Cecal ligation and puncture (CLP) in mice

Experiments involving animals were approved by the Institutional Animal Care and Use Committee of Kagoshima University, Kagoshima, Japan, and complied with the ARRIVE guidelines. Male C57BL/6J mice (Kyudo) were anesthetized with isoflurane at 10 weeks of age and subjected to CLP-induced sepsis as described previously (Rittirsch et al. 2009), with slight modifications. Briefly, the cecum of each volatile-anesthetized mouse was ligated at a site 1.5 cm distant from the tip of the cecum. For a mild model of sepsis, the tip of the cecum was punctured once with a 25-gauge needle. For a severe model of sepsis, the midway point between the ligation and the tip of the cecum was penetrated by a single through-and-through puncture with a 25-gauge needle. The cecum was returned to the abdominal cavity, and the abdominal wall was sutured. In some experiments, 2 mg/kg of HMGB1, desHMGB1, or saline was administered into the tail vein at 6 and 24 h after CLP, and the survival rate was monitored for 7 days. The general condition of the mice was evaluated every 6 h, and the mice were sacrificed when moribund. In another set of experiments, 5 mg/kg of rTM (Asahi Kasei Pharma) was administered into the tail vein at 6 h after CLP. Blood samples were then collected from volatile-anesthetized mice at 0, 6, 12, and 24 h after CLP, and were anticoagulated with one-tenth volume of 3.2% sodium citrate and centrifuged at 1500×g for 10 min. Plasma samples were stored at − 80 °C until further analysis.