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 genetic neuron ablation studies26. It can be modified to measure responses to a variety of rates of cooling using different thicknesses of glass, and the size of the cold stimulus applied can be altered using different sized syringes. 
The most critical step for performing this assay properly is cooling or warming the glass plate evenly during testing. Carefully arranging the aluminum boxes so that they are evenly spaced on either side of the animal enclosures will help generate more uniform temperatures. Marking the glass plate to standardize aluminum box placement between experiments will also improve the reproducibility of the assay, although these markings may have to be periodically recalibrated to account for seasonal variations in building temperature.
It can also be challenging to maintain a consistent glass plate temperature over the course of the experiments. Ensuring that the aluminum boxes are always evenly filled with dry ice/wet ice/warmed water will yield experiments with stable plate temperatures. Additionally, under the 17 &#176;C testing condition it may become more difficult to maintain the temperature as the wet ice melts. We recommend draining excess water from the melted ice out of the aluminum boxes before refilling with more wet ice. 
The main limitation is that the mouse paw must be in contact with the glass surface in order to ensure a consistent cold stimulus. This can be an issue in some neuropathic models, such as the chronic constriction injury, which can cause paw curling. Other challenges to obtaining reproducible results with this assay include that the mice must be fully at rest, which requires adequate acclimation periods. If the mice are too active for testing, it may help to allow longer acclimation periods. Finally, groups that are planning to utilize this assay must also ensure that there is sufficient ventilation in the room in order to prevent CO2 buildup when using large amounts of dry ice.

