机械冲突避免测定测量小鼠疼痛行为
Summary
机械避免冲突测定用作小鼠疼痛敏感性的非反射性读数，可用于更好地理解各种小鼠疼痛模型中的情感动机反应。
Abstract
疼痛包括感觉（伤害感受）和情感（不愉快）两个方面。在临床前模型中，传统上使用反射测试来评估疼痛，这些测试可以推断疼痛的伤害感受成分，但很少提供有关疼痛的情感或动机成分的信息。因此，开发捕获这些疼痛成分的测试在翻译上很重要。因此，研究人员需要使用非反射性行为测定来研究该水平的疼痛感知。机械冲突避免（MCA）是一种已建立的自愿非自反行为测定，用于在3室范式中研究对有害机械刺激的动机反应。当面对相互竞争的有害刺激时，小鼠位置偏好的变化被用来推断强光与爪子的触觉刺激的感知不愉快。该协议概述了MCA测定的修改版本，疼痛研究人员可以使用该版本来了解各种小鼠疼痛模型中的情感动机反应。虽然这里没有具体描述，但我们的示例MCA数据使用体内完全弗氏佐剂（CFA），幸免于神经损伤（SNI）和骨折/铸造模型作为疼痛模型来说明MCA程序。
Introduction
疼痛是一种具有感觉和情感成分的复杂体验。疼痛知觉阈值的降低和对热和/或机械刺激的超敏反应是这种体验的关键特征，刺激诱发的疼痛行为测试可以捕获（如Hargreaves的热敏感性测试和von Frey的机械敏感性测试）1，2。虽然这些测试给出了稳健且可重复的结果，但它们受到依赖从感知的有害刺激中反射性戒断的限制。这使人们质疑疼痛研究仅依靠这些测试的持续依赖。为此，疼痛研究人员多年来一直在探索用于啮齿动物疼痛模型的替代/补充行为测试，以捕获疼痛的更多情感和/或动机成分。这些非诱发性、自愿性或非反射性措施（例如，车轮运行、挖洞活动、条件位置偏好3，4，5）正在实施，以提高临床前疼痛研究的可转化性。
机械冲突避免（MCA）测定最初由Harte等人在20166中描述，主要用于大鼠7，8，并代表了早期方法的修改 – 逃逸 – 避免范式。在这种方法中，后爪的有害刺激在其他理想的（黑暗）室中进行，以驱动动物的有目的行为以逃避/避免这种刺激9，10。MCA测定不是依靠观察者对后爪的手动有害刺激，而是迫使小鼠通过潜在的有害刺激来逃避令人厌恶的环境并到达暗室。使该测定法得名的冲突/回避源于这两种相互竞争的动机：逃离明亮的区域并避免对爪子的有害刺激。MCA测定还与条件性的地方偏好测试共享特征，其中疼痛缓解与环境线索的配对推动了行为的变化，反映了对缓解疼痛/奖励环境的偏好11。
从根本上说，所有这些测定都有一个相似的方法：使用动物对一种厌恶环境的偏好相对于另一种环境的偏好的转变，作为其情感/动机状态的指标。MCA测定是一种3室范式，由一个明亮的腔室组成，然后是一个带有可调节高度探针的黑暗中间室和一个没有任何厌恶刺激的黑暗第三室。一只未受伤的老鼠通常有动机逃到一个黑暗的房间，因为啮齿动物天生厌恶明亮的光12。在这个例子中，逃离明亮环境的自然动机克服了遇到后爪刺激（可调节高度探头）的倾向，后者只发生在黑暗的环境中。相反，经历疼痛（例如，由于炎症或神经病变）的小鼠可能会选择在明亮的环境中花费更多时间，因为在持续的触觉超敏反应的情况下，有动机避免机械探针的不愉快的触觉体验。
本文介绍了MCA测定的修改版本。我们已经将原始方法（在大鼠6中进行）用于小鼠。我们还将测试的探头高度从 6 个减少到 3 个（高于地板高度 0、2 和 5 mm），以简化数据采集。这种方法已经在多个疼痛模型中进行了测试，并用已知的镇痛药进行了验证，表明疼痛超敏反应和/或相关的情感和动机变化正在推动这些行为变化。与其他非反射性措施相比，这种方法的进行速度相对较快且适应性强，后者可能需要许多天的习惯化和训练1，2。与其他疼痛测量方法一起，MCA可以对疼痛的情感和动机方面产生有价值的见解。
Protocol
Representative Results
Discussion
与所有行为测试一样，正确处理，随机化和对动物治疗的盲法至关重要。鉴于复杂行为和决策的多因素输入，必须尽可能一致地处理，习惯和测试动物，同时最大限度地减少痛苦。还应注意重现鼠标放置在腔室1中，打开LED灯并消除障碍物的时间，因为此处的差异可能会影响后续行为。
应该注意的是， 图1A 中描述的不同结局指标是相互关联的。例如，?…
Declarações
The authors have nothing to disclose.
