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

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.