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><tbody><tr><td>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><td>Glass plate</td><td>Local glass company (in St. Louis, Stemmerich Inc)</td><td></td><td>We use pyrex glass (borosilicate float). Our lab generally uses 1/4&#039;&#039;, but 3/16&#039;&#039; and 1/8&#039;&#039; are also useful</td></tr><tr><td>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&amp;amp;prodid=408)</td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td>The top is cut off, and dry ice is compressed in the syringe to generate a cold probe</td></tr><tr><td>Computer</td><td>If using Extech logger, any Pcwill work</td><td></td><td></td></tr><tr><td>Aluminum boxes</td><td>Washington University in St. Louis machine shop</td><td></td><td>boxes are 3&#039; long, 4.5&#039;&#039; wide, and 3&#039;&#039; tall with a sealed lid.&amp;nbsp; There is a 1/2&#039;&#039; 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><td>Heated water circulator</td><td>VWR</td><td></td><td>Any water circulator model with a pump will work</td></tr><tr><td>21 G needle</td><td>BD</td><td>305165</td><td>The exact needle size is not important</td></tr><tr><td>Hand timer</td><td></td><td></td><td>Any hand timer will work</td></tr><tr><td>Mirror</td><td></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，但应注意的是，在整个行为罩（ 图5B）2，产生一个冷梯度。外壳是靠近干冰上机箱的两侧的部分是冷却器，而中央部分是稍暖（ 图5B）2。在最寒冷的气温üSED，小鼠花大部分时间在机箱的中部地区。一旦玻璃板的温度已经稳定，焦点干冰刺激被放置在爪/热电极下方的玻璃。基于来自此设置中记录的温度的痕迹，很显然，使用该CPA中产生的冷刺激是在各温度范围（ 图4C）20的高度可重复的。 在CPA中产生的冷刺激，使用三种不同厚度的玻璃，以改变冷却（ 图3）的强度也测量。冷却速率是负相关的玻璃的厚度，和任何这些厚度可以根据需要使用（ 图3）来测量冷敏感性。 以前的工作已经表明，该CPA可以检测镇痛和超敏反应小鼠。 1.5毫克/公斤吗啡皮下注射后30分钟，小鼠有显著升onger延迟停药比给予生理盐水皮下注射小鼠（ 图6A:双因子ANOVA主效应* P &lt;0.05和Bonferroni事后检验; 30分钟** P &lt;0.01; N = 12每组）1。吗啡/生理盐水后60分钟，就有了生理盐水和吗啡注射组，这与吗啡代谢的小鼠的速率一致的没有区别。 完全弗氏佐剂（CFA）先前已显示后爪注射21后引起的炎症和超敏反应。后CFA注射，会计师退缩潜伏期降低2和3小时后喷射（ 图6B:双因子ANOVA主效应P &lt;0.001和Bonferroni事后检验; 2小时* P &lt;0.05，3小时** P &lt;0.01 N = 12％组）。 4小时CFA注射后，对小鼠给予1.5毫克/公斤吗啡皮下注射。吗啡注射剂，既CFA-和生理盐水注射的小鼠增加了withdr后30分钟回历在3小时的延迟相对于它们的延迟（ 图6B:1单向ANOVA和Dunnett的事后检验; CFA 3小时与 CFA 4.5小时$$$ P &lt;0.001，盐水3小时对生理盐水4.5小时$$$ P &lt;0.001）。一小时后，一旦吗啡已代谢，所述CFA注射的小鼠再次有较低的退缩潜伏期比盐水注射的对照小鼠（ 图6B:2单向ANOVA和Bonferroni事后检验; ** P &lt;0.01） 1。 大多数哺乳动物物种具有适应其温度敏感性，以满足他们的环境的能力。 在体外 ，研究表明，这种适应过程是依赖于画中画2水解22- 24，但以前的行为的工具无法验证此假设在体内的 CPA能够以两种不同的方式量化这种适应。通过测试小鼠的退缩潜伏期作为玻璃冷却（ <stroNG&gt;图7A，C），注册会计师可以测量冷适应，因为它发生2。在正常条件下的退缩潜伏期是不变的板冷却，这表明冷适应发生比可以与CPA（ 图7B来量化更快:0分钟= 12.13±0.8秒时，30分钟= 12.1±1.6秒时，60分钟= 13.2 ±1.1秒时，90分钟= 10.8±1.2秒1单向ANOVA和Bonferroni事后检验P&gt; 0.05，N = 6）2。然而，当小鼠中的板之前给出足底内的磷脂酶C抑制剂U73122 25的注射被冷却（ 图7C）的退缩潜伏期被减少，这表明适应受损（ 图7D:基线= 11.29±0.53秒，30分钟= 8.09±1.17秒; 1路ANOVA与Dunnett的事后检验，主要功效P = 0.02，单个基准主场迎战 30分钟，P = 0.02，N = 9）。 注册会计师还可以测量ABility以适应寒冷的环境温度以上的很长一段时间。当野生型小鼠使用CPA在30℃，23℃，17℃，或12℃下被驯化3小时后测试的退缩潜伏期是相同的，在所有的起始温度下，这表明与野生型老鼠适应寒冷的环境温度（ 图2A:WT 30°C = 13.23±0.5秒，23°C = 12.8±0.7秒，17°C = 12.3±0.9秒，12°C = 12.8±0.5秒，1单向ANOVA和Bonferroni事后检验，p&gt; 0.05，N = 6进行30℃，15例为23℃，17℃，12℃）20。不同于野生型小鼠中，作为起始温度降低TRPM8-KO小鼠的退缩潜伏期降低，这表明它们是无法适应他们的响应阈值，以适应其环境（ 图8:1-方式重复测量ANOVA和Bonferroni后hoc检验;男性主要作用P = 1.5×10-5，12℃，VS。 