2.2. Establishment of the PI-IBS Rat Model

Since PI-IBS is an inflammatory immune disease, only newborn male rats were used. Based on previously published methods [28, 29], the rat model for PI-IBS was established using a multistimulation paradigm composed of EPSD, TNBS, and CUMS. Briefly, the litters were moved from the maternity cages to the adjacent cages at 9:00–12:00 AM during postnatal day 2 (PN2) to PN14. After pentobarbital anesthesia, colitis was induced in PN28 rats through intrarectal administration of 0.8 mL TNBS solution (20 mg per rat) in 50% ethanol, following a previously described method [30]. In contrast, the control rats were administered with 0.8 mL 50% ethanol as a vehicle. All solutions were delivered via a soft catheter and introduced 8 cm above the anus. After recovery from TNBS treatment for 2 weeks, the following procedures were performed: (1) water-fasting for 24 h; (2) fasting for 24 h; (3) reverse day/night cycle (dark from 7:00 to 19:00 and light from 19:00 to 7:00 the next day); (4) induction of cold stress for 5 min (the rats were placed in a transparent barrel containing ice water at 4°C at a depth of 15 cm); (5) induction of heat stress for 5 min (the rats were placed in a thermostat at 45°C); (6) pain induction (the rats were placed in an observation cage and their tails were clipped 1 cm from the distal tip, with appropriate strength to make the rat scream); (7) horizontal oscillation for 15 min (the rats were placed in a high-speed horizontal oscillator [110 r/min]). Each procedure was performed daily for 21 consecutive days (Figure 1) [31, 32].

Experimental protocol.