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.

所有的鼠标协议均符合卫生指引国家机构，并批准了华盛顿大学医学院的动物研究委员会（圣路易斯，MO）。 1.准备测试板和外壳<ol><li>清洁过的玻璃表面。 </li><li>固定在T型丝热电偶探头的表面在玻璃板与实验室磁带的中间。 </li><li>放置在玻璃板上的动物罩在沿板的中间一行。 </li><li>通过该中心的动物外壳螺纹热电偶探头和插入数据记录器。转动而停用自动关机功能上的数据记录器，并附加数据记录仪到计算机提供的电缆。 <ol><li>如果在实验过程中记录板温度，打开数据记录软件开始录制板温度。 </li><li>如果有必要，调整软件重新线板温度每秒一次。 </li><li>开始录制用打包带的热数据记录器软件温度。 </li></ol></li><li>与黑色插入机箱分离，以防止老鼠之间的视觉交互。 </li><li>位置镜玻璃，使得所述外壳的下侧是从舒适的就座位置可见的下方。 </li></ol> 2.变暖/冷却玻璃板<ol><li>填充铝框用温热水，湿冰或干冰，并将它们在玻璃板适当地定位（填充有干冰铝箔包也可以被用于冷却玻璃; 图1）2。 <ol><li>用于测试在30℃下，约定位铝框0.25&#39;&#39;远离动物罩（ 图2B）2。 <ol><li>在玻璃板的两侧设置了加热水循环。设置在环行器45 - 60℃，我们Ë它填补了铝合金箱用热水（ 图1C）2络绎不绝。 </li><li>定位循环，使得热水从铝框水渠直接返回到循环器的每一侧（ 图1C）的贮存器2。 </li></ol></li><li>用于测试在室温，离开拖曳空（ 图2）2。 </li><li>为了测试在17°C，大约放置箱子0.25&#39;&#39;离两边的动物外壳和填充湿冰（ 图2）2。 </li><li>为了测试在12°C，大约放置箱子1.25&#39;&#39;离两边的外壳和填充用干冰（ 图2）2。 </li><li>为了测试在5℃，放置盒约0.25&#39;&#39;离两边的外壳和填充用干冰（ 图2）2。<ol><li>当冷却的玻璃用干冰，确保有足够的通风，以防止二氧化碳积聚在房间里。 </li></ol></li></ol></li><li>等待玻璃以达到所需的温度范围。 </li><li>老鼠添加到上盘的外壳。 注：白噪声发生器可用于减少噪音干扰。 </li><li>等待老鼠适应。 注：在我们的设施这需要大约2.5小时，但是这可能基于动物住房和操作条件显著不同。 </li><li>通过确保拖曳保持充分的温热水，湿冰或干冰保持玻璃在所需的温度范围内。 注：使用我们的设备的箱子需要用冰，大约每90分钟进行补充。 注意：对于17℃的条件下，它是有帮助的冰再填充之前清空大部分的水从铝框通过排水孔。这将稳定的温度更好，且Pr事件溢出 注：在干冰的确切量会随季节变化，但在一般保持拖曳多于四分之一满沿盒子的整个长度将保持温度恒定。 </li></ol> 3.测试小鼠在固​​定温度<ol><li>行为的房间外，填补冰桶约半满的干冰。 </li><li>用锤或槌，粉碎干冰成细粉。 注意：过量加载斗将使它难以完全粉碎干冰成粉末。 </li><li>使用直刀片或剪刀，削顶过3毫升注射器。 </li><li>使用21g的针，戳在反对注射器（共6孔）两侧3个孔。 注意：这些孔会降低通过升华所产生的压力而压缩干冰。截止注射器可重复用于多个实验。 </li><li>取注射器，干冰粉末，和一个手持秒表进行为房间。 </li><li>填充注射器室半满的干冰粉末。靠住平坦物体的注射器的切断端，以及使用该柱塞牢牢压缩的粉末。当心;塑料柱塞可弯曲或从压力破裂。如果发生这种情况，从一个新的注射器更换柱塞。 </li><li>扩展压缩干冰丸粒的前端越过注射器的边缘。 </li><li>测试小鼠完全处于静止状态。 <ol><li>在30℃，23℃和17℃，有所有4个爪子在玻璃上，而不是测试老鼠感动，但没有完全睡着了19。 </li><li>在12°C和5°C，试验小鼠都在2爪子或4爪子，而不是移动或跳跃。 </li></ol></li><li>用镜子为目标，轻轻但坚定地按压鼠标后肢（ 图1A）2下的玻璃表面的平坦球平齐。启动手定时器。 </li><li>停止定时器并取出粒料当鼠标移动远离冷却的玻璃。 注：退回运动可以垂直或水平。 <ol><li>如果鼠标非常简要地移动爪子，然后它返回到冷却面，继续定时和刺激直到小鼠使一个永久搬走。 注：我们的实验室使用20秒的最大刺激时间的小鼠在大多数情况下。 </li></ol></li><li>重复此测试过程，直到对每个动物的每个爪的至少3个值被收集。独立的试验中在相同的小鼠由至少7分钟，和单独的连续试验至少15分钟上的任何单一的爪检测相反的爪子。 </li><li>如果需要的话，可以使用不同厚度的玻璃来产生的冷却（ 图3）1不同的速率。 注：冷却速率成反比与玻璃的厚度相关。 </li></ol> 4.测试小鼠在冷适应注意：这是一个替代的协议，它允许测试作为玻璃高原ê冷却，而不是一旦板块企稳和老鼠已经完全适应了寒冷的环境。 <ol><li>按照第1节设立的设备列出的说明。 </li><li>按照第3节所列采取基线测量在室温（ 图7A）2的说明。 </li><li>预冷却的铝框用干冰。 </li><li>一旦基线退缩潜伏期已测，上盘约1.25&#39;&#39;远离机箱两侧放置预冷箱（ 图7A，箭头标记为“干冰加上”）2。 </li><li>按照第3节所列进行测量的玻璃板冷却后，进行测量尽可能多的指令。 </li></ol>

