Development of a chronic rat model of perianal fistulizing Crohn's disease

Abstract Objective Perianal fistulizing Crohn's disease (pfCD) is a common and debilitating complication of Crohn's disease for which clinically relevant and reproducible experimental models remain limited. This study aimed to develop and characterize a stable rat model that recapitulates key features of human pfCD. Methods Chronic colitis was induced in rats by repeated administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). A fistulous tract was subsequently created using a 2-mm-diameter steel wire, followed by local TNBS instillation to sustain fistula-associated inflammation. Model establishment was evaluated by colonoscopy, perineal ultrasonography (PU), and magnetic resonance imaging (MRI). Histopathological and molecular characterization included hematoxylin and eosin (H&E) staining, Alcian blue-periodic acid–Schiff (AB-PAS) staining, immunofluorescence, and Western blotting for intestinal barrier-associated proteins, including MUC2, ZO-1, and Occludin. Serum and colonic levels of IL-23, IL-12, TNF-α, and neutrophil gelatinase-associated lipocalin (NGAL) were measured by ELISA. Fistula-associated inflammatory features were further compared with fistula specimens obtained from 10 treatment-naïve patients with pfCD. Results Rats subjected to the two-stage induction protocol developed persistent clinical and endoscopic features of chronic colitis. Colonoscopy demonstrated mucosal erosions and ulceration accompanied by hyperemia and edema, while histological examination revealed marked mucosal injury and disruption of intestinal barrier integrity. Expression of MUC2, ZO-1, and Occludin was reduced, whereas serum and colonic levels of IL-23, IL-12, TNF-α, and NGAL were significantly increased (P < 0.01). Both PU and MRI confirmed the formation of persistent fistulous tracts. Immunofluorescence further demonstrated increased CD86 and MPO expression within fistula tissues of the rat model, with spatial expression patterns similar to those observed in fistula specimens from treatment-naïve patients with pfCD. Conclusion We developed a reproducible chronic rat model of pfCD using a two-stage induction strategy combining chronic TNBS-induced colitis with mechanically created and locally inflamed fistulous tracts. The model reproduces key endoscopic, imaging, histopathological, inflammatory, and barrier-associated features of human pfCD and may provide a useful preclinical platform for investigating disease mechanisms and evaluating therapeutic interventions.