Intrathecal catheterization of rats

Anesthesia was administered through an intraperitoneal injection of 2% pentobarbital sodium (11,715, 60 mg·kg^− 1, Sigma Corp., San Francisco, CA, USA) in rats placed in a supine position. Hair from the neck and lumbar regions was removed and covered with aseptic hole-towel after iodine disinfection. An incision (1 cm) was made on the iliac crest L 5–6 space, and the intervertebral space was punctured with a thick needle. A polyethylene (PE)-10 pipe (Becton, Dickinson and Company, NJ, USA) was put into the subarachnoid space (3.5 cm) and was fixed on the subcutaneous fascia, resulting in an outflow of clear cerebrospinal fluid. The PE pipe was placed and firmly fixed 2-3 cm outside of the neck incision, preventing the catheter from folding. Folded. Physiological saline (20 μL) was used to wash the catheter and tissue forceps were used to close the catheter to prevent leakage. The rats were fed eight hours later in a warm and quiet environment with lower lighting. After a day, 20 μL of 2% Lidocaine (L5783, Sigma Corp., San Francisco, CA, USA) was slowly injected into the rats. The rats weren’t able to lift their left and right hind limbs 10 s after the injection and showed no retraction reflex during acupuncture. The left and right hind limb movements were recovered 10 min later, indicating catheter was rightly placed and the model was successfully established.

On the second day, behavioral tests were conducted for familiarization training and basic value measurement. On the third day, the rats in the control group were injected with 10 μL physiological saline at 8:00–9:00 a.m. and 16:00–17:00 p.m. every day for seven days. The rats in the morphine tolerance group were injected with 10 μL morphine (10 mg/mL morphine injection liquid diluted with physiological saline to 1 mg/mL, Qinghai Pharmaceutical Co., Ltd., Qinghai, China) in the morning and in the afternoon for seven days (rats were injected with 10 μL physiological saline 30 min before the morphine administration). The rats in the miR-365 mimic group were injected with 10 μL morphine in the morning and afternoon every day for seven days in addition to being injected with 10 μL of miR-365 mimics (5’-UAAUGCCCCUAAAAAUCCUUAU-3′) before morphine administration in the morning. In the miR-365 inhibitor group, the rats were injected with 10 μL morphine in the morning and afternoon every day for seven days as well as being administered with 10 μL of miR-365 inhibitors (5’-AUAAGGAUUUUUAGGGGCAUUA-3′) before morphine administration in the morning. Finally, in the miR-365 NC group, the rats were injected with 10 μL morphine in the morning and afternoon every day for seven days and were injected with 10 μL of miR-365 NC (5’-UUCUCGAACGUGUCACGUUUU-3′) before morphine administration in the morning. Compared with the control group, the analgesic effect of the morphine tolerance group was markedly reduced after seven days, indicating that the model for chronic morphine tolerance had been successfully established [4, 14].