**Background:** Sepsis is a life-threatening condition caused by a dysregulated host response to infection, accounting for approximately 11.0 million deaths globally in 2017. The cecal ligation and puncture (CLP) model is the standard preclinical sepsis model, but it has significant limitations: low clinical relevance to human sepsis, inconsistency in severity grading, and an unknown proportion of animals meeting the modern Sepsis-3 criteria (based on Sequential Organ Failure Assessment [SOFA] score). Many promising treatments in CLP models fail to translate to human patients, partly due to these model deficiencies. This study aimed to develop a modified CLP (M-CLP) model that better satisfies Sepsis-3 criteria and provides more consistent disease severity.
**Methods:** Adult male Sprague-Dawley rats were randomly assigned to three groups: M-CLP, traditional CLP, or sham operation. In the M-CLP group, the cecum was ligated at a standardized 3-mL volume, punctured twice with 9-gauge needles (creating four 3-mm holes), and wrapped with a glove finger containing seven 4-mm zigzag incisions to slow cecal content leakage. Additionally, 0.25 mL of cecal content was harvested and injected into the abdominal cavity, and rats received postoperative fluid resuscitation with normal saline and 5% glucose. Traditional CLP involved ligation at the cecal midpoint and a single puncture with a 16-gauge needle. All rats received butorphanol analgesia, imipenem (25 mg/kg), and fluid resuscitation. Organ function was evaluated at 24 hours postoperatively via blood pressure, behavioral testing (Barnes maze, neurobehavioral scoring), histopathology, and blood tests (creatinine, total bilirubin, platelets, oxygenation index). Cytokine levels (IL-6, IL-17, TNF-α, IL-1β, IFN-γ, IL-10) were measured by ELISA, and T-cell subsets (CD3+, CD4+, CD8+) by flow cytometry at days 1, 7, and 14. Model stability was assessed by comparing survival rates across three independent experiments (n=10 per group per experiment) over 14 days. The primary outcome was the percentage of rats meeting Sepsis-3 criteria (dysfunction of ≥2 organs based on rat-specific thresholds). Sample size calculation (64 rats) provided 80% power at α=0.05.
**Key Results:** Significantly more rats in the M-CLP group met Sepsis-3 criteria at 24 hours compared to the CLP group (53.1% vs. 21.9%, P=0.01). M-CLP rats developed more severe organ dysfunction: higher total bilirubin, aspartate aminotransferase, alanine aminotransferase, creatinine, and urea levels (all P<0.05); lower platelet counts (P<0.05); greater decreases in oxygenation index and mean arterial pressure (both P<0.05). Barnes maze testing showed M-CLP rats had longer escape latencies and more errors (median 5 errors vs. 1.5 in CLP, P<0.05). Histopathology revealed more severe liver (focal necrosis, hemorrhage), kidney (tubular necrosis), and lung (alveolar inflammation) injury in M-CLP rats (all P<0.05). Plasma inflammatory cytokines (IL-6, IL-17, TNF-α, IL-1β, IFN-γ, IL-10) were significantly elevated in M-CLP rats at days 1, 7, and 14 compared to sham and CLP groups (P<0.05). Immunosuppression was evidenced by lower CD3+ T-cell proportions and higher CD4+/CD8+ ratios in M-CLP rats at days 1 and 7 (P<0.05). Survival consistency was markedly improved: in three independent experiments, CLP group 24-hour survival rates were 100%, 80%, and 60% (SD=0.2, P=0.048 for difference), while M-CLP rates were 70%, 60%, and 60% (SD=0.058, P=0.81). Overall 14-day mortality was 73.33% in M-CLP vs. 46.67% in CLP (P=0.049). M-CLP rats showed sustained weight loss over 14 days, while CLP rats began recovering weight after 7 days.
**Clinical Implications:** This M-CLP model addresses key limitations of traditional CLP by producing a higher proportion of animals meeting modern Sepsis-3 criteria, more severe and consistent organ dysfunction, chronic inflammation, persistent immunosuppression, and stable mortality across experiments. The improved consistency (smaller standard deviation in survival rates) may reduce the number of animals needed in preclinical sepsis studies and enhance the reliability of therapeutic evaluations. The model's ability to mimic both acute and chronic phases of human sepsis (including sustained weight loss and immunosuppression) makes it a valuable tool for studying sepsis pathophysiology and testing new interventions. However, limitations include use of only male rats, lack of blood culture, absence of necrotic tissue removal, and greater surgical complexity. Future studies should explore this model in aged animals and those with comorbidities to further increase clinical relevance.