出，例如脚印模式（模仿墨水爪测定6）或相对于身体轴的站立阶段。重要的是，由于其开源性质，可以通过更新 MATLAB 脚本包来提取新参数。
在这里，更新了以前发布的 MouseWalker23 系统组件。提供了如何设置它的说明，以及实现最佳视频质量、跟踪条件和参数采集所需的所有步骤。还共享了其他量化后工具，以增强对MouseWalker（MW）输出数据集的分析。最后，通过在脊髓损伤（SCI）背景下获得一般运动性能的可量化值，特别是步道周期和前肢 – 后肢协调，证明了该工具的有用性。
Protocol
Representative Results
Discussion
在这里，通过分析SCI后的运动行为来证明MouseWalker方法的潜力。它为步进、足迹和步态模式的特定变化提供了新的见解，否则其他标准测试会错过这些变化。除了提供 MW 包的更新版本外，还使用提供的 Python 脚本描述了数据分析工具（请参阅步骤 5）。
由于MW生成了一个大型数据集和反映高维运动过程的运动学参数集合，因此采用了PCA;事实上，PCA已被广泛用于其他运动学数据?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢Laura Tucker和Natasa Loncarevic对手稿的评论以及Instituto de Medicina Molecular João Lobo Antunes的啮齿动物设施给予的支持。作者希望感谢Prémios Santa Casa Neurociências – 脊髓损伤研究奖Melo e Castro（MC-36/2020）对L.S.和C.S.M.的财政支持。这项工作得到了科学与技术基金会（FCT）（PTDC/BIA-COM/0151/2020）、iNOVA4Health（UIDB/04462/2020和UIDP/04462/2020）和LS4FUTURE（LA/P/0087/2020）的支持，得到了CEEC个人首席研究员合同（2021.02253.CEECIND）的支持。AFI得到了FCT（2020.08168.BD）的博士奖学金的支持。AMM得到了FCT博士奖学金的支持（PD/BD/128445/2017）。I.M.得到了FCT的博士后奖学金（SFRH / BPD / 118051 / 2016）的支持。DNS得到了FCT博士奖学金的支持（SFRH / BD / 138636 / 2018）。
Materials
| 45º Mirror | |||
| 2 aluminum extrusion (2 x 2 cm), 16 cm height, 1 on each side | Misumi | ||
| 2 aluminum extrusion (2 x 2 cm), 23 cm, @ 45° , 1 on each side | Misumi | ||
| 1 aluminum extrusion (2 x 2 cm), 83 cm long | Misumi | ||
| 87 x 23 cm mirror | General glass supplier | ||
| black cardboard filler | General stationery supplier | We used 2, one with 69 x 6 cm and another with 69 x 3cm to limit the reflection on the mirror | |
| Background backlight | |||
| 109 x 23 cm plexiglass (0.9525 cm thick) | General hardware supplier | ||
| 2 lateral aluminum extrusion (4 x 4 cm), 20 cm long, 1 on each side | Misumi | ||
| multicolor LED strip | General hardware supplier | ||
| white opaque paper to cover the plexyglass | General stationery supplier | ||
| fTIR Support base and posts | |||
| 2 aluminum extrusion (4 x 4 cm), 100 cm height | Misumi | ||
| 60 x 30 cm metric breadboard | Edmund Optics | #54-641 | |
| M6 12 mm screws | Edmund Optics | ||
| M6 hex nuts and wahers | Edmund Optics | ||
| fTIR Walkway | |||
| 109 x 8.5 cm plexyglass (1.2 cm thick) | General hardware supplier | 109 x 8.5 cm plexyglass (1.2 cm thick) | |
| 109 cm long Base-U-channel aluminum with 1.6 cm height x 1.9 cm depth thick folds (to hold the plexyglass) | General hardware supplier | ||
| 2 lateral aluminum extrusion (4 x 4 cm) 20 cm length, 1 on each side | Misumi | ||
| black cardboard filler | General stationery supplier | we used 2 fillers on each side to cover the limits of the plexyglass, avoiding bright edges | |
| 12 mm screws | Edmund Optics | M6 | |
| High speed camera (on a tripod) | |||
| Blackfly S USB3 | Blackfly | USB3 | This is a reccomendation. The requirement is to record at least 100 frames per second |
| Infinite Horizon Impactor | |||
| Infinite Horizon Impactor | Precision Systems and Instrumentation, LLC. | ||
| Lens | |||
| Nikkon AF Zoom-Nikkor 24-85mm | Nikkon | 2.8-4D IF | This lens is reccomended, however other lens can be used. Make sure it contains a large aperture (i.e., smaller F-stop values), to capture fTIR signals |
| Software | |||
| MATLAB R2022b | MathWorks | ||
| Python 3.9.13 | Python Software Foundation | ||
| Anaconda Navigator 2.1.4 | Anaconda, Inc. | ||
| Spyder 5.1.5 | Spyder Project Contributors | ||
| Walkway wall | |||
| 2 large rectagular acrilics with 100 x 15 cm | Any bricolage convenience store | ||
| 2 Trapezian acrilic laterals with 6-10 length x 15 cm height | Any bricolage convenience store | ||
| GitHub Materials | |||
| Folder name | URL | ||
| Boxplots | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Boxplots | Script to create Boxplots | |
| Docs | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Docs | Additional documents | |
| Heatmap | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Heatmaps | Script to create heatmap | |
| Matlat script | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Matlab%20Script | MouseWalker matlab script | |
| PCA | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/PCA%20plots | Script to perform Principal Component Analysis | |
| Raw data Plots | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Rawdata%20Plots | Script to create Raw data plots | |
| Residual Analysis | https://github.com/NeurogeneLocomotion/MouseWalker/tree/main/Residual_Analysis | Code to compute residuals from Raw data |
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