Acknowledgements
GM由NDSEG研究生奖学金提供支持。VLT由NIH NIGMS赠款#GM137906和Rita Allen基金会提供支持。AJS 由国防部拨款 W81XWH-20-1-0277、W81XWH-21-1-0197 和 Rita Allen 基金会提供支持。我们感谢华盛顿大学医学院的Alexxai Kravitz博士为2室地板和探针板设计和免费提供3D打印机文件。
Materials
| 32.8ft 3000K-6000K Tunable White LED Strip Lights, Dimmable Super Bright LED Tape Lights with 600 SMD 2835 LEDs | Lepro | SKU: 410087-DWW-US | For lighting chamber 1. https://www.lepro.com/32ft-dimmable-tunable-white-led-strip-lights.html |
| 3D printed 'spike bed' and 'chamber 2 floor' | Shapeways | N/A | Optional, for mechanical probes as an alternative to blunted map pins. |
| 70% ethanol | Various | N/A | To clean MCA between mice. |
| Acryl-Hinge 2 | TAP Plastics | N/A | for attaching chamber lids to rear walls. https://www.tapplastics.com/product/plastics/handles_hinges_latches/acryl_hinge_2/122 |
| Chemcast Cast Acrylic Sheet, Clear | TAP Plastics | N/A | 3mm thick. For front wall of chamber 1. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_cast_clear/510 |
| Chemcast Cast Transparent Colored Acrylic, Transparent Dark Red – 50% | TAP Plastics | N/A | 3mm thick. 50% light transmission. For walls and lids of chambers 2 and 3. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_transparent_colors/519 |
| Chemcast Translucent & Opaque Colored Cast Acrylic, Sign Opaque White – 0.1% | TAP Plastics | N/A | 3mm thick. For side walls and lid of chamber 1. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_color/341 |
| Disinfectant (e.g. Quatricide) | Pharmacal Research Laboratories, Inc. | 65020F | To disinfect MCA at the end of a testing session. |
| Dry-erase markers and board | Various | N/A | To add experimental info to the beginning of video footage. |
| Map pins | Various | N/A | Optional, for mechanical probes. Use sandpaper to blunt sharp points before use. Can be used in place of 3D-printed parts. |
| Paper towels | Various | N/A | To clean/disinfect MCA. |
| SCIGRIP Weld-On #3 Acrylic Cement | TAP Plastics | N/A | For assembling acrylic sheets into chambers and affixing hinges. https://www.tapplastics.com/product/repair_products/plastic_adhesives/weld_on_3_cement/131 |
| Stopwatch | Various | N/A | To record escape latencies/dwell times in real-time or from recorded video. |
| Timer | Various | N/A | To ensure LED turn-on, barrier removal and test completion are timed consistently. |
| Video camera | Various | HDRCX405 Handycam Camcorder | To record mouse behavior in the MCA device. Can be substituted with any consumer-grade video camera capable of 1080p resolution. |
| Tripod | Famall | N/A | Any tripod that can hold the camera at bench height for recording MCA footage is acceptable. |
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