Hargreaves Test
Objective: Assessment of thermal hyperalgesia and allodynia by applying heat stimuli to measure pain-related withdrawal responses in rodents
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Protocol Steps
Animal placement and acclimation
Place rodents individually in testing environment to allow acclimation before heat stimulus application
Note: Animals must be tested individually
View evidence from paper
“Rodents are placed individually in small cages”
Baseline temperature establishment
Begin heat stimulus application starting at 32°C baseline temperature
Note: This is the standard starting temperature for heat threshold testing
View evidence from paper
“applying a metal probe to the skin that increases in temperature (starting at 32°C)”
Temperature increase application
Gradually increase temperature of the metal probe applied to the skin at a controlled rate until withdrawal response is observed
Note: Rate of heating influences heat thresholds and should be controlled
View evidence from paper
“rate of heating (1–10°C/s), the type (hairy or glabrous) and location of test skin”
Warm sensation threshold determination
Record temperature at which warm sensation threshold is reached
Note: Warm sensation is typically elicited before pain sensation
View evidence from paper
“Typically, the sensation of warm is elicited at temperatures of 34–37°C”
Heat-pain threshold determination
Continue temperature increase until heat-pain threshold is reached and withdrawal response occurs
Note: Pain sensation occurs at higher temperatures than warm sensation
View evidence from paper
“while the sensation of pain is elicited at temperatures of 42–48°C”
Withdrawal response recording
Record the temperature at which the animal exhibits withdrawal response, which indicates heat-pain threshold
Note: Withdrawal response is the primary outcome measure for thermal pain assessment
View evidence from paper
“withdrawal from a nociceptive stimulus, which is the most commonly used method to quantify nociception”