测定冷灵敏度和适应小鼠一种简单廉价的方法
Summary
The Cold Plantar Assay (CPA) measures cold responsiveness between 30 °C and 5 °C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.
Abstract
Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.
Introduction
在啮齿类动物中测量冷响应为正常和病理条件下改善的冷敏感性的潜在机制的理解在人类重要的。冷足底测定（CPA），最初开发几年 前1，被设计为生成可再生的，明确的小鼠的行为反应，以冷的刺激在RT递送。更近的本测定法的改进已经允许冷灵敏度的再现性的测定在一个宽温度范围内的2。这两个版本也被设计成相对高的通量，且廉价使用。
了很大的进步已取得理解冷灵敏度的机制使用其他行为的方法。一种方法是将丙酮蒸发试验，这涉及到涂抹或鼠标爪子喷洒丙酮和测量的时间，该鼠标花费轻弹爪3,4的量。不幸的是，该反应的丙酮蒸发由湿感和丙酮的气味混淆。此外，即在丙酮蒸发试验施加冷刺激可以基于施加的丙酮的量而变化，并且是难以量化。最后，未受伤的小鼠具有最小响应丙酮在基线，使它不可能测量镇痛在没有敏感症的这种方法。
另一个经典测定法冷的反应是在甩尾试验中，在那里的等待时间停药后的尾部浸入冷水5,6被测量。而在此测定的行为反应是明确的和测定测量的响应，以特定的温度，动物必须在测试期间被限制，从而可以改变通过充分描述应力诱发镇痛机构7冷响应能力。
另一种常用的工具是冷板试验，其测量行为小鼠的应答后，便被放置在一个珀尔帖冷却板8-10。虽然这种工具提供有关在特定温度下的动物的反应的信息，它也被不一致地使用;不同的群体有不同的测量类型的反应，包括一些跳跃8,11的，延迟到第一反应8,11- 13，和爪的数量提升11,13,14具有非常不同的结果。冷板试验是还可以在一个时间进行测试比较低的吞吐量，因为只有一只动物，它需要使用昂贵和脆弱的珀尔帖装置。
2-板温度偏爱测试是冷板试验，其测量时间的相对量，动物花费在不同温度9,15- 17的2连板的常用衍生物。另一个类似的常用分析方法是热梯度测定中，时间在那里的小鼠花费在不同温度区的数量在一个长的金属板之间5℃和45℃的范围内进行测量16。虽然这些测定法允许的温度比较，目前还不清楚的行为是否代表温度厌恶或对温度的偏好。
最后，将动态冷板试验已被用来测量小鼠对不断变化的环境温度下18如何响应。该方法包括将小鼠RT Peltier装置上斜坡下来，以1°C，同时测量小鼠多少跳或舔爪子在不同的板温度。虽然这种测试的小鼠如何适应冷却的环境中，它不提供一个方法来测试在冷却器环境温度的设定，以冷的刺激小鼠如何作出反应。另外，它需要昂贵的设备来执行，并且不提供一种方法测量其冷敏感性之前驯化小鼠的检测设备。
为了配合这些试验，注册会计师测试ACCLIMA泰德响应一个明确定义的冷刺激，在各种温度范围，或在适应寒冷的环境温度下的过程。它可以测试多达14只小鼠在现今的装置的时间，与要廉价地扩大规模用于高通量试验的潜力。
Protocol
Representative Results
Discussion
The CPA can be used to assess cold sensitivity and cold adaptation in mice. It provides an affordable, efficient way to measure cold responses in unrestrained, acclimated animals at a wide variety of temperature ranges. It also provides an unambiguous behavioral response with an easily quantified and analyzed output variable. It has already been used to assess changes in cold sensitivity induced by inflammation1, neuropathic injury1, analgesics1, genetic knockouts20, and ge…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge contributions from the entire Gereau Lab for manuscript editing. This work is supported by NINDS funds 1F31NS078852 to DSB and NINDS fund NS42595 to RWG.
Materials
| Name | Company | Catalog Number | Comments |
| T-type thermocouple probe | Physitemp | IT-24p | Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/) |
| Glass plate | Local glass company (in St. Louis, Stemmerich Inc) | N/A | We use pyrex glass (borosilicate float). Our lab generally uses 1/4'', but 3/16'' and 1/8'' are also useful |
| Thermal Data logger | Extech | EA15 | Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&prodid=408) |
| 3mL Syringe | BD | 309657 | The top is cut off, and dry ice is compressed in the syringe to generate a cold probe |
| Computer | If using Extech logger, any Pcwill work | N/A | |
| Aluminum boxes | Washington University in St. Louis machine shop | N/A | boxes are 3' long, 4.5'' wide, and 3'' tall with a sealed lid. There is a 1/2'' hole drilled into one short side of each box, near the bottom. These holes are filled with rubber stopcocks when the boxes are filled with wet ice or hot water. |
| Heated water circulator | VWR | Any water circulator model with a pump will work | |
| 21 gauge needle | BD | 305165 | The exact needle size is not important |
| Hand timer | N/A | Any hand timer will work | |
| Mirror | N/A | Any flat mirror will work |
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