Experimental robustness and reproducibility of the murine cecal ligation and puncture sepsis model

Sepsis, a dysregulated host response to infection leading to organ failure, remains a leading cause of death worldwide, reflecting the failure to translate preclinical findings into clinical therapies. This translational gap is partly driven by substantial variability and limited reproducibility of preclinical models, particularly the cecal ligation and puncture (CLP) model, widely regarded as the gold standard among experimental sepsis models. Although multiple biological and methodological variables influence outcomes, their relative contributions remain incompletely defined, and standardized approaches for integrated immune and hematopoietic profiling are lacking.

We established and validated a standardized experimental and analytical framework for CLP-induced sepsis, enabling controlled disease severity and integrated assessment of local and systemic immune responses. A total of 88 C57BL/6 mice underwent mid-grade sepsis (target survival ~ 70%) across 19 independent experiments performed by different operator teams. The primary endpoint was time to death or predefined humane endpoints within 48 h, aiming to identify biological and experimental variables influencing survival and immune responses. Immune cell populations, hematopoietic stem and progenitor cell subsets, and cytokines were quantified by multiparameter flow cytometry, and tissue responses were assessed histologically. Survival dynamics and the influencing variables were analyzed using Kaplan-Meier estimates and Cox proportional hazards regression.

The framework yielded consistent mid-grade sepsis, with a 48 h survival rate of 67.2% (95% CI 54.6-78.2%). Survival outcomes and immune readouts were reproducible across operators and experiments, demonstrating robust model performance under standardized conditions. Among tested variables in a multivariate Cox regression analysis, experiments performed during summer were associated with increased mortality (HR 3.178, 95% CI 1.164-8.681, P = 0.024), whereas sex, age, body weight, day time, and cage density had no significant effect on survival (all P > 0.05). Integrated immune and hematopoietic profiling revealed sex-dependent immune responses that were not associated with altered survival.

This standardized CLP framework enables reproducible survival outcomes that are largely independent of common biological and procedural variables. Consistent results can be obtained across approximately three independent experiments, each including around 8 animals per group. Systematic assessment of biological and environmental variability may improve reproducibility and support more consistent interpretation of CLP-based sepsis studies.