转棒实验用于评估偏侧帕金森综合征大鼠行走运动中的运动功能状态。
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勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
高速视频分析可以提供有关行走运动定性特征的详细信息。当动物在转棒上行走时，其位置基本保持不变，进行重复的踏步运动。因此，该任务能够提供丰富的足部踏步运动细节信息。实验对象为偏侧帕金森大鼠模型，通过向右侧黑质纹状体束注射6-羟基多巴胺（6-OHDA）以耗竭黑质纹状体多巴胺（DA）而制备。本报告展示了此前从前方、侧面和后方视角拍摄的动物行走视频的分析示例（15）。通过评分量表及对踏步行为视频记录的逐帧回放分析发现，偏侧帕金森大鼠在多巴胺耗竭对侧的姿势控制和肢体使用方面存在慢性功能障碍。与同侧肢体相比，对侧肢体在启动和维持推进运动中的参与程度较低。这种由单侧多巴胺耗竭引起的缺陷表明，转棒实验可作为分析踏步运动的有效任务。
方案
转棒行走装置
转棒仪是一个直径为4 cm的旋转圆柱体，固定在距地面35 cm的高度，周围由透明有机玻璃包围。转棒仪的圆柱体表面覆盖有纹理橡胶涂层，以增强抓地力。仪器底部铺有一层泡沫材料，以防止动物跌落时受伤。小型电动机通过橡胶皮带驱动转棒旋转。转棒设定为每5秒旋转一圈。
视频记录与运动学分析
使用高速数码摄像机拍摄动物运动，快门速度为每秒1/1000。在动物于转棒上行走时，分别从前方、侧面和后方进行拍摄。记录过程中采用尼康双臂MII冷光源及一套光纤照明设备，以提供充足的光照条件。录像通过索尼DSR 20 DV CAM录像机逐帧进行分析。视频帧使用图像采集卡和Macintosh计算机进行捕获。
测试步骤与分析
为了评估6-OHDA模型大鼠与对照组大鼠在行走运动上的差异，两名独立观察者在不知晓实验分组的情况下，对术后第15至30天（此时损伤后的恢复过程基本已完成）的视频记录进行了分析。黑质纹状体的6-OHDA损伤诱导于右侧半球，导致左侧前肢和后肢出现持续性功能障碍。研究采用了一种评分量表来评估大鼠的姿势以及前肢的迈步动作（Whishaw等，2003）。迈步动作的评估包含以下五个方面：
- 姿势，通过观察大鼠在滚筒上的位置来评定其姿势；
- 抬足与释放，步态周期中爪子从滚筒上抬起的部分；
- 悬空，大鼠向前移动时肢体抬离滚筒所保持的阶段；
- 前伸，肢体向前伸展以重新接触滚筒的运动阶段；以及
- 放置，爪子重新放回滚筒上的阶段。
采用以下标准来定义正常运动：
- 姿势： 大鼠身体位于滚筒上方，头部保持抬起状态。
- 抬升与释放： 肩关节、肘关节和腕关节屈曲，使前爪离开滚筒表面。
- 摆动： 前爪悬于滚筒表面之上，脚趾轻度屈曲，脚趾尖对准身体中线。推进：肢体向前伸展，脚趾从半屈曲状态逐渐变为伸展并张开状态。
- 放置： 肩关节、肘关节和腕关节伸展，脚趾张开，将前爪向前置于滚筒表面，第二趾（从中线起第二个脚趾）大致位于大鼠身体中心位置。
每种运动行为采用3分制进行评分：1分表示运动表现与对照组大鼠相似，0.5分表示运动表现略显异常，0分表示运动明显异常（Whishaw等，2003）。该评分标准用于评估每只大鼠在术后第1、3、5、7、9和15天的五种踏步运动。每天对每只大鼠的五项评分取平均值，所得结果以天数作为重复测量因素进行方差分析（ANOVA）。
6-OHDA 大鼠和对照大鼠转棒行走实验概述
运动评分量表被应用于术后测试阶段，以进行定性运动分析。在测试过程中，6-OHDA 损伤组的得分显著低于对照组，F(1,8)=33.2，p<0.001，但测试日效应不显著，组别与测试日之间的交互作用也不显著。随后，以前肢作为变量，分别对踏步得分进行分析（对于 6-OHDA 大鼠，为损伤对侧前肢与损伤同侧前肢；对于对照组大鼠，为左爪与右爪）。前肢主效应显著，F(1,40)=15.8，p<0.01，且组别与前肢之间存在显著交互作用，F(1,40)=15.0，p<0.01。事后 t 检验表明，6-OHDA 组损伤对侧前肢的得分显著低于其损伤同侧前肢，也显著低于对照组大鼠的前肢得分，而对照组大鼠两侧前肢之间无显著差异。为了确定哪些运动成分导致 6-OHDA 组得分较差，将 6-OHDA 组同侧和对侧前肢的姿势指标以及踏步周期各个组成部分的测量值与对照组大鼠的相应数值进行了比较。结果显示，6-OHDA 组在姿势以及对侧前肢的所有踏步成分上均存在显著差异，但在同侧前肢的任何成分上均无显著差异（p<0.05）。
对照组与6-OHDA大鼠在姿势上的定性与描述性差异
在对照组大鼠中，头部保持水平位置，支撑前肢和后肢分开位于滚筒两侧，形成支撑弓。6-OHDA 大鼠通常低头行走，其受损的前肢（面向观察者）比同侧肢体或对照组大鼠的肢体更靠近且位置更高地置于滚筒表面（参见视频中姿势评分为“1”的对照大鼠，以及评分为“0”的6-OHDA损毁动物）。通常，通过大鼠头部的相对位置即可一目了然地观察到姿势异常。
前肢迈步
