2.5. Morphine analgesia and tolerance

Morphine hydrochloride was diluted in saline (0.9% NaC1). Melatonin was dissolved in saline with 0.5% ethanol (v/v), and saline with 0.5% ethanol was used as a vehicle. We chose the concentrations of melatonin (0.5 mg/kg) and morphine (10 mg/kg) that had the best effect according to our previous study [21]. Melatonin was injected intraperitoneally 30 min before subcutaneous injection of morphine [21]. Analgesia was assessed using the radiant heat tail-flick latency (Tail-Flick Unit 37360, UGO Baseline, Comerio, Italy) and a hot platform (YLS-21A, Beijing Dameida Technology Co., Ltd, China) as previously described [28]. For the tail-flick test, mice were placed in Plexiglas cages (9 × 6 × 3 cm) on a modified Hargreaves Device. Mice were habituated to the device for 2 min before each test session. A halogen lamp was focused on the tail and the withdrawal reflex time was determined by a photocell. We measured tail flick latency at IR30 and IR50 (decimal selector of heat intensity) every two days from day 0 (baseline) to day 7, 14 and 21. We used 20 s as a cutoff time to avoid damaging the tail. Baseline responses were determined for each mouse before drug injection. For morphine hot plate tolerance, repeated morphine injections (10 mg/kg subcutaneously) were given daily for 7, 14 and 21 days. The hot plate test was performed on a platform heated to 47.5 °C, 50 °C and 52.5 °C with a cutoff of 30 s, and the latency to paw lick or jump was recorded. The baseline response was determined for each animal before treatment. The analgesic response to morphine was assessed by the hot plate test or tail-flick test at 30 min or 60 min after morphine injection (10 mg/kg) [21,29]. For cross-tolerance test, the tolerance was established by repeated injections of either melatonin (or morphine) alone or the saline (or vehicle) twice a day for 2 days. On day 3, melatonin or morphine was injected to mice, followed by the tail-flick test and hot plate test as previously described [30].