Macroscopic and microscopic assessment of colon injury
After the mice were sacrificed by cervical dislocation, the abdominal wall was opened, and the intestine was exposed. Then, the entire segment of the colon from the rectum to the cecocolic junction was removed, opened and rinsed thoroughly with normal saline, after which the isolated colon was examined for macroscopic damage. Scores were assessed by using the following damage scoring system [16]: 0: no damage; 1: localized hyperaemia without ulcers; 2: linear ulcers with no significant inflammation; 3: linear ulcer with inflammation at one site; 4: ulcer and inflammation at two or more locations; and 5: two or more major sites of inflammation and ulceration or one major site of inflammation and ulceration extending more than 1 cm along the colon. Then, colon specimens were fixed in 10% paraformaldehyde for hour(s), after which paraffin sections were generated and haematoxylin and eosin (H&E) staining was performed. The pathological sections of the colon were observed under an optical microscope (Leica, GER). Histological damage was assessed using the criteria of Wallace and Keenan [17]: 0: intact tissue construction with no apparent damage; 1: damage limited to surface epithelium; 2: localized ulcer confined to mucosa; 3: focal, transmural inflammation and ulceration; 4: extensive transmural ulceration and inflammation adjacent to normal mucosa; and 5: extensive transmural ulceration and inflammation involving entire section. All data were obtained from three separate experiments, and the scores were assigned by three observers without knowing the state of the mice (Table 1).