Modified Method for Inducing Acute Intestinal Fibrosis in Rats Using 2,4,6-Trinitrobenzene Sulfonic Acid

doi:10.12300/j.issn.1674-5817.2021.147

Abstract

Abstract:

Objective To explore 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute intestinal fibrosis in rats and the effect of different drug ratios. Methods Thirty female Sprague-Dawley (SD) rats were randomly divided into five groups with six rats in each group. Groups A to D were the model groups, in which the rats were induced by modified retention enemas using solutions with different drug ratios: group A, 5% TNBS + 50% ethanol (1∶1 v/v); group B, 5% TNBS + 75% ethanol (1∶1 v/v); group C, 5% TNBS + 100% ethanol (1∶1 v/v); group D, 5% TNBS + 50% ethanol (2∶1 v/v); and the control group was induced by normal saline (0.9% sodium chloride solution) enema. The symptoms, signs, and body mass changes of the animals were observed within one week after modeling, and scored using the disease activity index (DAI). At 7th d and 14th d after model establishment, half of the rats in each group were randomly selected for sampling to observe the degree of gross damage to the colon tissue and scored using the colon macroscopic damage index (CMDI). Pathological sections of colon tissue were stained with hematoxylin-eosin (HE) to observe the severity of enteritis, and Masson staining was used to observe collagen fiber deposition. Results The rats in each model group showed enteritis and intestinal fibrosis lesions of different severities, of which 5% TNBS + 75% ethanol solution (1∶1 v/v) did not lead to death during the observation period. At 24 h after model establishment, the rats had significantly decreased body weight, loose stool, and bloody stool, significant colonic wall fibrosis lesions, and increased DAI and CDMI scores compared with the control group (P＜0.05). The degree of inflammation was transmural and more severe, as seen under the light microscope, and Masson staining showed that the intestinal wall at the model site was significantly thickened, and diffuse collagen fiber deposition occurred in the submucosa, muscular layer, and serosal layer. Conclusion The modified TNBS retention enema method can effectively construct a rat model of intestinal fibrosis, and the model established using 5% TNBS + 75% ethanol solution (1∶1 v/v) can simulate the two major characteristics of Crohn’s disease fibrosis, namely transmural inflammation and intestinal wall fibrosis. This method is simple and efficient, and the mortality rate of animals is low.

Key words: Inflammatory bowel disease, Crohn's disease, Intestinal fibrosis, 2,4,6-trinitrobenzene sulfonic acid, Sprague-Dawley rats

CLC Number:

R-332

Yiru WANG, Xiaoying JIANG, Ruoxi DONG, Yibin PAN, Xianghui HAN, Yongqing CAO. Modified Method for Inducing Acute Intestinal Fibrosis in Rats Using 2,4,6-Trinitrobenzene Sulfonic Acid[J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 284-293.

Figures/Tables 5

Figure 1 Schematic diagram of retention enema

Figure 2 Changes in rat body mass （A） and disease activity index (B) after model establishmentNote：*shows the modeling day. The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema. There're 6 rats contained in each group， but one of which died on days 2 and 6 in group C and two of which died on day 3 in group D after modeling.

Figure 3 Colonic tissue of rats after model establishmentNote: (A) Seven days after model establishment. (B) Fourteen days after model establishment. The upper panel shows the overall appearance of the whole colon and the lower panel shows the longitudinal section of the colon model site. The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema.

Figure 4 The length of rat colon after model establishment (A) and colon macroscopic damage index (B) in rats after model establishmentNote: The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema. There're 6 rats contained in each group， but one of which died on days 2 and 6 in group C and two of which died on day 3 in group D after modeling. Compared with the control group, *P < 0.05, **P < 0.01.

Figure 5 HE staining （A） and Masson staining （B） of rats’ colons after model establishment (×100)Note：The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema.

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