The hot plate test is a gold standard thermal pain test in rodents and serves as a useful screening tool for interventions of analgesia. Placing a mouse or rat into a chamber with a heated floor with surrounding clear acrylic walls. Two key behaviors are measured: paw licking and jumping.
Price & Dimensions
Hot Cold Plate
- Acrylic Wall: Diameter 24 cm × 29 cm
- Volume: Length: 45 cm × Width: 32 cm × Height: 48 cm
- Temperature setting range: -10°C to 80°C, adjustment stepwise 0.1°C
Hot/Cold Plate test is a blend of the Hot and Cold plate test. These tests are usually performed individually on their standalone apparatuses. The Hot/Cold Plate subjects the animal to either noxious hot temperatures or innocuous cold temperatures to evoke nocifensive behaviors. The Cold plate is useful in the assessment of cold-induced hyperalgesia and allodynia. The measure of cold temperature sensitivity allows observation and evaluation of unilateral pain. By combining the two pain response tests, experimenters can easily compare results between the two conditions and perform both tests using the same apparatus. Another temperature-based assay is the Thermal Gradient test.
The Hot/Cold Plate apparatus consists of a plantar surface that can be set to a constant temperature or programmed to increase or decrease the temperature gradually. When subjected to warm or cold temperatures, subjects can show varied nocifensive behaviors such as jumping, licking paw and stamping. However, hind paw withdrawal and licking are considered better indications of nociception than forepaw related response in hot plate trials, since forepaws of the animals are usually busy in exploration and grooming.
Apparatus & Equipment
The Hot/Cold Plate apparatus is composed of a transparent glass cylinder and a hot/cold plate. The plantar surface is a metal floor equipped with adjustable temperature control. The glass cylinder (diameter 24 cm, height 29 cm) is used to limit the subject to the plate and prevent it from jumping off the apparatus while allowing an unobstructed view.
Before beginning the experiment, the entire apparatus must be cleaned thoroughly with 70% ethanol solution. Ensure the apparatus is cleaned in between trials as well.
Set the metal plate temperature to 5°C. Bring the subject into the test area and allow it at least 30 to 60 minutes to acclimate. Once the subject has familiarized with the test area, place it on the apparatus and immediately start the timer. Each trial lasts until 5 minutes have expired. Trials are repeated as required with at least 10-minute inter-trial intervals.
Pre-heat the metal plate to a maximum temperature (between 52° to 55°). Bring the subject into the test area and allow it at least 30 to 60 minutes to acclimate. Once the subject has familiarized with the test area, place it on the hot plate and immediately start the timer. Remove the subject immediately after it has shown a nocifensive response such as hind paw licking, hind paw flicking, vocalization or jumping. If the subject shows no such response within 30 seconds, remove the subject from the apparatus and terminate the test. Repeating trials may not be advantageous.
To assist observation, tracking and video system such as Noldus EthoVision XT may be used.
Differentiating thermal allodynia and hyperalgesia
Yalcin et al., 2009 used Male C57Bl/6J mice and Sprague-Dawley rats in their investigation differentiating thermal allodynia and hyperalgesia. The subjects were treated with capsaicin or dimethylsulfoxide (DMSO) to induce sensitization of capsaicin-sensitive C-fibers or carrageenan-induced hind paw inflammation. For the hot plate test, the initial temperature was maintained at a 30° ± 0.1°C which was increased gradually to 43°C for mice and 45°C for rats. On the other hand, for the cold plate test, the initial temperature was set to 20° ± 0.1°C which was reduced to 1°C for mice and 5°C for rats. For both the test the rate of temperature change was 1°C/min. Observation of nocifensive behavior showed that appropriate nocifensive parameter were escape behaviors in C57B1/6J mice and paw licks and withdrawal for Sprague-Dawley rats. The usage of hot and cold plate allowed distinction of thermal allodynia and thermal hyperalgesia in rodents.
Investigation of the analgesic effect of edible brown seaweed Ecklonia cava
Kim et al., 2014 explored the possible use of Ecklonia cava as an analgesic for plantar incision and spared nerve injury (SNI) in rats. In their study, male Sprague-Dawley rats underwent plantar incision and spared nerve injury and were treated with 300 mg/kg E. cava extracts. Hypersensitivity was evaluated on the hot plate (50 °C) or on the cold plate (0 °C). Treatment with E. cava extract resulted in increased paw withdrawal latency in the plantar incision rats suggesting the potential use of E. cava extract as pain relief treatment.
