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

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

Measuring cold responsiveness in rodents is important for improving understanding of the potential mechanisms of cold sensitivity in humans under both normal and pathological conditions. The Cold Plantar Assay (CPA), originally developed several years ago¹, is designed to generate reproducible, unambiguous murine behavioral responses to a cold stimulus delivered at RT. More recent enhancements of this assay have allowed the reproducible measurement of cold sensitivity at a wide range of temperatures². Both versions are also designed to be relatively high-throughput, and inexpensive to use.

A great deal of progress has ....

Protocol

All mouse protocols were in accordance with National Institutes of Health guidelines and were approved by the Animal Studies Committee of Washington University School of Medicine (St. Louis, MO).

1. Preparing the Testing Plate and Enclosures

1. Clean off the glass surface.
2. Secure the T-type filament thermocouple probe to the surface in the middle of the glass plate with laboratory tape.
3. Place the animal enclosures on the glass plate in a single line along the middle of the plate.
4. Thread the thermocouple probe through the center animal enclosure and plug into the data logger. Turn the data logger on whi....

Results

The behavioral responses elicited from mice starting at 30 °C, 23 °C, 17 °C, and 12 °C are highly reproducible (Figure 4A)²⁰. In order to measure the cold stimulus being generated under the hindpaw, mice were anesthetized with a ketamine/xylazine/acepromazine cocktail and their paws were secured on the glass on top of a T-type filament thermocouple (Figure 4B)²⁰. The glass was cooled or warmed to the desired testing range. Although the plate is cooled uniformly alon.......

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.......

Materials

List of materials used in this article

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)		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)
3 ml 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
Aluminum boxes	Washington University in St. Louis machine shop		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 G needle	BD	305165	The exact needle size is not important
Hand timer			Any hand timer will work
Mirror			Any flat mirror will work