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).