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).

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
<ol>
	<li>Select male C57BL/6J mice (weight: 20-25 g; age: 8-12 weeks) and house for 3 days before performing any procedures.</li>
	<li>Weigh the mouse.</li>
	<li>Anesthetize the mouse with 1.5% isoflurane by inhalation and pinch the toes to check the depth of anesthesia.</li>
	<li>Fix ...

<ol>
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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>21G needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#8217;,5,5&#8217;-tetramethylbenzidine&#160;</td>
			<td>R&#38;D Systems Inc</td>
			<td>DY999</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CitH3 monoclonal antibody</td>
			<td>laboratory self developed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CitH3 polyclonal antibody</td>
			<td>Abcam</td>
			<td>ab5103</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-003</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Xiangya School of Medicine, Central South University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cl-amidine</td>
			<td>Sigma Aldrich</td>
			<td>SML2250</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma Aldrich</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>ketoprofen</td>
			<td>Sigma Aldrich</td>
			<td>PHR1375</td>
			<td></td>
		</tr>
		<tr>
			<td>silk sutures (4-0 &#38; 6-0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>surgical instruments&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>YW3-56</td>
			<td>GLPBIO</td>
			<td>GC48263</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of a reliable biomarker in a cecal ligation and puncture-induced mouse model of sepsis

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.

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...

Watch this Scientific Journal Video about Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis at JoVE.com

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

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.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...

The main purpose of this study is to use mouse model of cecal ligation and puncture-induced sepsis to evaluate reliable blood biomarker of sepsis. Cecal ligation and puncture, or CLP in short, is a clinically-relevant model of sepsis. CLP is commonly used model in sepsis research.Citrullinated histone H3, or CitH3, originates from sepsis-induced NETosis. Our previous study demonstrates that CitH3 is a reliable diagnostic biomarker of sepsis. Select male C57BL/6 mice.Weigh the mouse, and anesthetize the mouse by inhalation of 1.5%isoflurane. Fix the mouse on a heating pad. Apply depilatory cream to the abdomen and leave for no longer than one minute before removing the cream and hair.Prepare sterile surgical instruments suitable for rodent animal surgery. Disinfect the abdominal skin with iodine at least three times. Cover the operative surgical area with the proper sterile surgical drapery.Use sterile surgical scissors to make an approximately two centimeters incision in the abdominal wall, along the linea alba. Identify and isolate the cecum out of the peritoneal cavity with sterile tweezers. Ligate 75 volume of the cecum with 4-0 silk suture.Do not ligate the mesenteric blood vessels. To be noted, different percentage of cecum ligated determines the severity of sepsis. 25%leads to a low-grade sepsis, 50%leads to the mid-grade sepsis, and 75%leads to a high-grade sepsis.Puncture the cecum by one through-and-through perforation with a 21 gauge needle at the midpoint between the tail end and the ligation. Gently squeeze the feces out of the cecum through penetration holes. To be noted, the amount of feces squeezed into the peritoneal cavity should be consistent, since feces also determines the severity of sepsis.Replace the cecum as well as feces into the abdominal cavity gently. Close the abdominal muscle layer and the skin layer with 6-0 silk suture. Disinfect the incision with iodine.Injection of ketoprofen, five milligrams per kilograms, should be performed on the lower left quadrant of the abdomen to avoid the cecum. Place the mouse on a heating pad till full recovery. The mouse is housed in a temperature-controlled room and is given free access to food and water.Animals will be euthanized by carbon dioxide overdose with symptoms of sepsis, relevant to our predefined endpoints based on our study presented. Mice are randomly divided into the sham group, CLP group, and the CLP plus Cl-amidine or YW3-56 treatment group. Mice in sham group receive all CLP procedures except cecum ligation and puncture.Harvest peripheral blood at 24 hours post-operatively and prepare serum through centrifugation. CitH3 concentration was determined by western blotting and a self-developed ELISA kit. As shown in figure two, no CitH3 was detected in sham group.CitH3 levels were elevated after CLP insert, which was attenuated by PAD inhibition significantly. Based on our data, we conclude that circulating CitH3 is a reliable diagnostic biomarker of sepsis.

evaluation-reliable-biomarker-cecal-ligation-puncture-induced-mouse

Research

JoVE Journal

Immunology and Infection

2.9K visualizações.  Central South University. O principal objetivo deste estudo é utilizar modelo de camundongo de ligadura cecal e sepse induzida por punção para avaliar biomarcadores sanguíneos confiáveis de sepse. A ligadura e punção cecal, ou CLP em suma, é um modelo clinicamente relevante de sepse. A FLP é um modelo comumente usado na pesquisa de sepse.A histona H3 citrulinada, ou CitH3, origina-se da NETose induzida por sepse. Nosso estudo anterior demonstra que o CitH3 é um biomarcador diagnóstico confiável de s...