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The authors have nothing to disclose

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...

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。

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Name</td>
			<td style="width:320px;">Company</td>
			<td style="width:353px;">Catalog Number</td>
			<td style="width:303px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-type thermocouple probe</td>
			<td>Physitemp</td>
			<td>IT-24p</td>
			<td>Used to measure the surface temperature of the glass (http://www.physitemp.com/products/probesandwire/)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass plate</td>
			<td>Local glass company (in St. Louis, Stemmerich Inc)</td>
			<td>N/A</td>
			<td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#39;&#39;, but 3/16&#39;&#39; and 1/8&#39;&#39; are also useful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal Data logger</td>
			<td>Extech</td>
			<td>EA15</td>
			<td>Thermologger to keep track of glass temperature (http://www.extech.com/instruments/product.asp?catid=64&#38;prodid=408)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3mL Syringe</td>
			<td>BD</td>
			<td align="right">309657</td>
			<td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td>If using Extech logger, any Pcwill work</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum boxes</td>
			<td>Washington University in St. Louis machine shop</td>
			<td>N/A</td>
			<td>boxes are 3&#39; long, 4.5&#39;&#39; wide, and 3&#39;&#39; tall with a sealed lid.&#160; There is a 1/2&#39;&#39; 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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated water circulator</td>
			<td>VWR</td>
			<td></td>
			<td>Any water circulator model with a pump will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">21 gauge needle</td>
			<td>BD</td>
			<td align="right">305165</td>
			<td>The exact needle size is not important</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hand timer</td>
			<td>N/A</td>
			<td></td>
			<td>Any hand timer will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mirror</td>
			<td>N/A</td>
			<td></td>
			<td>Any flat mirror will work</td>
		</tr>
	</tbody>
</table>

a simple and inexpensive method for determining cold sensitivity and adaptation in mice

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 &#176;C to 5 &#176;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.

从小鼠引起开始在30℃，23℃，17℃，和12℃下的行为反应是高度可再现的（ 图4A）20。为了测量后爪下所产生的冷刺激，将小鼠用氯胺酮/甲苯噻嗪/乙酰丙嗪鸡尾酒和它们的爪子被固定在上一个T型热电偶丝（ 图4B）20顶部的玻璃。玻璃被冷却或加热至所需的测试范围。虽然该板被均匀地冷却沿所述板（ 图5A）2的长度，但应?...

Watch this Scientific Journal Video about A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice at JoVE.com

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

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14.6K 次观看.  Washington University in St. Louis. 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.