23℃，P = 6×10 -5，17℃， 与 23℃，P = 0.004;女性主要作用P = 3.6×10 -5，12℃， 与 23℃，P = 9.25×10 -5，17℃， 与 23℃，P = 0.0005; DF = 1，N = 11名男性和11名女性）20。 <img alt="图1" src="/files/ftp_upload/52640/52640fig1.jpg" /> 图1.冷足底分析（CPA）装置2。（A）示意图执行注册会计师。小鼠被驯化的塑料外壳行为玻璃板，直到他们都在休息。干冰丸粒被施加到后爪下的玻璃的下侧，并且延迟到从冷却玻璃测量撤回。 （B）中的图片CPA装置中，在配置于冷却板至5℃。热数据记录器是在外壳的中心和铝框侧翼在任一侧的外壳。 （C）的图像的CPA装置中，在配置中的地暖板到30℃。水循环流动热水到铝框，然后流出的一侧的漏回循环器的贮存器。从布伦纳等人 2014年2许可复用。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52640/52640fig2.jpg" /> 图中的玻璃板的CPA 2（A）的期间2温度的平均值的玻璃板的温度描记过程中行为实验在CPA。 30℃，N = 1，23℃，N = 5，17℃的N = 7，12℃，N = 7，4℃，N = 5（B）原理图，数字高程模型onstrating如何产生不同的温度条件下在CPA。从布伦纳等人 2014年2许可复用。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52640/52640fig3.jpg" /> 图3.玻璃厚度成反比关系与冷却1的速率。（A）的示意图图表的实验设计（BD）。在爪下方冷刺激脚底的温度，测定在所有三个玻璃厚度在正常条件下，而其中泡沫聚苯乙烯衬垫支撑爪远离玻璃表面。在所有情况下，支撑爪远离玻璃造成在爪测得的冷的刺激（每组n = 6玻璃厚度）显着降低。 （ <strong&gt;的E）的爪子底下的温度在与撑起了聚苯乙烯泡沫塑料的爪子哈格里夫斯辐射热刺激测量。不像冷跖测定中，在测定哈格里夫斯的热刺激是很大程度上未改变时，脚爪撑着远离玻璃（N = 6）。布伦纳等人 2012 1的重复使用许可<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52640/52640fig4.jpg" /> 图4. CPA退缩潜伏期是一致的20。（A）的平均潜伏期为23℃，17℃，或12℃，开始小鼠。 （B）的配置来衡量CPA冷的刺激。麻醉小鼠的爪固定在玻璃板上用实验室磁带上顶一个T型热电偶丝。注册会计师的刺激被放置在这两个爪和热电偶下方玻璃的下侧。在CPA产生的（C）温度为30℃，23℃，17℃，或12℃的开始。黑色箭头代表平均退缩潜伏期为清醒小鼠在每个条件。重复使用从Brenner 等权限。 2014年2。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52640/52640fig5.jpg" /> 图5.玻璃板温度是在CPA 2（A）的热电偶T1（黑色） 相一致被放置在板的中心。热电偶T2（红色）置于行为外壳最靠近板的右边缘。温度TRAcings和图形最右侧（T1-T2）示出几乎相同的温度，在t1和t2在整个实验过程中。 （B）的热电偶T1（黑），放置在板的中心。热电偶T2（红色）被放置在中央的行为的外壳，在壁更靠近干冰填充铝框。温度描记和图形最右侧（T1-T2）表明，有t1和t2之间的大致3℃的差异，一旦板已经达到了稳定的温度。重复使用从Brenner 等权限。 2014年2。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52640/52640fig6.jpg" /> 图6.注册会计师可以测量镇痛和过敏1。（A）Subcutaneous注射1.5毫克/公斤吗啡增加小鼠的30分钟后注射的退缩潜伏期（2单向ANOVA和Bonferroni事后检验; 30分钟后喷射** P &lt;0.01）。 60分钟注射后，有吗啡注射和盐水注射的小鼠之间没有显著差异。 （B）的足底内注射10微升完全弗氏佐剂（CFA）降低小鼠2和3小时后喷射的退缩潜伏期（2单向ANOVA和Bonferroni事后检验; * P &lt;0.05，** P &lt;0.01）。所有小鼠给予吗啡皮下注射在4小时，并都退缩潜伏期在4.5小时均显著高于3小时进行比较（1单向ANOVA与邓尼特的事后检验; $$$ P &lt;0.001）。注射CFA（吗啡注射后1.5小时）后5.5小时，CFA注射的小鼠仍然有较低退缩潜伏期比盐水注射小鼠（2单向ANOVA和Bonferroni事后检验; ** P &lt;0.01）。重复使用的许可布伦纳等人2012年1。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52640/52640fig7.jpg" /> 图7.测量冷适应作为玻璃板动态冷却20。（A）的示意图，用于执行注册会计师作为玻璃板被冷却。基线的温度是在室温测量时，所述干冰的容器被添加到板，并撤出延迟被测量为在玻璃板冷却。 （B）的野生型小鼠具有相同的退缩潜伏期作为玻璃板冷却，这表明它们适应冷却温度的速度比可以与CPA（基线来测量= 12.8±0.3秒时，30分钟= 13.67±0.9秒，60分= 11.03±1.0秒，90分钟= 11.31±0.6秒，N = 3只;单因素方差分析和Bonferroni事后检验，各组间无显著差异）。 （C）的示意图，用于执行注册会计师作为玻璃板被冷却时，PLC抑制剂U73122或对照化合物U73343的足底内注射后。 （D）的小鼠有显著下退缩潜伏期，而板被U73122注射后的冷却，这表明U73122干扰，以适应冷却的环境温度的能力。从布伦纳等人 2014年20许可复用。该图已被复制与疼痛的研究国际协会（IASP）的许可。图中， 不得转载用于未经允许任何其他目的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8"SRC ="/文件/ ftp_upload / 52640 / 52640fig8.jpg"/&gt; 图8. TRPM8-KO小鼠不太适应环境冷却20 TRPM8-KO小鼠测（2路ANOVA和Bonferroni事后检验全部开始温度有较高的退缩潜伏期比野生型窝; *** P &lt; 0.001）。 TRPM8-KO小鼠的退缩潜伏期也减小作为起始温度降低（1单向ANOVA和Bonferroni事后检验; ## P &lt;0.01，### P &lt;0.001），而没有在撤回没有显著变化的野生型同窝作为起始温度降低延迟。从布伦纳等人 2014年20许可复用。该图已被复制与疼痛的研究国际协会（IASP）的许可。图中，不得转载用于未经允许任何其他目的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52640/52640fig8large.jpg" target="_blank">请CLIC</a>ķ此处查看该图的放大版本。

