Thermal withdrawal latency was first described by Hargreaves et al (1988). It is a widely used test for thermal stimuli response. In this procedure, a rodent hindpaw is exposed to a beam of radiant heat through a transparent glass surface using the plantar analgesia meter. The latency to withdrawal to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws.
The infrared (I.R.) heat intensity of the plantar test instrument can be adjusted in increments as per specifications below, usually set at average paw withdrawal latency to approx 10 seconds in rats.
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Hargreaves Plantar test was first described in the literature by Hargreaves and colleagues in 1988 (Hargreaves et al., 1988). Hargreaves et al. used the test to evaluate cutaneous hyperalgesia resulting from thermal stimulation in rats. The test was designed to assess thermal pain sensations in a specific part of the plantar surfaces of animals. The test has been widely employed in evaluating pain sensitization and pain response in subjects recovering from neural injuries and regeneration. The apparatus and assays similar to Hargreaves Plantar test include the Tail Flick test, Hot-Cold Plate test, and Thermal Gradient test.
The Hargreaves apparatus consists of glass-floored chambers in which the subject is placed, and a radiant or infrared heat stimulus is applied to the center of its plantar surface from underneath. The tests usually last for a maximum of 35 seconds to avoid tissue damage and unnecessary harm to the animals. To ensure that the temperature of the heat stimulus is not affected, urine must be cleaned up during trials. The use of glass flooring helps minimize the heat-sink effects thus allowing more accurate heat stimulus. In comparison to other thermal stimulus assessments, the Hargreaves test does not subject the entire plantar surface to the heating effect. This allows overcoming issues of heating irrelevant parts of the subject, and that could potentially influence the response of the subject.
Apparatus and Equipment
Hargreaves Plantar test apparatus is composed of emitter/detector vessel controlled by a controller that allows the manipulation of the intensity of heat, a glass panel, and animal enclosures. The size of the enclosures varies depending on the animal being used. For mice, a compartment of 10 x 10 cm and a height of 15 cm is usually used. The entire apparatus is usually supported at a height on a platform to allow the placement of the emitter/detector vessel.
Before beginning the experiments, the apparatus must be thoroughly cleaned. The performance
can be recorded and observed using a tracking and recording system such as the Noldus EthoVision XT.
Gently place the subject within its compartment on the Hargreaves test apparatus. Allow it at least 30 to 60 minutes of exploration time. Repeat this process for at least two days before starting with the Hargreaves test. To help with the habituation process, food or sucrose pellets can be used within the compartments.
Place the subject in its compartment and allow it to acclimate for 15 to 20 minutes. Setting the desired infrared intensity, position the emitter/detector vessel directly under the center of the paw being tested. Start the timer and the infrared light at the same time and observe the subject for pain elicited behaviors until the cut-off time (usually 20 to 35 seconds) for experiment has been reached. Repeat trials as necessary with at least 5-minute inter-trial interval. Licking or shaking of the stimulated paw signs behavior associated with pain.
Determination of the plausible role of Nf1 heterozygosity in pain and itch behaviors
O’Brien et al., 2013 investigated whether Nf1 heterozygosity resulted in increased pain and itch behaviors in mice. For their study, they used seven-to-ten week old male Nf1+/− and wild-type littermates. Subjects were placed on a pre-warmed (~30°C) glass plate in their individual chambers. The radiant heat stimulus was applied at an active intensity of 17% maximal for 20 seconds. The next day, subjects received an inflammatory mediator injection, and withdrawal latency was obtained at hourly time points by averaging two measurements per paw during that hour. Data analysis revealed no influence of Nf1 heterozygosity in increasing pain intensity in the subjects.
