Key factors in developing the trinitrobenzene sulfonic acid-induced post-inflammatory irritable bowel syndrome model in rats

Table 1 Study design and the treatments

Groups	Treatment (mg/mL per rat, i.col.)	Ethanol (%)	Position (cm to anus)	Time points for evaluation
				5 d	4 wk	8 wk
1	TNBS, 20/0.4 per rat	50	8	IE	IE	-
2	TNBS, 20/0.8 per rat	50	8	IE	IE	-
3	TNBS, 10/0.8 per rat	50	8	IE	IE	-
4	TNBS, 5/0.8 per rat	50	8	IE	IE/PE	PE/EE
5	TNBS, 5/0.8 per rat	50	4	IE	IE/PE	PE
6	TNBS, 5/0.8 per rat	25	8	IE	IE/PE	PE
7	Saline, 0.8/rat	-	8	IE	IE/PE	PE/EE

TNBS: Trinitrobenzene sulfonic acid; IE: Inflammation evaluation; PE: Pain evaluation; EE: Enterochromaffin cell evaluation.

Table 2 Criteria for macroscopic scoring of colonic ulceration and inflammation

Score	Appearance
0	Normal appearance
1	Ulceration with inflammation at 1 or 2 sites
2	More sites of ulceration and inflammation
3	Major sites of damage extending > 1.5 cm along length of colon
4	Major sites of damage extending > 3 cm along length of colon

Table 3 Criteria of histological scoring in colon tissue

Variables	Severity and scoring
	0	1	2	3
Ulceration	No ulcer	Ulcerations not exceeding lamina muscularis mucosae	Ulcerations not exceeding submucosa	Ulcerations exceeding submucosa
Edema	Normal thickness	Submucosal expansion < 30%	Submucosal expansion 30%-100%	Submucosal expansion > 100%
Inflammatory cells	No infiltration	Few scattered cells	Distributed but not dense	Dense
Fibroplasia	Normal collagen	Increase < 30%	Increase 30%-50%	Increase > 50%

Table 4 Effects of trinitrobenzene sulfonic acid concentration on body weight and mortality rate

Treatment	TNBS	Body weight (g)		Mortality rate
	concentration	5 d	4 wk	(%)
Saline	-	249.6 ± 26.7	343.0 ± 28.9	0
TNBS 20 mg/0.8 mL per rat	25 mg/mL	214.5 ± 16.8b	324.5 ± 24.8	0
TNBS 20 mg/0.4 mL per rat	50 mg/mL	179.2 ± 21.4d	291.8 ± 42.6a	33

^aP < 0.05,

^bP < 0.01 vs saline-treated group;

^dP < 0.01 vs TNBS (20 mg/0.8 mL)-treated group. TNBS: Trinitrobenzene sulfonic acid.

Table 5 Detailed protocols in developing trinitrobenzene sulfonic acid induced post-inflammatory irritable bowel syndrome model in Sprague-Dawley rats

Procedures	Cautions
1. 7-wk-old male Sprague-Dawley rats, fast 24 h before TNBS administration
2. Mix 1 volume of 12.5 mg/mL TNBS-saline solution with 1 volume of absolute ethanol	Fresh preparation
3. Weight and anesthetized rats with chloral hydrate (350 mg/kg, i.p.)	Anesthetization may last for 3-4 h
4. Insert a catheter into the colon at 8 cm from anus	Proceed carefully to avoid damage the colon wall
5. Keep the rat with head-down vertical position, instill 0.8 mL TNBS solution slowly into the colon lumen within 1 min	Handle slowly to avoid TNBS leakage
6. Keep the rat in head-down vertical position for 1 min before gently removing the catheter
7. Put the rats in a mound of bedding in head-down position until consciousness recovery	Ensure TNBS solution remains completely in the colon; Keep the rats warm
8. On day 3, 7, 14, 21 and 28, observe and weight the rats	On day 3, body weight decreased by 10%-20% with unformed bloody stool;
	On day 7, body weight regained and reached the original level, the stool become formed but still soft;
	From day 7 to day 28, body weight increased smoothly, and finally reached the control level

TNBS: Trinitrobenzene sulfonic acid.