Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Summary

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Abstract

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

Introduction

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection¹, and septic shock is the leading cause of death in severe cases of sepsis². Sepsis and septic shock cause millions of deaths worldwide each year³. The key to improving the outcome of patients with sepsis is the prompt initiation of treatments such as antibiotics⁴. The gold standard method for the diagnosis of sepsis is microbial culture; however, microbial culture is time-consuming and can lead to false-positive and false-negative results, which greatly limit the cli....

Protocol

All animal experiments were performed in accordance with the guidelines approved by theAnimal Review Committee at Xiangya Hospital and Central South University (No. 202103149).

1. Preparation

1. Select male C57BL/6J mice (weight: 20-25 g; age: 8-12 weeks) and house for 3 days before performing any procedures.
2. Weigh the mouse.
3. Anesthetize the mouse with 1.5% isoflurane by inhalation and pinch the toes to check the depth of anesthesia.
4. Fix the mouse on the heating pad. Apply depilatory cream to the abdomen and leave for no longer than 1 min before removing the cream and hair.
   R....

Results

As shown in Figure 2A, no CitH3 was detected in the sham group by western blotting. The serum CitH3 concentration significantly increased after CLP, and this increase was blocked by the inhibition of NET formation via the administration of YW3-56, a PAD inhibitor¹⁰. Figure 2B shows the serumCitH3 concentrations determined by ELISA. At 24 h after CLP, the serum concentration of CitH3 was increased in the CLP groups compared with t.......

Discussion

CLP introduces pathogens into the abdomen to create a preclinical model of sepsis. When performing CLP, it is important to use sterile conditions to eliminate the interference of exogenous bacteria and to use accurate dosages of anesthetics¹⁶. The three technical aspects of CLP that affect the severity and replicability of the sepsis model are the percentage of the cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. Ligation of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
21G needle
3,3’,5,5’-tetramethylbenzidine	R&D Systems Inc	DY999
anti-CitH3 monoclonal antibody	laboratory self developed
anti-CitH3 polyclonal antibody	Abcam	ab5103
anti-rabbit secondary antibody	Jackson ImmunoResearch	111-035-003
C57BL/6 mice	Xiangya School of Medicine, Central South University
Cl-amidine	Sigma Aldrich	SML2250
depilatory cream
Dnase I	Sigma Aldrich	11284932001
isoflurane	Sigma-Aldrich	26675-46-7
ketoprofen	Sigma Aldrich	PHR1375
silk sutures (4-0 & 6-0)
surgical instruments
YW3-56	GLPBIO	GC48263