Investigation of thermal hyperalgesia resulting from the plantar incision with or without fentanyl administration
Sprague-Dawley rats were divided into 4 treatment groups: normal saline, fentanyl, incisional and fentanyl + incisional. Fentanyl (60 µg/kg) or normal saline injections were given subcutaneously at intervals of 15-minutes, 4 times in volumes of 1.2 ml/kg. Behavioral testing of thermal induced pain was performed on the Hargreaves apparatus. Subjects were placed in their compartments on the glass plate. A 30 W intensity beam was directed at the sole near to toes, ipsilateral and contralateral to the surgical site. The observation period was set to 20-seconds. Paw withdrawal latencies revealed that perioperative fentanyl and surgery-induced ipsilateral thermal hyperalgesia was more severe than that induced by fentanyl only. Further, perioperative fentanyl led to the aggravation of postoperative ipsilateral thermal hyperalgesia induced by surgery. However, thermal hyperalgesia in contralateral sites was weaker than in ipsilateral sites in incisional and fentanyl + incisional groups. (Chang et al., 2018)
Evaluation of anti-nociceptive effect of agrimonia eupatoria
Lee and Rhee investigated agrimonia eupatoria extract for its anti-nociceptive effects. Sprague-Dawley rats were used as cisplatin-induced neuropathic pain model to test the effectiveness of argrimonia eupatoria extract. Subject’s thermal stimulated pain response was calculated before and after the injections. In the plantar test, each hind paw was tested thrice with at least a minute’s interval. The results suggested that the extract showed a significant increase in paw withdrawal latencies in the first week of treatment. However, there was no consequent increase in the effect in the following weeks. In comparison to gabapentin treated group, argrimonia eupatoria extract proved to be more potent in preventing thermal hyperalgesia induced by cisplatin.
The data collected using the Hargreaves test is straightforward. Withdrawal latencies, the time taken to withdraw paw as a response to heat, and the average reaction time are recorded. It is important to ensure that observed behavior is a reflex behavior and not a voluntary one. In case an ambiguous behavior is observed, repeat the experiment and record the correct withdrawal latency.
Strengths & Limitations
The Hargreaves test evaluates thermal nociceptive responses of the subject. As opposed to the Hot plate test that subjects the entire plantar surface of the subject to the heat stimulus, the Hargreaves test uses a directed heat stimulus that tests a particular point on the plantar surface. This allows unilateral pain response assessments. The apparatus is also simple and straightforward to use. The Hargreaves test uses a compartmentalized area that enables testing multiple subjects at once. The use of glass bottom for the compartments minimizes the errors arising from heat sink effects. The Hargreaves test is widely used in investigations of neural injuries and regeneration to evaluate pain sensitization or recovery of thermal pain response.
While testing in the Hargreaves apparatus, it is important to ensure the apparatus is cleaned before and after trials, and as required during trials since the presence of urine can affect the temperature of the heat stimulus. Care must also be taken to observe and differentiate voluntary behaviors from reflex behaviors. In case of voluntary behaviors, the experiment should be repeated to obtain accurate results. It is also essential to not exceed the cut-off time to prevent unnecessary damage and harm to the animals used. The test may require longer periods of habituation and acclimation depending on the species and strains as some are known to have an excited exploratory drive. The experiments should also be performed while the subject is awake. It is important to minimize any external stimulus in the test arena to reduce their impact on the subject’s performance. The Hargreaves test also does not allow the direct measure of paw withdrawal temperatures. Handling and consistency of the surgical process can also potentially influence paw withdrawal responses.
Summary and Key Points
- Hargreaves test was first described in the literature by Hargreaves and colleagues in 1988.
- Only a specific part of the plantar surface of the animal is subjected to heat stimulus.
- Withdrawal latencies are observed as the time taken to respond to the heat stimulus.
- Different strains and species showcase a range of exploratory drive thus habituation time may be increased or decreased to acclimate the subject to the apparatus.
- Voluntary movements should be clearly distinguished from heat-stimulus generated reflex movements.
Chang L, Ye F, Luo Q, Tao Y, Shu H (2018). Increased Hyperalgesia and Proinflammatory Cytokines in the Spinal Cord and Dorsal Root Ganglion After Surgery and/or Fentanyl Administration in Rats. Anesth Analg. 2018 Jan;126(1):289-297. doi: 10.1213/ANE.0000000000002601.
Cheah M, Fawcett JW, Andrews MR (2017). Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test. Bio Protoc. 7(16). pii: e2506. doi: 10.21769/BioProtoc.2506.
Hargreaves K, Dubner R, Brown F, Flores C, Joris J (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988 Jan; 32(1):77-88.
Lee KH, Rhee KH (2016). ANTI-NOCICEPTIVE EFFECT OF AGRIMONIA EUPATORIA EXTRACT ON A CISPLATIN-INDUCED NEUROPATHIC MODEL. Afr J Tradit Complement Altern Med. 13(5):139-144. doi: 10.21010/ajtcam.v13i5.18.
O’Brien DE, Brenner DS, Gutmann DH, Gereau RW 4th (2013). Assessment of pain and itch behavior in a mouse model of neurofibromatosis type 1. J Pain. 14(6):628-37. doi: 10.1016/j.jpain.2013.01.770.