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 been made in understanding the mechanisms of cold sensitivity using other behavioral methods. One method is the acetone evaporation test, which involves dabbing or spraying acetone on the mouse paw and measuring the amount of time that the mouse spends flicking the paw^3,4. Unfortunately, the responses to acetone evaporation are confounded by the wet sensation and the smell of the acetone. Also, the cold stimulus that is applied in the acetone evaporation test can vary based on the amount of acetone applied, and is difficult to quantify. Finally, uninjured mice have minimal responses to acetone at baseline, making it impossible to measure analgesia in the absence of hypersensitivity with this method.

Another classical assay for cold responses is the tail flick assay, where the latency to withdrawal is measured after the tail is immersed in cold water^5,6. While the behavioral responses in this assay are unambiguous and the assay measures responses to a specific temperature, the animals must be restrained during testing, which can alter cold responsiveness through well-described stress-induced analgesic mechanisms⁷.

Another commonly used tool is the cold plate test, which measures the behavioral responses of mice after they are placed on a peltier-cooled plate^8-10. While this tool provides information about animal responses at specific temperatures, it has also been inconsistently used; different groups have measured different types of responses including number of jumps^8,11, the latency to first response^8,11-13, and the number of paw lifts^11,13,14 with very different results. The cold plate assay is also relatively low throughput as only one animal can be tested at a time, and it requires an expensive and fragile peltier device.

The 2-plate temperature preference test is a commonly used derivative of the cold plate test that measures the relative amount of time that animals spend on 2 connected plates of different temperatures^9,15-17. Another similar commonly used assay is the thermal gradient assay, where the amount of time that mice spend in different temperature zones ranging between 5 °C and 45 °C on a long metal plate is measured¹⁶. While these assays allow comparison of temperatures, it is unclear whether the behavior represents temperature aversion or to temperature preference.

Finally, the dynamic cold plate assay has been used to measure how mice respond to changing ambient temperatures¹⁸. This method involves placing mice on a RT peltier device and ramping it down to 1 °C while measuring how much the mice jump or lick their paws at different plate temperatures. While this tests how mice adapt to a cooling environment, it does not provide a way to test how mice respond to a cold stimulus in the setting of a cooler ambient temperature. Additionally, it requires expensive equipment to perform and does not provide a way to acclimate mice to the testing equipment before measuring their cold sensitivity.

To complement these assays, the CPA tests the acclimated responses to a well-defined cold stimulus at a variety of temperature ranges, or during the process of adapting to cold ambient temperatures. It can test up to 14 mice at a time with our current apparatus, with the potential to be inexpensively scaled up for high-throughput testing.

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 while deactivating the auto-shutdown feature, and attach the data logger to the computer with the provided cable.
   1. If recording the plate temperature during the experiment, open the data logger software to begin recording plate temperatures.
   2. If necessary, adjust the software to record the plate temperature once every second.
   3. Begin recording temperatures using the software packaged with the thermal data logger.
5. Separate the enclosure with black inserts to prevent visual interaction between mice.
6. Position mirrors underneath the glass such that the underside of the enclosures is visible from a comfortable seated position.

