Visceral Sensitivity Assessment

Visceral hypersensitivity was evaluated using the AWR score at PND78, as described previously (Xu et al., 2009; Wang et al., 2018). Briefly, a flexible balloon (6 cm) constructed from a surgical glove finger was attached to Tygon tubing and inserted 8 cm into the descending colon via the anus and held in place by taping the tubing to the tail. Rats were placed in transparent plastic cubicles (8 × 10 × 20 cm) and allowed to adapt for 10 min. colorectal distension was then established by rapidly inflating the balloon to constant pressure. Pressure was measured using a sphygmomanometer connected to a pressure transducer. The balloon was inflated to various pressures (20, 30, 40, 50, 60, 70, 80, 90, and 100 mm Hg) for a 10-s stimulation period followed by a 1-min rest period. Behavioral responses to colorectal distension were measured by visual AWR observations by two blinded observers, and AWR score assignments were as follows: 0, normal behavior without response; 1, slight head movement without abdominal muscle contraction; 2, contraction of abdominal muscles; 3, lifting of abdominal wall; and 4, body arching and lifting of pelvic structures. The minimal distention pressures that evoked abdominal visceromotor responses corresponding to AWR scores of 2, 3, and 4 were recorded by both observers and defined as the distension threshold.