Procedure-Matched Comparison of TNBS/Ethanol Enema and DSS Free-Drinking Models of Ulcerative Colitis in Rats: Immune Endotype Profiling and a Translational Model-Selection Framework

ObjectiveChoosing between TNBS/ethanol enema and DSS free-drinking water protocols for establishing rat ulcerative colitis(UC) models remains a practical challenge in preclinical research. Beyond confirming that both methods induce colitis, no prior study has quantified the independent contribution of enema procedural trauma to inflammatory readouts, reported sex-stratified cytokine responses, or formally linked model-specific immune profiles to human UC endotypes. MethodsTen SPF-grade SD rats(half male and half female) were allocated to a normal control, a procedure-matched saline enema control(anesthesia and enema without chemical agent), a TNBS group(100 mg/kg TNBS + 50% ethanol enema), and a DSS group(3% DSS free-drinking, 7 days). Endpoint assessments included body weight, colon length, macroscopic and histological scoring(modified Macpherson criteria, 0-10), and serum TNF- and IL-6 by ELISA. Post-hoc exploratory analyses included sex-stratified cytokine sub-analysis and coefficient of variation(CV%) for each readout. ResultsBoth model groups successfully induced UC(P<0.05 vs. controls for all endpoints). Critically, the procedure-matched saline enema control confirmed that colonic inflammation in the TNBS group was attributable to the chemical agent rather than mechanical trauma. TNBS induced significantly higher TNF-(90.2{+/-}10.5 vs. 73.5{+/-}9.8 pg/mL, P<0.05) and IL-6(66.5{+/-}8.3 vs. 53.2{+/-}7.6 pg/mL, P<0.05) than DSS,consistent with Th1-polarized vs. innate barrier-disruption immune endotypes, respectively. Coefficient of variation analysis indicated greater intra-group variability in TNBS versus DSS, reflecting the procedural dependency of enema-based models. ConclusionA procedure-matched control design is essential for valid TNBS-vs.-DSS comparisons and has been systematically underutilized in prior studies. Based on immune endotype alignment, TNBS is recommended for screening T-cell-targeted therapies and acute anti-inflammatory agents, while DSS is recommended for epithelial barrier repair and microbiome-modulating interventions. A model-selection decision framework derived from these data is provided to guide preclinical study design.