Summary

处理技巧,以减轻老鼠的压力

Published: September 25, 2021
doi:

Summary

本文描述了小鼠的处理技术,即3D处理技术,它通过减少类似焦虑的行为来促进日常处理,并介绍了两种现有相关技术(隧道和尾部处理)的细节。

Abstract

实验室动物受到科学家或动物护理提供者的多次操纵。这种引起的压力会对动物的福祉产生深远的影响,也可以成为焦虑措施等实验变量的混淆因素。多年来,人们开发出能够最大限度地减少处理相关压力的处理技术,尤其侧重于大鼠,而很少关注小鼠。然而,已经表明,老鼠可以习惯使用处理技术操纵。习惯小鼠处理可减轻压力,便于日常处理,改善动物健康,降低数据变异性,提高实验可靠性。尽管处理有有益的效果,但特别紧张的尾部拾取方法仍然被广泛使用。本文详细介绍了新开发的鼠标处理技术,旨在最大限度地减少动物在人际交往中承受的压力。这种手动技术在3天(3D处理技术)中执行,并侧重于动物适应实验者的能力。这项研究还显示了以前建立的隧道处理技术(使用聚碳酸酯隧道)和尾部拾取技术的效果。具体研究的是它们对焦虑行为的影响,使用行为测试(提升加迷宫和新奇抑制喂养),与实验者的自愿互动和生理测量(皮质激素水平)。3D处理技术和隧道处理技术减少了焦虑类表型。在第一个实验中,使用6个月大的雄性小鼠,3D处理技术显著改善了实验者的相互作用。在第二个实验中,使用2.5个月大的女性,它降低了皮质激素水平。因此,在需要或首选与实验者交互或在实验期间可能无法进行隧道处理的情况下,3D 处理是一种有用的方法。

Introduction

小鼠和大鼠是临床前研究的重要资产1,2用于多种目的,包括内分泌、生理、药理学或行为研究2。从越来越多的动物研究中,出现了包括人际交往在内的不受控制的环境变量影响生物医学研究的各种结果。这造成了实验和研究实验室4、5的显著变异性,对动物研究提出了重大警告。

实施了各种方法,目的是限制环境压力源的影响,减少对人类相互作用的反应。例如,为了限制环境压力源的影响,标准化住房条件和自动化住房系统6,7已在实验室实施。关于与人类的互动,通常用来处理和运输动物的方法很少考虑动物的不适和压力。例如,用尾巴或用钳子拾取动物8会增加基线焦虑9,10,11,减少探索9,12,并大大有助于个体间的变异,在研究13,14。因此,还开发了其他方法,例如适用于小鼠和大鼠的杯子处理技术。在这种方法下,动物被”杯”出笼子,由实验者用双手握住,形成一个9、10、11的杯子。另一个有用的尾部处理方法是使用聚碳酸酯隧道将老鼠转移9、10、15。此方法消除了鼠标和实验者之间的直接相互作用。杯子和隧道的方法都显示出有效的减少焦虑的行为和对实验者的恐惧,可以夸大反向处理技术,如尾部拾取/尾部处理9,10。

因此,越来越多的证据表明,适当的老鼠处理有助于减少个体之间的变异性9,11,并改善动物福利10。然而,上述技术仍然面临局限性。杯具处理技术已经实施,时间表从10天(10次超过2周16)到15周17,这是相当长的时间设施工作人员和实验者。此外,杯子处理的有效性因应变9而不同,而传统的杯子处理在张开手中可能导致天真的小鼠或特别跳跃的菌株从手9,18跳。隧道处理结果更一致,一般更快的结果,在绅士19。隧道也被用作家庭笼子浓缩。它们帮助动物习惯于快速处理,并提供丰富的额外好处。然而,隧道处理在设备之间转移动物时有局限性。有趣的是,赫斯特和西9号,亨德森等人20日证明,使用温和而简短的手动处理方式将动物从隧道转移到机器上并不影响它们的表型。

为了提供现有方法的替代方案,在短时间内实现习惯,本文描述了一种新颖的技术,扩展了杯具处理技术,因此不需要特定的设备。此方法使用里程碑来衡量小鼠在处理过程中的舒适程度。它显示了减少小鼠反应和压力(在行为和荷尔蒙水平)的功效,促进日常处理,并有助于减少动物之间的变异性。此处提供了此技术的详细信息,与隧道处理(正控)和尾部处理技术(负控制)相比,在减少焦虑行为、改善与实验者互动和限制外周压力激素(皮质激素)释放方面的功效在两项独立的研究(雄性小鼠和雌性小鼠)中得到了证明。

