小鼠应用慢性约束应激的低成本足迹分析和吊箱试验协议
Summary
由约束应力后的足迹分析和吊箱测试组成的低成本协议可用于评价小鼠模型的运动障碍。
Abstract
在运动障碍患者中经常观察到步态障碍。在用于运动障碍的小鼠模型中, 步态分析是重要的行为测试, 以确定小鼠是否模仿患者的症状。当小鼠模型中没有观察到自发运动表型时, 压力往往会引起运动缺陷。因此, 步态分析, 其次是应力负荷将是一个敏感的方法来评价电机表型的小鼠模型。然而, 研究人员面临着昂贵的仪器的需求, 以自动获得定量结果的步态分析。对于应力, 不需要在触电和强制运行所需的昂贵设备的情况下, 采用简单的方法进行应力加载。因此, 我们引入了一种简单、低成本的协议, 包括用纸张和墨水进行足迹分析, 通过吊箱试验来评估电机的功能, 以及用锥形管约束定义的应力载荷。该方法成功地检测了小鼠的运动缺陷。
Introduction
运动障碍被定义为神经系统的紊乱, 显示自愿或自动运动的过度或缺乏1。特别是, 在运动障碍2、3、4 的患者中, 步态障碍经常被记录在案。因此, 步态分析是一种适合运动障碍动物模型验证的行为测试。在小鼠中, 通过 6, 7级跑步机对以自然速度5 和可调速度行走进行了自动步态分析。这些分析自动提供步态的定量结果。另一种检测步态干扰的方法称为足迹分析。用墨水标记脚底后, 老鼠在纸上行走, 并对脚印进行分析。最初, 凡士林和粉状木炭被用来显示足迹8, 然后被测谎仪纸9上的墨水和摄影纸上的摄影开发商取代.与其他方法相比, 使用墨水和纸张的一种更便宜、毒性更小的方法至今仍是11。与自动分析5、6、7相比, 足迹分析的成本较低, 对于没有充足研究资金的研究人员评估小鼠模型中的运动障碍将是有用的.
吊箱试验是一种使用铁丝笼盖12和金属丝网13的四肢悬挂试验.盒子是一种在盒子顶部沿中心条旋转网盖的装置。除了步态分析外, 测试还可以低成本、轻松地进行。因此, 除了本协议中的占地面积分析外, 我们还进行了吊箱测试, 以评估抓地力和平衡。
压力会诱发运动障碍的症状14,15。即使在16、17、18的运动障碍小鼠模型中没有观察到自发运动表型, 也往往是由几个慢性应力引起的。约束是小鼠压力负荷的常用方法之一, 因为动物身体没有受到伤害, 与专用装置电击、使用跑步机强制运行等其他方法相比, 成本较低。通过将老鼠限制在一个洞50毫升锥形管中进行的控制, 比其他方法 (如丝网过滤器、带状肢体和用纱布包裹动物) 更容易.本文总结了管道约束后的占地面积分析和吊箱试验的方案。该协议将帮助我们使用没有自发运动表型的运动障碍的小鼠模型。
Protocol
所有动物实验都是以人道的方式进行的。吉吉医科大学机构动物实验委员会批准了这项研究。这项研究是根据《动物实验制度条例》和《日本 mext 管辖下的学术研究机构动物实验和相关活动的适当进行的基本准则》进行的。本协议中使用的老鼠已经在前面描述过21。 1. 吊箱测试 记录每个鼠标的重量。标记尾巴的笔的个人歧视 (例如,一条线, 双?…
Representative Results
本方案采用了at1a3 (at1a3+/-)的杂合雄性小鼠模型, 作为快速发病肌张力障碍帕金森病和野生类型同学的模型。atp1a3+/–显示 前肢和后肢的步幅明显短于4周大的野生类型 (图 5a和图 5A, 开放圆和正方形)。”压力” atp1a3+/-显示两个肢体的步幅长度明显短于8周大的”无应力” atp1a3+/-…
Discussion
足迹分析和吊箱测试是对小鼠运动功能的简单而廉价的行为测试。通过这些测试, 成功地检测出了几种小鼠模型中的神经行为表型。例如, 肌萎缩侧索硬化症24的步长缩短, 在华支扩张25中增加了不对称步幅的长度, 增加了亨廷顿病26和肌张力障碍27的重叠长度,通过足迹分析, 显示了共济失调 28、<sup class…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了日本科学促进协会 (jsps) kakenhi (科学研究赠款 c)、18k07373 (h. s.) 赠款和私立大学补贴的支持。
Materials
| Hanging box | O’hara & Co. | http://ohara-time.co.jp/products/wire-hanging-test/ | |
| Marking pen | ZEBRA | MO-120-MC-BK | |
| Goal box | O’hara & Co. | http://ohara-time.co.jp/products/balanced-beam-test/ | Accessory for apparatus of balanced beam test |
| Boxes | O’hara & Co. | – | Side wall of runway |
| Black ink | Shin-asahi | – | |
| Red ink | Maruyamakogyo | BC-6 | |
| Disposable Petri Dish | Corning | 351008 | Petri dishe (35 mm in diameter) |
| Askul Multipaper Super White J Monochrome A3 | Askul | 701-712 | White paper (29.7 cm x 42 cm x 0.09mm) |
| 50 mL Conical tube | Corning | 430829 | |
| Square drill | KAKURI Corporation | DIY FACTORY (K32-0313) |
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Cite This Article
Sugimoto, H., Kawakami, K. Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress. J. Vis. Exp. (143), e59027, doi:10.3791/59027 (2019).