The Thermal Assay Test is a widely used behavioral task used for the study of nociception, the neural process of encoding actual or potential tissue damaging stimuli.

When given the choice to stay in areas with different temperatures, animals will show preference for the zone with the most comfortable temperature, as determined by their thermal sensitivity, influenced by their nociceptive state. By measuring the time spent in each temperature zone, differences in thermal sensitivity can be assessed and be used as an index of nociception.

Key Features include:

• Eliminate run time data entry mistakes by defining entire study in advance
• Programmable parameters include:

Features

Specifications

- 10 Zones for individual Control
- -5 Degrees C to +70 Degrees C, 1 degree stepwise
- Base Plate: Aluminum alloy
- Walls: Grey PPC
- Top Cover: Transparent PPC

Data Excellence

- Browse and Easily print any result set. Export via Excel
- Autocalculation of mean, std dev
- Data Inputs

Gradient Test

Dimension Length: 120 cm x 10 cm x 15 cm (L x W x H)

• The test apparatus consists of a platform made up of 10 adjacent plates in 2 rows, allowing for 20 total temperature zones
• Temperature from -5Degrees C to +70 Degrees C
• 120 cm long base plate

Documentation

Introduction

The thermal gradient test is a widely used behavioral task in neuroscience to infer pain in rodents. Pain is defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (IASP, 1994). Given that pain is a subjective emotional experience that animals cannot communicate, animal behavioral tests used for the study of pain assess nociception, the neural process of encoding actual or potential tissue damaging stimuli

The Thermal Gradient Test is based on the fact that thermal sensitivity is a common feature of many painful states. When in pain, rodents may show sensitization to thermal stimuli, be it heat, cold, or both (Jensen and Finnerup, 2014). This type of sensitization, wherein pain is felt as a response to a stimulus that normally would not provoke pain, is known as allodynia. When given the choice to stay in areas with different temperatures, animals will show preference for the zone with the most comfortable temperature, as determined by their thermal sensitivity, influenced by their nociceptive state. By measuring the time spent in each temperature zone, differences in thermal sensitivity can be assessed and be used as an index of nociception

The test apparatus consists of a platform containing adjacent plates that can be set at different temperatures, forming a temperature gradient. The temperature of each plate can be adapted to fit the experimental design. The Thermal Gradient Test therefore allows the assessment of both cold and heat allodynia

The Thermal Gradient Test (in its automatized version) was described by Moqrich et al in 2005. This test allows for a simpler and more precise assessment of thermal sensitivity in rodents by allowing them to move freely and choose their area of comfort with no imposed restraints. The Thermal Gradient Test allows for the evaluation of pain states and can also be used to assess the effect of other disease models, genetic manipulation, or drugs on thermal sensitivity (e.g., Carlisle et al, 1999; Lee et al, 2005). The sensitivity of the Thermal Gradient Test for the assessment of the effect of pharmacological interventions has also been validated (Balayssac et al, 2014)

Apparatus & Equipment

The test apparatus consists of a platform made up of 10 adjacent plates in 2 rows, allowing for 20 total temperature zones. Each plate can be set at different temperatures, forming a temperature gradient. The temperatures can range from -5 to 70°C, such as to fit the experimental design

The apparatus can be monitored by the Noldus EthoVision® XT video tracking system software, which allows the measurement of the time each animal spends in each temperature zone

Training Protocol

The purpose of the Thermal Gradient Test is to assess nociceptive behavior in rodents in a control vs. disease model/intervention group by assessing their spontaneous preference for the most comfortable temperature zone

Typically, when on a painful state, such as neuropathic pain, rodents may show sensitivity (allodynia) to heat or cold. Standing on areas whose temperatures would normally be perceived as comfortable may become painful, leading to a preference for other temperatures, more adequate to their current pain state

These changes in temperature preference will be indicative of nociception, from which pain can be inferred

There are several versions of protocols to be used with the Thermal Gradient Test, varying mainly in the temperature intervals according to the experimental design

Pre-training for the Thermal Gradient Test

Since pain perception can be influenced by stress (Ossipov et al, 2010), stress-inducing variables should be controlled. Therefore, the animals must be habituated to the environment of the testing facilities. Animals should also be habituated to the apparatus in the days preceding the test and before the beginning of the test, so that their zone preference is not influenced by their exploratory behavior

Evaluation of nociceptive behavior using the Thermal Gradient Test

One of the plates is set at a neutral temperature (usually 25°C). The other plates are set at varying temperatures that may range from -5 to 70°C, such as to form a temperature gradient

The animal is placed in the neutral temperature plate and allowed to move freely for 10-30 min

The time spent on each plate is automatically measured by the monitoring software, allowing for the determination of the preferred temperature and of the time spent in each zone

Modifications

In the original description of this version of the Thermal Gradient Test (Moqrich et al, 2005), the apparatus consisted of a rectangular platform divided into 16 temperature zones with a surface temperature gradient of 15°C to 55°C along the length

An earlier version had been described using a chamber with Plexiglas walls and an aluminum floor on which a temperature gradient was maintained along the long axis with resistive heaters (Lee et al, 2005). The current version allows for a better precision in establishing the temperature gradient, and consequently, for a better reproducibility of the results

The Thermal Gradient Test has been used with a different temperature ranges and different numbers of plates creating the thermal gradient

Strengths and Limitations

Most tests used to assess thermal sensitivity require the restraint of the animals in order to apply a noxious stimulus. This is a situation that induces stress and can cause bias in the assessment of the behavioral outcomes

The main advantage of the Thermal Gradient Test is that it can be used on freely moving rodents, thereby decreases the influence of stress factors on nociceptive behavior

Furthermore, from a bioethical standpoint, this test has the advantage of, unlike other tests that assess thermal sensitivity, not requiring the application of potentially painful stimuli. The animals are free to avoid what would be a painful temperature zone by staying in a comfortable area. This thereby minimizes pain and distress

Summary and Key Points

• The Thermal Gradient Test is a simple method to assess nociceptive behavior
• This test evaluates the animals’ preference for comfortable temperature zones, as determined by their thermal sensitivity due to pain
• Animals are allowed to move freely and choose their area of comfort with no imposed restraints
• The characteristics of the apparatus can be adapted to evaluate different types of thermal sensitivity (heat, cold, or both)
• Animals with heat allodynia will spend less time in hotter regions, whereas animals with cold allodynia will spend less time in colder zones
• The Thermal Gradient Test has been used to study models of pain, as well as the effect of diseases and treatments thermal sensitivity

References

Balayssac D, et al (2014). Assessment of thermal sensitivity in rats using the thermal place preference test: description and application in the study of oxaliplatin-induced acute thermal hypersensitivity and inflammatory pain models. Behav Pharmacol, 25(2):99-111

Carlisle HJ, et al (1999). Thermal preference behavior following clonidine, norepinephrine, isoproterenol, and ephedrine. Physiol Behav, 66(4):585-9

IASP Task Force on Taxonomy (1994). Part III: Pain Terms, A Current List with Definitions and Notes on Usage. Classification of Chronic Pain, Second Edition, edited by H. Merskey and N. Bogduk (pp 209-214), IASP Press, Seattle, USA

Jensen TS and Finnerup NB (2014). Allodynia and hyperalgesia in neuropathic pain: clinical manifestations and mechanisms. Lancet Neurol, 13(9):924-35

Lee H, et al (2005). Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4. J Neurosci, 25(5):1304-10

Moqrich A, et al (2005). Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science, 307(5714):1468-72

Ossipov MH, et al (2010). Central modulation of pain. J Clin Invest, 120(11):3779-87