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 ...

a-simple-inexpensive-method-for-determining-cold-sensitivity

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

14.7K 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.

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

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

简单和计算机辅助测试嗅觉的小鼠

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

科研

行为学

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

免疫与感染

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

神经科学

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

使用轻度厌恶研究小鼠偏头痛样行为

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature.
Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli.
This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

测量小鼠和大鼠的骨骼肌产热

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

尿激酶型纤溶酶原激活剂诱导的小鼠背痛模型

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

探索棕色脂肪组织中的代谢物交换

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

作者聚焦:阐明脂肪组织代谢和疾病预防的新途径 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

生物化学

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

测定冷灵敏度和适应小鼠一种简单廉价的方法

摘要

探索更多视频

此视频中的章节

简单和计算机辅助测试嗅觉的小鼠

小鼠量度本地过敏反应

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

使用轻度厌恶研究小鼠偏头痛样行为

测量小鼠和大鼠的骨骼肌产热

尿激酶型纤溶酶原激活剂诱导的小鼠背痛模型

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换

新的分析冷痛觉在

测定冷灵敏度和适应小鼠一种简单廉价的方法

摘要

探索更多视频

此视频中的章节

简单和计算机辅助测试嗅觉的小鼠

小鼠量度本地过敏反应

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

使用轻度厌恶研究小鼠偏头痛样行为

测量小鼠和大鼠的骨骼肌产热

尿激酶型纤溶酶原激活剂诱导的小鼠背痛模型

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

新的分析冷痛觉在

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换