The Efffect Of Different Concentrations Of Ethanol On Mice Crohn’s Disease Model Induced By TNBS

Objective The incidence of Crohn’s disease increased year by year gradually.As well,the incidence of the intestinal fibrosis was as high as 33% in CD patients as one of the serious complications.However, 40% of the patients recurred stenosis after the first surgery. At present, there is no effective radical treatment. This study intends to set up 2,4,6-trinitrobenzene sulfonic acid induced mice CD intestinal fibrosis mice model. By exploring the influence of different concentration of ethanol on each model group, choose the best ethanol concentration needed for modeling, establish animal model suitable for experimental requirements, so as to provide some theoretical basis for our later research related to anti-intestinal fibrotic drugs.Methods Divide 60 BALB/c mice nto: normal control group, TNBS/20% ethanol group, TNBS/40% ethanol group and TNBS/ 50% ethanol group randomly after breeding experimentally for one week, each group have 12 mice. After mice were fasted for solids but not liquids for 24 h, lightly anesthetized them by an intraperitoneal injection of 3% chloral hydrate. Model groups were administered intracolonically respectively with different concentration(20%, 40%, 50%) of ethanol containing 2.0mg TNBS solution enema-mixture, the normal control group was administered intracolonically with the same amount of saline enema. All mice were examined for sighs of colitis including DAI、weight loss、stool consistency, and feces occult blood, we calculated the survival rate since the second day of the delivery, and calculated the gross survival rate on week 7. A 3.5 F catheter was inserted into the model group mice’ colonsuch that the tip was 3.5cm proximal to the anus from anal. The insertion depth is about 3.5 cm colon(from the anal edge), mice were inverted upside down, slowly administered the enema into the colon, the enema volume was 100 ul for each mice. gently pull out the catheter after 30 s, kept the mice in a vertical position for about 60 s. In order to prevent the flow of the enema liquids, stuff the finger with mice’anus during the delivery, then returned them into their cages with their natural waking up, and TNBS was replaced with saline in the control group. The delivery consisited for six weeks, then breeded mice normally. Mice were killed under anaesthesia on week 7th. Separated the colon, cut the colon along the side of the lumen mesenteric, flushed stool by using saline or PBS rinses. When there was a 3 centimeter distance apart from the anus, collectted the most obvious part of the lesions, preserved them in 10% formaldehyde fixed liquid. observe the macroscopic performance. experiment mucosa morphological changes under light microscopy and the level of collagenous fiber. in intestinal mucosal, analysis the relevant result.Results Mice appearred common clinical manifestations of Crohn’s disease in varying degrees in TNBS/20% ethanol group, TNBS/40% ethanol group and TNBS/ 50% ethanol group. DAI in all groups(in order of normal control group, TNBS/20% ethanol group, TNBS/40% ethanol group and TNBS/50% ethanol group)respectively was 1.27±0.78, 3.90±0.56, 5.50±0.52, 8.56±1.13, we could find that with the increasing in ethanol concentration, the DAI score increased gradually in model groups compared with normal control group, the score was highest in the 2.0TNBS/50% ethanol group(P<0.01), there were statistical difference between 2.0TNBS/20% ethanol group 、2.0TNBS/40% ethanol group, and TNBS/50% ethanol group(p<0.01). There was statistical difference between 2.0TNBS/20% ethanol group and normal control group(p<0.05); inflammatory score was 0.36±0.50, 1.40±0.51, 2.00±0.81, 3.22±0.66, we could find that with the increasing in ethanol concentration, the inflammatory score increased gradually in model groups compared with normal control group, the score washighest in the 2.0TNBS/50% ethanol group(P<0.01). there were statistical difference between 2.0TNBS/20% ethanol group、2.0TNBS/40% ethanol group, and TNBS/50% ethanol group(p<0.01). There was statistical difference between 2.0TNBS/20% ethanol group and normal control group(p<0.05); the survival rate respectively was 91.7%, 83.3%, 83.3%, 75.0%. We examined the level of collagenous fiber in each model groups by V-G stainning, and according to the intestinal fibrosis score, we could find that with the increasing in ethanol concentration, the intestinal fibrosis score increased gradually in model groups compared with normal control group, the score was highest in the 2.0TNBS/50% ethanol group(P<0.01), there were statistical difference between 2.0TNBS/20% ethanol group 、 2.0TNBS/40% ethanol group, and 2.0TNBS/50% ethanol group(p<0.05).1. Conclusion With the increase in concentration of ethanol, mice’s DAI score、intestinal pathology and fibrosis score increased gradually, the score was highest in the 2.0TNBS/50% ethanol group, this model was similar with human CD in pathological and histological changes.2. The CD animal model induced by 2.0 mg TNBS in 50% ethanol mixed solution enemas could simulate human CD performance in pathology. Animal model induced by this method had a moderate survival rate, high rate of replication and repeatability.3. Colonic mucosal inflammation lasted long, and the model was stable, also it could reflect the dynamic pathological process of acute intestinal mucosal inflammation to chronic, it was in accordence with human CD evolution regulation, it was well suitable for research related to the pathogenesis of CD and new targets of drugs in treating CD.