建立小鼠睡眠剥夺装置
Summary
本方案概述了一种用于建立用于诱导小鼠睡眠剥夺的基于摇杆平台的具有成本效益的摇杆平台的装置的方法。该设备已被证明可有效导致脑电图 （EEG） 证明的睡眠模式中断，以及诱导与睡眠剥夺相关的代谢和分子变化。
Abstract
昼夜节律紊乱是指外部环境或行为与内源性分子钟不同步，严重损害健康。睡眠不足是昼夜节律紊乱的最常见原因之一。据报道，各种方式（例如，水上平台、轻柔处理、滑动杆室、旋转鼓、轨道振动器等）可诱导小鼠睡眠剥夺，以研究其对健康的影响。目前的研究引入了一种小鼠睡眠剥夺的替代方法。设计了一种基于自动摇杆平台的设备，该设备具有成本效益，并且可以在可调节的时间间隔内有效地扰乱群居小鼠的睡眠。该装置以最小的应激反应诱导睡眠剥夺的特征性变化。因此，这种方法可能对有兴趣研究睡眠剥夺对多种疾病发病机制的影响和潜在机制的研究人员有用。此外，它还提供了一种经济高效的解决方案，特别是当需要多个睡眠剥夺设备并行运行时。
Introduction
昼夜节律紊乱是指外部环境或行为与内源性生物钟之间的不同步。昼夜节律紊乱的最常见原因之一是睡眠剥夺1.睡眠不足不仅会对人类健康产生负面影响，还会显着增加患多种疾病的风险，包括癌症2 和心血管疾病3。然而，睡眠剥夺的有害影响背后的机制在很大程度上仍然未知，建立睡眠剥夺模型对于加强我们在这方面的理解至关重要。
已经报道了各种剥夺小鼠睡眠的方法，例如使用水平台4，轻柔处理5，滑动杆室6，旋转鼓7和笼式搅拌协议5,8,9。滑动杆室自动扫过笼子底部的杆，迫使老鼠走过它们并保持清醒。笼子搅拌方案包括将笼子放在实验室轨道振动器上，从而有效地中断睡眠。虽然这些方法是自动且有效的，但当需要多个设备并行运行时，它们可能会很昂贵，特别是对于涉及昼夜节律基因分析所需的大量睡眠不足小鼠的特定研究设计。另一方面，水平台和温和的处理方案是通常用于诱导睡眠剥夺的更便宜、更简单的方法。然而，水平台不允许自动控制预先指定的剥夺-休息周期10,11，并且轻柔的处理需要研究人员持续保持警惕以扰乱睡眠。此外，其他方式，如旋转鼓，可能会因社会孤立或压力而混淆 12.
受基于轨道振荡器的方法的启发，我们旨在引入一种协议，用于建立基于摇臂平台的小鼠睡眠剥夺装置。这种方法便宜、有效、压力最小、可控且自动化。根据我们的可访问性，当前的协议允许我们以比轨道振动器便宜约十倍的成本创建基于摇杆平台的设备。该装置有效地扰乱了群居小鼠的睡眠，并以最小的应激反应诱导了睡眠剥夺的特征性变化。对于有兴趣研究睡眠剥夺对多种疾病发病机制的影响和潜在机制的研究人员来说，这将特别有用，特别是当该研究同时涉及多组睡眠剥夺时。
Protocol
本研究所有动物实验方案均获得上海交通大学医学院仁济医院实验动物福利伦理委员会批准。该研究使用了年龄在8至10周之间的雄性C57BL / 6J小鼠。这些动物是从商业来源获得的（见 材料表）。 图1A列出了建立设备所需的主要部件。 1.睡眠剥夺装置的制备 用螺钉将 50 cm 开槽钢槽的一端固定在 40 cm 开槽钢槽的中间（见<stro…
Representative Results
图1D显示了用于小鼠睡眠剥夺的既定装置。在睡眠剥夺开始后的第 7 天，脑电图 （EEG） 和肌电图 （EMG） 监测16 表明该装置显着缩短了小鼠的睡眠持续时间并增加了清醒持续时间（图 2A-D）。同时，目前的方案显着增加了大脑中 Homer1a 的腺苷积聚和 mRNA 水平（图 2E，F?…
Discussion
睡眠剥夺的小鼠模型对于研究睡眠中断对各种疾病的影响至关重要，包括心血管疾病21、精神疾病22 和神经系统疾病23。在小鼠现有的睡眠剥夺策略中，涉及重复短期睡眠中断的物理方法是最常用的5,7,12。这些物理方法包括使用水平台4、轻柔处理<sup class="x…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了国家自然科学基金（82230014,81930007,82270342）、上海市杰出学术带头人计划（18XD1402400）、上海市科学技术委员会（22QA1405400,201409005200,20YF1426100）、上海浦江人才计划（2020PJD030）、上海老龄医学临床研究中心（19MC1910500）和蚌埠医学院研究生创新计划资助（Byycxz21075）。
Materials
| 1.5 mL microcentrifuge tube | Axygen | MCT-150-C-S | |
| 50 mL centrifuge tube | NEST | 602002 | |
| Adenosine ELISA kit | Ruifan technology | RF8885 | |
| Animal cage | ZeYa tech | MJ2 | |
| Blood glucose meter | YuYue | 580 | |
| C57BL/6J Mice | JieSiJie Laboratory Animal | N/A | Age: 8-10 weeks |
| Connecting rod | ShengXiang Tech | N/A | Length: 20 cm |
| Cooling fan | LiMing | EFB0805VH | Supply voltage: 5 V; Power consumption: 1.2 W; Air flow: 26.92 cfm; Dimensions: 40 mm * 40 mm * 56 mm |
| Corticosterone ELISA kit | Elabscience | E-OSEL-M0001 | |
| EEG/EMG recording and analysis system | Pinnacle Technology | 8200-K1-iSE3 | |
| Isoflurane | RWD | 20071302 | |
| mosquito hemostats | FST | 13011-12 | Surgical instrument |
| Motor and motor mount | MingYang | MY36GP-555 | Supply voltage: 24 V dc; Shaft diameter: 8 mm; Maximum output torque: 100 Kgf.cm; Maximum output speed: 10 rpm |
| NanoDrop 2000c | Thermo Scientific | NanoDrop 2000c | |
| Power brick adapter | MingYang | QiYe-0243 | Input voltage: 110-220V ac; Output voltage: 24 V dc; Outputcurrent: 2 A; Cable length: 2 m |
| qPCR commercial kit | Vazyme | Q711-02 | |
| qPCR measurement equipment | Roche | 480 | |
| Rectangle platform attached with a screw-compatible steel cylinder | Customized | N/A | Width: 20 cm; length: 25 cm; length of the cylinder: 30 cm, thickness: 2 mm |
| Reverse RNA to cDNA commercial kit | Vazyme | R323-01 | |
| Screw and nut | Guwanji | N/A | Inner diameter: 6 mm, 12 mm |
| Screw-compatible steel cylinder | Customized | N/A | Length: 300 mm |
| Slotted steel channels | Customized | N/A | Length: 400 mm or 500 mm, thickness: 2 mm |
| Time contactor | LiXiang | DH48S-S | Supply voltage: 110-220 V ac; Units measured: hours, minutes, seconds; Contact configuration: DPDT |
| TRIzol | Vazyme | R401-01 |
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Cite This Article
Chen, J., Wei, J., Ying, X., Yang, F., Zhao, Y., Pu, J. Establishing a Device for Sleep Deprivation in Mice. J. Vis. Exp. (199), e65157, doi:10.3791/65157 (2023).