2.5. Tail flick test

Tail flick test is focusing the light burning on the middle one-third of the animal's tail. In this experiment, 30 mice in five groups (6 mice in each group) were treated with different concentrations of CuNPs (25, 50, and 75 mg/kg), morphine (positive control) as well as morphine along with 25 mg/kg of CuNPs in tail flick test and their termal pain threshold was evaluated. Light intensity of the tail flick apparatus (Sparco, Iran) was adjusted to make a 2–4 s latency time in the intact animal. A cutoff time of 10 s was considered to prevent any possible tissue damage. Latency time was recorded thrice with 15-min interval for each set of the tail flick test; the mean was considered as a thermal pain threshold (tail flick latency) (20).