Histological Assessment of Mouse Colon Damage

Colon tissues from mice were analyzed in accordance with a previously published protocol^{59 (link)}. In short, the tissues were excised, distal colonic segments (0.5 cm) were removed and fixed with 4% neutral-buffered formalin (Hepeng Biology, Shanghai, China) for 24 h and subsequently embedded in paraffin. Cross sections of the colon (4 μm) were cut and mounted on slides. The cross sections were then stained with hematoxylin and eosin solution (Sigma-Aldrich). Tissue damage was evaluated by using a score for the severity of epithelial injury, the extent of inflammatory cell infiltration and goblet cell depletion, according to a previously described protocol^{59 (link)}. Colonic damage was assigned scores as shown in Table 1^{26 (link)}.Table 1

Colonic damage score criteria used to evaluate the severity of epithelial injury, the extent of inflammatory cell infiltration and goblet cell depletion in mouse tissue.

Score	Colonic damage	Inflammatory cell infiltration	Submucosa	Muscle/serosa
0	Normal	Normal	Normal	Normal
1	Hyperproliferation, irregular crypts, goblet cell loss	Mild	Moderate to severe	Moderate to severe
2	Mild to moderate cypt loss (10–50%)	Modest	Severe
3	Severe crypt loss (50–90%)	Severe
4	Complete crypt loss, surface epithelium intact
5	Small to medium-sized ulcers (<10 crypt widths)
6	Large ulcers (≥10 crypt widths)