Sepsis model

A cecal ligation and puncture (CLP) sepsis model was established as previously described (31–33). The mice were anesthetized in the same manner as for the burn protocol, and their abdominal fur was shaved. A 1 cm skin and peritoneal incision was made vertically in the midline of the abdomen, and the cecum was gently exposed after resection of the adipose tissue on both sides (34). Ligation was performed at 50% of the cecum, with two punctures with a 21-gauge needle 5 mm from the blind end. The method yielded a survival rate equivalent to the sepsis survival rate in humans (65–80%) (35–37). The cecum was returned to the abdominal cavity, whereafter the peritoneum was sutured with five stitches and the skin with three stitches using a 4-0 nylon thread. Resuscitation was performed via subcutaneous injection of 1 ml 0.9% pyrogen-free saline after CLP. The CLP procedure was performed on a hot plate (Heater Mat KN-475, Natsume Seisakusho, Co., Ltd., Tokyo, Japan) and maintained at approximately 38°C. To prevent surgical site infection, post-operative mice were kept in the supine position until the anesthetic effect wore off and they regained consciousness. To prevent post-operative hypothermia, the cage was placed on a hot plate until the mice woke up from anesthesia (38). Antibiotic treatment began 2 h after the CLP procedure, with a subcutaneous injection of imipenem/cilastatin (25 mg/kg) repeated every 12 h for the first 3 days (for a total of six injections) (39).