2. Warming/Cooling the Glass Plate

1. Fill aluminum boxes with warmed water, wet ice, or dry ice and position them appropriately on the glass plate (aluminum foil packets filled with dry ice can also be used to cool the glass; Figure 1)².
   1. For testing at 30 °C, position the aluminum boxes approximately 0.25’’ away from the animal enclosures (Figure 2B)².
      1. Set a heated water circulator on either side of the glass plate. Set the circulator to 45 - 60 °C, and use it to fill the aluminum boxes with a steady stream of hot water (Figure 1C)².
      2. Position the circulators such that the hot water from the aluminum boxes drains directly back into the reservoir of the circulator on each side (Figure 1C)².
   2. For testing at RT, leave the boxes empty (Figure 2)².
   3. For testing at 17 °C, position the boxes approximately 0.25’’ away from the animal enclosures on either side and fill with wet ice (Figure 2)².
   4. For testing at 12 °C, position the boxes approximately 1.25’’ away from the enclosures on either side and fill with dry ice (Figure 2)².
   5. For testing at 5 °C, position the boxes approximately 0.25’’ away from the enclosures on either side and fill with dry ice (Figure 2)².
      1. When cooling the glass with dry ice, make sure there is sufficient ventilation to prevent CO₂ buildup in the room.
2. Wait for the glass to reach the desired temperature range.
3. Add the mice to the enclosures on the plate.
   NOTE: A white noise generator may be used to decrease noise disturbances.
4. Wait for the mice to acclimate.
   NOTE: In our facility this takes roughly 2.5 hr, but this may vary significantly based on animal housing and handling conditions.
5. Maintain the glass at the desired temperature range by ensuring that the boxes are kept full of warmed water, wet ice, or dry ice.
   NOTE: With our apparatus the boxes need to be refilled with ice roughly every 90 min.
   NOTE: For the 17 °C condition, it is helpful to empty most of the water from the aluminum boxes through the drain holes before refilling it with ice. This will stabilize the temperature better, and prevent overflow
   NOTE: The exact amount of the dry ice will vary seasonally, but in general keeping the boxes more than ¼ full along the entire length of the box will keep the temperature constant.

3. Testing the Mice at Fixed Temperatures

1. Outside of the behavioral room, fill an ice bucket about half full of dry ice.
2. Using a hammer or mallet, crush the dry ice into a fine powder.
   NOTE: Overfilling the bucket will make it difficult to fully crush the dry ice into powder.
3. Using a straight razor blade or scissors, cut the top off a 3 ml syringe.
4. Using a 21 G needle, poke 3 holes on opposing sides of the syringe (total of 6 holes).
   NOTE: These holes will decrease the pressure generated by sublimation while compressing the dry ice. The cut-off syringe can be reused for multiple experiments.
5. Take the syringe, dry ice powder, and a hand-held stopwatch into the behavioral room.
6. Fill the syringe chamber half full of dry ice powder. Hold the cut end of the syringe against a flat object, and firmly compress the powder using the plunger. Be careful; the plastic plunger may bend or break from the pressure. If this happens, replace the plunger from a new syringe.
7. Extend the tip of the compressed dry ice pellet past the edge of the syringe.
8. Test mice that are fully at rest.
   1. At 30 °C, 23 °C and 17 °C, test mice that have all 4 paws on the glass and not moving, but not fully asleep¹⁹.
   2. At 12 °C and 5 °C, test mice that are on 2 paws or 4 paws and not moving or jumping.
9. Using the mirrors for targeting, gently but firmly press the flat pellet flush against the glass surface underneath the mouse hindpaw (Figure 1A)². Start the hand-timer.
10. Stop the timer and remove the pellet when the mouse moves away from the cooled glass.
    NOTE: The withdrawal movement can be vertical or horizontal.
    1. If the mouse very briefly moves the paw and then returns it to the cooling surface, continue timing and stimulating until the mouse makes a permanent move away.
       NOTE: Our lab uses a maximum stimulus time of 20 sec for mice in the majority of cases.
11. Repeat this testing procedure until at least 3 values on each paw of each animal are collected. Separate trials testing opposite paws on the same mouse by at least 7 min, and separate consecutive trials on any single paw by at least 15 min.
12. If needed, use different thicknesses of glass to generate different rates of cooling (Figure 3)¹.
    NOTE: The rate of cooling is inversely correlated with the thickness of the glass.

4. Testing the Mice During Cold Adaptation

NOTE: This is an alternate protocol which allows testing as the glass plate cools, rather than once the plate has stabilized and the mice have fully adapted to the cold environment.

1. Follow the instructions listed in Section 1 to set up the apparatus.
2. Follow the instructions listed in Section 3 to take baseline measurements at RT (Figure 7A)².
3. Pre-cool the aluminum boxes with dry ice.
4. Once baseline withdrawal latencies have been measured, position the precooled boxes on the plate approximately 1.25’’ away from the enclosures on either side (Figure 7A, arrow labeled “Dry ice added”) ².
5. Follow the instructions listed in Section 3 to take measurements as the glass plate cools, taking measurements as often as possible.

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