Combined anti-allodynic effects of synthetic cannabinoid and a selective noradrenaline reuptake inhibitor
Male Balb-c mice were used as partial sciatic nerve ligation (PSNL) model of neuropathic pain to assess the combined effect of synthetic cannabinoid and selective noradrenaline reuptake inhibitor. Cannabinoid agonist WIN 55,212-2 was dissolved in 1% ethanol, 1% Tween 80, 20% DMSO and 78% in physiological saline solution, while noradrenaline reuptake inhibitor maprotiline was dissolved only in physiological saline. The administration of drugs was done 30 minutes’ prior the assessments and was co-administered as two separate injections for synergy studies. The mice were tested on a 4° ± 0.1°C cold plate and observed for withdrawal latencies of the injured paw. Data analysis revealed the plausible advantage of the combination as a treatment for neuropathic pain. (Gunduz et al., 2016)
The data obtained from the Hot/Cold Plate test is straightforward. The latency to react to the pain associated with the heat/cold is recorded. The latency is recorded as the time between the placement of the subject on the plate and the first sign of nocifensive response such as paw licking or jumping. Other data that can be recorded is the duration of the nocifensive response and the temperature at which nocifensive behavior is shown in an increasing Hot/Cold Plate temperature test.
Strengths & Limitations
The Hot/Cold plate test combines both cold and hot stimulus apparatus into a single unit. The cold plate test allows an understanding of animal’s sensitivity to cold temperatures that enables assessment of cold-induced hyperalgesia and allodynia. The apparatus allows testing of unilateral pain quantification using the cold plate which isn’t possible with the hot plate test alone. The feature to increase or decrease the rate of cooling and heating allows observation of thermal stimulus temperatures that incite nocifensive responses. The apparatus is effective in the assessment of the effectiveness of analgesics.
The Hot/Cold Plate test performance can be affected by the anxiety arising from the enclosed space. The test also allows free exploration of the surface during the trial which may lead to incorrect observations of the nocifensive responses. The test also may not be advantageous when used at a constant temperature set-up due to learned behavioral responses which may lead to reduced response time. The gender of the subject may also be a factor in the response behavior.
Summary & Key Points
- Hot/Cold plate apparatus allows investigation of nocifensive responses to cold or hot temperatures within a single apparatus.
- Hot/Cold Plate test is one of the many nociception tests available to test for pain response behaviors.
- Hind paw withdrawal or licking is taken as the best indication of nocifensive response to thermal pain in hot plate test.
- Hot/Cold Plate test is commonly used in evaluating the effectiveness of analgesics.
- The cold plate allows assessment of cold-induced hyperalgesia and allodynia
Deuis JR, Dvorakova LS, Vetter I (2017). Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci. 10:284. doi: 10.3389/fnmol.2017.00284
Gunduz O, Topuz RD, Karadag CH, Ulugol A (2016). Analysis of the anti-allodynic effects of combination of a synthetic cannabinoid and a selective noradrenaline re-uptake inhibitor in nerve injury-induced neuropathic mice. Eur J Pain. 20(3):465-71. doi: 10.1002/ejp.752.
Jasmin L, Kohan L, Franssen M, Janni G, Goff JR (1998). The cold plate as a test of nociceptive behaviors: description and application to the study of chronic neuropathic and inflammatory pain models. Pain. 75(2-3):367-82.
Kim JG, Lim DW, Cho S, Han D, Kim YT (2014). The edible brown seaweed Ecklonia cava reduces hypersensitivity in postoperative and neuropathic pain models in rats. Molecules. 19(6):7669-78. doi: 10.3390/molecules19067669.
Yalcin I, Charlet A, Freund-Mercier MJ, Barrot M, Poisbeau P (2009). Differentiating thermal allodynia and hyperalgesia using dynamic hot and cold plate in rodents. J Pain. 10(7):767-73. doi: 10.1016/j.jpain.2009.01.325.