Inducing Experimental Polymicrobial Sepsis by Cecal Ligation and Puncture

Abstract

Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and biochemical responses similar to those seen in septic humans. Further, a transient numerical reduction of multiple immune cell types, followed by development of prolonged immunoparalysis, occurs in CLP-induced sepsis just as in humans. Use of the CLP model has led to a vast expansion in knowledge regarding the intricate physiological and cellular changes that occur during and after a septic event. This updated article details the steps necessary to perform this survival surgical technique, as well as some of the obstacles that may arise when evaluating the sepsis-induced changes within the immune system. It also provides representative monoclonal antibody (mAb) panels for multiparameter flow cytometric analysis of the murine immune system in the septic host. © 2020 Wiley Periodicals LLC. Basic Protocol: Cecal ligation and puncture in the mouse.

Keywords: CLP; cecal ligation and puncture; sepsis; septic shock; surgical murine model; systemic inflammatory response syndrome (SIRS).

Figure 1

Utilization of the cecal ligation and puncture (CLP) model over time based on publications. Total and murine CLP results were generated by PubMed searches of “cecal ligation and puncture” or “mouse cecal ligation and puncture,” respectively, performed on June 25, 2020. Indexing information was downloaded and used for evaluation. Total CLP results include 50 review articles, 26 published since 2010. Murine CLP results include 21 review articles, 10 published since 2010. (A) Synopsis of CLP publications, murine CLP publications, and number of unique authors, in total and since 2010. The number of unique authors that have only published a CLP paper since 2010 is also included. (B,C) Numbers of total and murine CLP publications per year from 1975 to 2019. (D) Representation of murine CLP publications among all CLP publications in 5-year periods from 1981 to present.

Figure 2

Prepping the mouse and exposing the cecum. (A) The mouse is positioned on heated surgical mat and the nose cone secured. (B) The abdominal fur is shaved and 5% povidone-iodine antiseptic is applied. (C) A small (~1 cm) paramidline incision is made through the skin. (D) The cecum is externalized.

Figure 3

Performing CLP. (A) The cecum is ligated using a ruler as a guide. (B) The cecum is punctured from the basolateral side into the lumen using a 25-G needle. (C) To verify puncture placement, a small amount of cecal material is extruded though the puncture using forceps. (D) The peritoneum is closed using two or three 4–0 absorbable polyfilament uninterrupted sutures. (E) The skin is closed using Vetbond tissue adhesive.

Figure 4

Increased morbidity/mortality in CLP-treated microbially experienced (dirty) mice correlates with an exacerbated cytokine storm. Specific pathogen–free (SPF) and dirty co-housed (CoH) B6 mice underwent sham or CLP surgery. (A) Survival was monitored over time (n = 8–17 mice/group; ** p ≤ .01). (B) Serum samples were obtained 6 hr after surgery and the amount of CXCL1, CXCL10, IL-1β, IL-6, IL-10, IFNγ, and TNF was determined by BioPlex. (C) Additional samples were collected at 12 and 24 hr post-surgery to quantify changes in IL-1β, IL-6, IFNγ, and TNF over time (n = 4–7 mice/group/time point; *p ≤ .05, ** p ≤ .01, *** p ≤ .005 for SPF-CLP vs. CoH-CLP at the indicated time points). Data are adapted from Huggins et al. (2019b).