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 clinical significance⁵. Thus, it is highly desirable to identify a blood biomarker of sepsis. Procalcitonin is recognized as an ideal sepsis biomarker but has limited diagnostic efficacy because it is unable to distinguish sepsis from sterile diseases⁶.

Mouse cecal ligation and puncture (CLP) is commonly used to create a model of sepsis in scientific research. CLP is one of the most widely used sepsis models because it mimics polymicrobial peritonitis, activating both proinflammatory and anti-inflammatory immune responses⁷. It is well accepted that CLP creates a more clinically relevant sepsis model than alternative techniques, such as the injection of bacterial endotoxin. Therefore, CLP is considered the classical sepsis model for use in research⁸. However, a major disadvantage of CLP is its reproducibility, as the model severity is affected by several factors such as the percentage of cecum ligated, needle size, number of punctures, and laparotomy technique. Therefore, there is a need to standardize the CLP-induced sepsis model. The present study describes the protocol details of the CLP-induced sepsis model to show the standardized procedure and increase its reproducibility.

The inflammatory response occurs in the early stage of sepsis, with neutrophils releasing excessive amounts of oxidants and proteases that cause organ damage⁸. A key factor in the pathophysiology of sepsis is the formation of neutrophil extracellular traps (NETs), which release nuclear and cytosolic components such as DNA, citrullinated histones, and antimicrobial proteinases⁹. Recent studies suggest that excessive generation of NETs mediates the pathology of sepsis; meanwhile, a decrease of NETs, through enzymatic inhibition of peptidyl arginine deiminase (PAD) by chemicals like YW3-56 or Cl-amidine, exerts a pro-survival effect in mouse models of sepsis^10,11. Citrullinated histone H3 (CitH3) was identified as a sepsis-specific protein in 2011¹², and subsequent publications have demonstrated that the circulating CitH3 concentration is a reliable diagnostic biomarker of sepsis^13,14. CitH3 is considered a more sensitive and long-lasting biomarker than procalcitonin, and is more specific in distinguishing sepsis than inflammatory cytokines¹³.

In this study, we have evaluated a reliable diagnostic biomarker of sepsis in a CLP-induced mouse model of sepsis.

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.
   ​NOTE: Normal body temperature should be maintained by placing a warm pad under the anesthetized mouse during surgery.

2. Operation

1. Prepare sterile surgical instruments suitable for rodent animal surgery. Wear sterile gloves, a face mask, and a surgical gown to ensure aseptic conditions.
2. Disinfect the abdominal skin with iodine wipes at least three times. Cover the operative surgical area with sterile surgical drapes.
3. Use sterile surgical scissors to make an approximately 2 cm incision in the abdominal wall along the linea alba.
   NOTE: Do not damage the organs.
4. Identify and isolate the cecum out of the peritoneal cavity with sterile tweezers.
5. Ligate 75% of the volume of the cecum with 4-0 silk sutures. Do not ligate the mesenteric blood vessels.
   NOTE: The percentage of the cecum that is ligated determines the severity of sepsis.
6. Puncture the cecum by creating one through-and-through perforation (two holes) with a 21 G needle (from one side through the cecal wall to the opposite side) at the midpoint between the tail end and the ligation set.
7. Discard the needle. Gently squeeze a small droplet of feces out of the cecum through the penetration holes.
   ​NOTE: The amount of feces squeezed into the peritoneal cavity should be consistent, as this also determines the severity of sepsis. Steps 2.5-2.7 should be skipped for sham mice.
8. Gently replace the cecum into the abdominal cavity.
9. Close the abdominal muscle and skin separately with 6-0 silk sutures.
10. Disinfect the incision with iodine.
11. Inject ketoprofen (5 mg/kg) in the lower left quadrant of the abdomen to avoid the cecum. Place the mouse on a warm pad until it has fully recovered from the anesthetic.
12. Place the mouse in a cage in a temperature-controlled room (22 °C) and give free access to food and water. Check the mouse every 6 h for the first week postoperatively.
    1. Euthanize the mouse by carbon dioxide overdose when symptoms of sepsis meet the pre-defined endpoints.

3. Treatment

1. Randomly divide mice into the sham group, CLP group, CLP + YW3-56 group (Figure 2a), and CLP + Cl-amidine group (Figure 1).
   NOTE: The sham group underwent the same procedures as the CLP groups except CLP. YW3-56 (5 mg/kg) or Cl-amidine (40 mg/kg) was administered via peritoneal injection 1 h after step 2.9 when suturing the muscle and skin layer.
2. Sample harvest
   1. Harvest peripheral blood from the retrobulbar plexus¹⁵ 24 h after the operation.
3. Prepare the serum by centrifugation (1,000 x g, 5 min) and store at -80 °C until use.
4. Measure the CitH3 concentration using an indirect sandwich ELISA kit as previously described¹³.
   1. Coat the anti-CitH3 monoclonal antibody on a 96-well plate to capture the CitH3 protein.
   2. Treat the serum with DNase I (150 unit/mL, 37°for 1 h) and incubate in the wells (20 µL, room temperature).
   3. Add anti-CitH3 polyclonal antibody (0.33 µg/mL, 100 µL, 2 h) to detect the captured CitH3 protein.
   4. Incubate the anti-rabbit peroxidase-labeled secondary antibody (0.02 µg/mL, 100 µl, 1 h) in the wells.
   5. After thorough washing, develop the wells with 3,3',5,5'-tetramethylbenzidine (100 µL, 20 min).
      NOTE: Further protocol details of the ELISA kit are provided in previous publication¹³.
5. Statistical analysis
   1. Perform one way ANOVA to analyze the differences among the three groups, followed by Bonferroni post hoc testing for multiple comparisons. Use a statistical software to perform the analysis. p values of 0.05 or less were considered significant.

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

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