Protocol

涉及动物主体的程序已得到CAMH动物护理委员会的批准,并符合加拿大动物护理理事会的准则进行。 注:此处描述的处理方法可用于各种鼠标菌株,包括非转基因(C57/BL6、BalbC、CD1、SV129 等)和转基因线。它也可以与年轻或老老鼠一起使用,指出年轻的成年(4-6周大)小鼠往往比成年或老老鼠稍微活跃一些,尤其是在第一天。 1. 实验准备 在研究开…

Representative Results

对C57BL/6小鼠进行了两项单独的研究。研究#1包括6个月大的男性,研究#2包括2.5个月大的女性(N=36/研究)从杰克逊实验室(猫#000664)。老鼠在2个月大的时候到达了工厂。虽然研究#2女性在抵达后两周被处理和测试,但研究#1男性只在6个月大时才被处理和测试(由于全球大流行关闭而延迟)。在此期间,一只来自研究#2老鼠在开始处理实验之前死亡。这项研究#1雄性小鼠由动物设施工作人员照顾。所…

Discussion

这项研究和方法开发基于这样一种观察,即科学界仍然忽视老鼠的处理技术,一些实验室仍然不愿意在实验前实施习惯或处理技术,以减轻其动物的压力和反应能力。动物处理在代表时间承诺的同时,为动物提供了有益的效果,可能有助于要进行的实验的成功,并防止由于数据变异或动物反应过度而不得不多次进行实验。使用3D处理技术减少了小鼠的逃生尝试。它还增加了与实验者的互动,并减?…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢CAMH动物护理委员会支持这项工作,以及CAMH的动物护理人员,他们提供了关于程序有用性的广泛反馈,激励了上述实验的执行,并为其他用户提交了详细的协议。这项工作部分由授予TP的CAMH突破挑战和CAMH的内部资金资助。

Materials

23 G x 1 in. BD PrecisionGlide general use sterile hypodermic needle. Regular wall type and regular bevel. BD 2546-CABD305145 Needles for Blood collection
BD Vacutainer® Venous Blood Collection EDTA Tubes with Lavender BD Hemogard™ closure, 2.0ml (13x75mm), 100/pk BD 367841 EDTA Coated tubes for blood collection
Bed’o cobs ¼” Corn cob laboratory animal bedding Bed-O-Cobs BEDO1/4 Novel bedding for novelty suppressed feeding
Centrifuge Eppendorf Centrifuge 5424 R For centrifugation of blood.
Corticosterone ELISA Kit Arbor Assays K003-H1W
Digital Camera Panasonic HC-V770 Camera to record EPM/Experimenter interactions
Elevated Plus Maze Home Made n/a Custom Maze made of four black Plexiglas arms (two open arms (29cm long by 7 cm wide) and two enclosed arms (29 cm long x7 cm wide with 16 cm tall walls)) that form a cross shape with the two open arms opposite to each other held 55 cm above the floor
Ethanol Medstore House Brand 39753-P016-EA95 Dilute to 70% with Distilled water, for cleaning
Ethovision XT 15 Noldus n/a Automated animal tracking software
Laboratory Rodent Diet LabDiet Rodent Diet 5001 Standard Rodent diet
Memory Card Kingstone Technology SDA3/64GB For video recording and file transfer
Novelty Suppressed Feeding Chamber Home Made n/a Custom test plexiglass test chamber with clear floors and walls 62cm long, by 31cm wide by 40cm tall .
Parlycarbonate tubes Home Made n/a 13 cm in length and 5cm in diameter
Purina Yesterday’s news recycled newspaper bedding Purina n/a Standard Bedding
Spectrophotometer Biotek Epoch Microplate Reader

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Marcotte, M., Bernardo, A., Linga, N., Pérez-Romero, C. A., Guillou, J., Sibille, E., Prevot, T. D. Handling Techniques to Reduce Stress in Mice. J. Vis. Exp. (175), e62593, doi:10.3791/62593 (2021).

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