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 sepsis10,11. 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 sepsis13,14. 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
- Select male C57BL/6J mice (weight: 20-25 g; age: 8-12 weeks) and house for 3 days before performing any procedures.
- Weigh the mouse.
- Anesthetize the mouse with 1.5% isoflurane by inhalation and pinch the toes to check the depth of anesthesia.
- 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
- Prepare sterile surgical instruments suitable for rodent animal surgery. Wear sterile gloves, a face mask, and a surgical gown to ensure aseptic conditions.
- Disinfect the abdominal skin with iodine wipes at least three times. Cover the operative surgical area with sterile surgical drapes.
- 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. - Identify and isolate the cecum out of the peritoneal cavity with sterile tweezers.
- 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. - 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.
- 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. - Gently replace the cecum into the abdominal cavity.
- Close the abdominal muscle and skin separately with 6-0 silk sutures.
- Disinfect the incision with iodine.
- 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.
- 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.
- Euthanize the mouse by carbon dioxide overdose when symptoms of sepsis meet the pre-defined endpoints.
3. Treatment
- 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. - Sample harvest
- Harvest peripheral blood from the retrobulbar plexus15 24 h after the operation.
- Prepare the serum by centrifugation (1,000 x g, 5 min) and store at -80 °C until use.
- Measure the CitH3 concentration using an indirect sandwich ELISA kit as previously described13.
- Coat the anti-CitH3 monoclonal antibody on a 96-well plate to capture the CitH3 protein.
- Treat the serum with DNase I (150 unit/mL, 37°for 1 h) and incubate in the wells (20 µL, room temperature).
- Add anti-CitH3 polyclonal antibody (0.33 µg/mL, 100 µL, 2 h) to detect the captured CitH3 protein.
- Incubate the anti-rabbit peroxidase-labeled secondary antibody (0.02 µg/mL, 100 µl, 1 h) in the wells.
- 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.
- Statistical analysis
- 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.
Representative 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 the sham group (p = 0.0008), and this CitH3 increase was significantly attenuated by Cl-amidine, a PAD inhibitor that limits NET formation (p = 0.0028).
Figure 1: Experimental grouping. Mice were randomly divided into the sham group, CLP group, CLP +YW3-56 group (A), and CLP + Cl-amidine group (B). CLP was performed as described in the protocol. YW-3-56 (5 mg/kg) or Cl-amidine (40 mg/kg) was administered via peritoneal injection 1 h after CLP. The sham group underwent the same procedures as the CLP groups except for CLP. Blood was collected at various timepoints, and serum was prepared and stored at -80 °C until use. YW3-56 and Cl-amidine are both peptidyl arginine deiminase inhibitors that significantly limit the formation of neutrophil extracellular traps. Abbreviations: CitH3 = citrullinated histone H3; CLP = cecal ligation and puncture. Please click here to view a larger version of this figure.
Figure 2: Serum CitH3 concentration was significantly increased in the CLP-induced sepsis model and was alleviated by PAD inhibition. (A) Western blotting was performed to test the serum CitH3 concentration. No CitH3 was detected in the sham group. The CitH3 concentration was significantly increased in the CLP groups, and this increase was blocked by YW3-56 treatment. (B) Serum CitH3 concentrations were measured by ELISA. Almost no CitH3 was detected in the sham group. The CLP groups showed marked increases in CitH3, which was attenuated by the administration of Cl-amidine. YW3-56 and Cl-amidine are both PAD inhibitors that significantly limit the formation of neutrophil extracellular traps. Abbreviations: CitH3 = citrullinated histone H3; CLP = cecal ligation and puncture; PAD = peptidyl arginine deiminase. Please click here to view a larger version of this figure.
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 approximately 75% of the cecum results in severe sepsis, 50% ligation results in moderate sepsis, and 25% ligation or less results in mild sepsis17. The number of punctures and the needle size determine the mortality rate18, and the best result is gained by through-and-through puncture. A small amount (a droplet) of feces is enough to cause bacterial peritonitis, and care is required when squeezing out the feces. CLP takes approximately 10 min when performed by an experienced individual.
The mouse model of CLP-induced sepsis described in this study has several limitations. First, the mouse may be too small to obtain enough blood samples for peripheral blood analysis and cytokine measurement. Second, CLP cannot be performed in newborn animals. Third, variability still occurs, and reproducible results may not be obtained. It is important to ensure that the surgeons have had adequate practice in performing CLP before conducting research tests.
CLP creates a clinically realistic model of sepsis that mimics the pathophysiological process and cytokine profiles and has been widely used in rodents. The severity of CLP can be manipulated to fulfill different study purposes by adjusting the size of the needle and the percentage of the cecum ligated. According to our results obtained using the CLP model, detecting the circulating CitH3 concentration enables the early diagnosis of sepsis in mice. The diagnostic value of serum CitH3 concentration enables the prompt initiation of anti-sepsis treatments that significantly improve the outcomes of septic individuals.
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Professor Wang Wei and Doctor Liu Shuai for helping with the experiments. This work was funded by grants from the Young Research Funding of Xiangya Hospital, Central South University (No. 2019Q10), From the National and Science Foundation of Hunan Province (No. 2020JJ4902), and from the National Natural Science Foundation of China (No. 82202394).
Materials
| 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 |
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