在对照序列中，前肢通过肩关节、肘关节和腕关节的屈曲而抬起。当肢体被抬起时，指部呈轻微屈曲状态，且肢体被抬高至滚筒表面之上较远的位置。当肢体前移时，肩关节、肘关节和腕关节逐渐伸展，而在肢体放置过程中，指部随之伸展并张开。与对照组大鼠相比，单侧黑质损伤所致多巴胺耗竭大鼠（DA耗竭大鼠）的对侧肢体各运动成分的幅度均减弱。其肢体释放滚筒的时间较晚，抬高程度较低，前移距离也短于对照组前肢。此外，当肢体被抬起时，指部屈曲更明显；而当肢体放置时，指部的伸展和张开程度均小于对照组大鼠。对照组前肢在到达滚筒顶部后即被抬起，腕关节屈曲以将足掌向上和向前提起，并在肢体前移过程中伸展腕关节。相比之下，6-OHDA大鼠的对侧前肢因滚筒转动而被进一步向尾侧带动，因此释放时间更晚，且此时腕关节仍处于伸展状态。腕关节的屈曲似乎是被动发生的，发生在指部因滚筒转动而向后移动的过程中；当前肢向前摆动时，足掌和前臂并未明显离开滚筒表面。正常对照大鼠的前肢能够充分向前伸展，且在前移过程中指部张开。当肢体着地时，以琶音式动作落地，即第5指首先接触滚筒表面，随后第4、3、2指依次接触。相比之下，6-OHDA大鼠的前肢抬高和伸展幅度均减小，指部伸展程度降低，各指几乎同时以“拍击”方式接触滚筒表面。视频展示了对照动物的前肢踏步动作，评分为“1”；以及6-OHDA损毁动物的前肢踏步动作，主要评分为“0”。
后肢迈步
在6-OHDA大鼠中，病灶对侧的后肢也存在功能障碍。当对照组大鼠的后肢向后移动时，其动作似乎会向转鼓施加推力，同时脚趾向外旋转；当肢体向前移动时，足部则向外摆动。而6-OHDA大鼠的后肢则随着转鼓旋转直接向后移动，若有旋转趋势，也仅轻微向内。当肢体前移时，足部也是直接向前推进。对照组大鼠的足部在摆动时向外展开，前移过程中脚趾高于踝关节，且脚趾呈伸展状态。在着地阶段，对照组大鼠通常由第5趾首先接触转鼓表面，随后体重均匀分布于各趾及足掌。相比之下，6-OHDA大鼠在摆动相中脚趾呈屈曲状态，着地时缺乏类似琶音（arpeggio）的逐个触地动作。对照组大鼠在抬足过程中，脚趾以相反的琶音模式依次抬起：第5趾最先抬起，第1趾最后抬起。这一动作在6-OHDA损毁大鼠中缺失，且在摆动过程中踝关节保持屈曲姿势。此外，6-OHDA大鼠的前移动作减弱，表现为肢体外旋程度降低、抬高幅度减小、前移距离缩短，均不及对照组大鼠。当对照组大鼠放置足部时，脚趾伸展并张开，使得第5趾相对于大鼠身体指向外侧；第5趾首先接触转鼓，随后其余脚趾依次着地，使体重主要由趾尖支撑。而对于6-OHDA大鼠，足部前移时脚趾略有屈曲，脚趾位置相对于踝关节更接近水平，足部着地时脚趾张开程度较小。其步长也略短于对照组大鼠。为了量化对照组与6-OHDA大鼠后肢运动之间的差异，对抬足与释放、摆动、前移和着地等动作成分采用3分制进行评分，前肢亦同样评分。评分依据术后第20天录制的视频资料，每只大鼠评估三个步态周期。组间差异具有显著性，F(1,8)=47.6，p<0.001。后续t检验结果显示，对照组大鼠在各项动作成分上的得分均显著高于6-OHDA大鼠。视频中显示的对照动物后肢踏步动作为评分为“1”的示例，而视频中6-OHDA大鼠的后肢踏步动作主要评分为“0”。
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讨论
本研究的目的是探讨用于啮齿类动物运动技能检测的转棒实验是否可用于神经系统损伤大鼠姿势、前肢迈步和后肢迈步的定性评估。在分析过程中，对单侧多巴胺（DA）耗竭的大鼠和对照组大鼠分别从前方、侧方和后方进行视频记录。姿势与前肢运动的评分量表显示，手术后大鼠的迈步运动长期受损。对肢体运动的进一步分析表明，尽管多巴胺耗竭大鼠能够使用病灶对侧肢体进行支撑，但在转移体重方面几乎无法有效利用。本研究结果表明，转棒实验除了可提供运动功能障碍的定量指标外，还能揭示其定性方面的功能缺陷。
转棒实验已被广泛用于评估啮齿类动物的运动功能。迄今为止，该实验主要采用终点指标，即动物能够行走的速度或持续时间。我们认为，该实验的某些特性可能使其非常适合对行走动作进行定性分析。首先，由于动物必须保持平衡并进行踏步，该任务涉及较高的技能要求，因此可能揭示出在简单的地面行走任务中不易察觉的功能障碍（Schallert et al., 1992；Olsson et al., 1995；Chang et al., 1999；Muir 和 Whishaw 1999b；Metz et al., 2005）。该任务及其视频分析方法...
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致谢
由加拿大卫生研究院、加拿大卒中网络以及阿尔伯塔医学研究遗产基金会的资助支持。
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重印与许可
勘误
Formal Correction: Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat
Posted by JoVE Editors on 4/01/2012. Citeable Link.
A correction was made to: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.
