Complete Freund's adjuvant (CFA)-induced inflammatory pain

For the CFA-induced pain, the mice were subcutaneously injected of 50% CFA (20 μl) into the plantar of the right hind paws. The CFA (50%) was prepared by isometric CFA (Sigma, F5881) and saline, and the mixture was emulsified by intensive mixing. The mechanical threshold and thermal sensitivity on the ipsilateral paw during chronic pain were tested by von Frey test and plantar test, respectively. For the von Frey test, the modified Dixon’s up-down method (Chaplan et al., 1994) was conducted by using a series of von Frey filaments to determine the 50% hind paw withdrawal threshold. For the plantar test, the Hargreaves apparatus (Ugo Basile, Comerio, Italy) was used to measure the paw withdrawal latency, a 20 s was set as the cut-off time to prevent injury. Both the thermal threshold and mechanical sensitivity were averaged from two trials, separated by 30 min intervals at each time point. To determine the morphine analgesia on CFA-induced inflammatory pain, morphine (10 mg/kg) was subcutaneously administrated on day 7 and day 14 after CFA injection, and then the mechanical threshold and thermal sensitivity were measured on 30, 60, 90, 120, 180 and 240 min, respectively. Both the mechanical threshold and thermal sensitivity were measured once at each time point for morphine test. To investigate the endogenous opioid function mediated by MOR expressed on the GABAergic neurons, β-funaltrexamine hydrochloride (β-FNA, 10 mg/kg), a specific MOR agonist, was intraperitoneally injected on day 42 after the tests on pain sensitivity in Vgat-Cre/Oprm1^KI/KI and Oprm1^KI/KI mice. The mechanical threshold was tested on the ipsilateral paw before and 15, 30, 45, 60, 90, 120, 150, 180 and 210 min after the β-FNA injection.