Behavioural testing to measure visceral hypersensitivity Abdominal withdrawal reflex (AWR)

Visceral hypersensitivity responses to colorectal distension (CRD) were measured by recording AWR scores, as described previously (3,10). Prior to testing, the rats were fasted and subjected to water deprivation, in order to reduce faeces. The rats were anaesthetized with isoflurane (1.5%) in a sealed cage (21), and a 6-cm deflated latex balloon was inserted into the distal colon up to 1 cm from the anal verge. After a 15-min recovery period, measurements of the visceral pain threshold were recorded when various pressure was applied to the abdominal walls by inflating the balloon (20, 40, 60 and 80 mmHg). Three measurements for each animal were taken at 5-min recovery intervals. A previously reported standard was used to evaluate the AWR score: 0, no response to given pressure; 1, slight head movement without abdominal muscle contraction; 2, contraction of the abdominal muscles; 3, lifting of the abdominal wall; and 4, body arching and lifting of pelvic structures (22).