Warm Water Tail-Flick Test for Pain Threshold

The warm water tail-flick test was used to determine pain threshold. Test was conducted before and during treatment. 4 cm of the rat-tail was placed in 50 ± 0.5°C warm water and the time between tail input and withdrawal from the water was recorded (three tests were conducted and the average in units of seconds was recorded). The latency was recorded with a sensitivity of 0.01 s. To perform a measurement, the rat had to remain calm, without unconditional unexpected movements of the tail. A maximum tail-flick latency of 10 s was used to minimize tissue damage to the tail (Liu et al., 2012 (link)).