EP562 - Establishment of a novel experimental model of perianal fistulizing Crohn’s disease

Purpose/Background: Crohn's disease (CD) is a chronic nonspecific inflammatory bowel disease with an unclear etiology and pathogenesis. Perianal fistula is a common complication of CD and remains a critical clinical challenge. Establishing an animal model of perianal fistula is essential for elucidating its pathogenesis and developing novel therapeutic strategies. This study aimed to develop a stable and reproducible rat model of perianal fistula Crohn's disease (pfCD).

Methods/Interventions: Rats were given TNBS to establish the model of chronic colitis and subsequently, a 2-mm-diameter steel wire was inserted at the 9 o'clock position (approximately 1 cm from the anal verge) in the lithotomy position to create a fistula tract, local TNBS instillation along the steel wire. After 9 weeks, the steel wire was removed, and the model group underwent colonoscopy, perineal ultrasound (PU), and perianal magnetic resonance imaging (MRI). Histopathological evaluation was performed using HE and AB-PAS staining to assess intestinal morphology. Immunofluorescence staining and Western blot were conducted to examine the expression of intestinal proteins (CD86, MUC2, ZO-1, and Occludin). ELISA was used to measure serum and colonic levels of inflammatory cytokines (IL-23, IL-12, TNF-α, and NGAL). Additionally, HE staining was performed for pathological assessment of the fistula, while immunofluorescence was used to detect macrophage (CD86) and neutrophil (MPO) infiltration in the fistula tissue.

Results/Outcomes: After 6-week TNBS enema, the pfCD rat model exhibited typical symptoms of chronic colitis, including weight loss, increased DAI score, bloody stools, decreased appetite and activity, and colon pathological injury. Colonoscopy revealed the formation of mucosal erosions or ulcers, along with hyperemia and edema. At the molecular level, a marked downregulation of key intestinal barrier proteins, MUC2, ZO-1, and Occludin, was observed, accompanied by elevated expression of CD86. Consistent with these changes, both serum and colonic tissues showed significantly increased levels of pro-inflammatory cytokines. After 3 weeks post-fistula induction, PU and MRI confirmed the formation of fistulous tracts. HE staining demonstrated substantial immune cell infiltration within the fistula tissue. Immunofluorescence analysis further revealed high expression of markers such as CD86 and MPO in fistula tissues, which was consistent with the histopathological morphology of fistula tissues from clinical pfCD patients.

Conclusion/Discussion: We successfully developed a composite rat model of pfCD, the model can simulate the human condition, and the intestinal and fistula lesions induced can be evaluated by endoscopic, PU, MRI and histological examinations. The establishment of this model provides an experimental platform for both clinical and basic research on pfCD.