Murine CLP sepsis model

Specific pathogen-free BALB/c female mice (Sankyo Labo Service, Tokyo, Japan) were allowed to acclimate for 1 wk in a temperature-controlled facility with 12/12 h light/dark cycles, and food and water ad libitum. All experimental procedures were approved by the Ethics Committee for the Use of Laboratory Animals of Juntendo University (300194, April 2018).

CLP was performed according to the previous description.²⁶ Briefly, mice (aged 7–8 wk) were anesthetized with isoflurane. The abdominal skin was disinfected with 70% ethanol, then a 1 cm midline laparotomy was performed. The cecum was exteriorized, the contents massaged to the tip, and the distal 7 mm of the tip was ligated with 3-0 silk suture. The ligated portion was perforated once with an 18G needle and a small amount of stool (1 mm in length) was extruded. The cecum was replaced back into the abdominal cavity and the midline incision was closed in two layers with 3-0 nylon suture. Mice were resuscitated by subcutaneous administration of 1 ml saline in the neck. In the sham group, mice underwent the same procedure without ligation and perforation. To evaluate the effect of LL-37 on ectosome release in CLP mice, 200 μl of LL-37 solution (3 μg) or PBS as a control was intravenously administered just prior to the CLP operation. To determine the humane endpoint, we employed the murine sepsis score that can be used as a surrogate marker of mortality²⁷ with some modifications; mice were euthanized when they manifested ruffled hair, no response to touch stimuli, completely closed eyes, and consistent labored breathing.