Peritoneal Cavity

Sepsis was induced following a modification of a previously published method of CLP [10 (link)]. Briefly, animals were anesthetized with intraperitoneal injection of ketamine and xylazine (250 and 10 mg/kg, respectively). After adequate anesthesia, the lower quadrants of the abdomen were shaved and the surgical area was disinfected. A longitudinal midline incision was made using a scalpel, and scissors were used to extend the incision into the peritoneal cavity. After intramuscular, fascial, and peritoneal incision, the cecum was located and exteriorized. In our experiments, the cecum was ligated at different lengths below the ileocecal valve to avoid bowel obstruction. Total cecal length was measured from the tip of the ascending cecum to the tip of the descending cecum. The cecum was then ligated at 5, 20, and 100 % of its total length. For the “100 %” group, the cecum was ligated to the longest possible without bowel occlusion (Fig. 1).Fig. 1

Description of cecal length ligation methods. The total length of the cecum is represented by the full line. Dotted arrows are placed at each level of cecal ligation: 5, 20, and 100 % of the total of cecum length

The cecum was then perforated by a single puncture midway between the ligation and the tip of the cecum with a 20-G needle. We chose this needle diameter to obtain mid-grade lethal sepsis [5 (link), 13 (link), 14 (link)]. After removing the needle, a small amount of feces was extruded. The cecum was relocated, after which the fascia, abdominal musculature, and peritoneum were closed via simple running sutures; the skin was also sutured. The control mice were anesthetized and underwent laparotomy without puncture or cecal ligation and served as the control. The animals were shocked or control-operated and euthanized at different times depending on the set of experiments.
Immediately post-procedure, 1 ml of saline was administered subcutaneously for fluid resuscitation (circa 0.045 ml/g) [8 (link), 14 (link)]. Pain control for CLP and sham mice was achieved with 0.05 mg/kg buprenorphine every 12 h.

The peritoneal cavity was opened during isoflurane anesthesia, and the cecum was exteriorized. To induce light-grade CLP ~10-15% of the cecum was ligated using a nonabsorbable 7-0 suture (Johnson and Johnson, New Brunswick, NJ, USA). The distal end of the cecum was then perforated using a 23 G needle, and a small drop of feces was extruded through the puncture. The cecum was relocated into the peritoneal cavity and the peritoneum was closed using a nonabsorbable 5-0 suture (Johnson and Johnson). Animals were resuscitated by s.c. injection of 1 mL of saline and pain medication (Buprenorphin, 0.15 mg/kg) was injected sc. (23G Terumo, Leuven, Belgium). Mice were infected with HSV-1 after 7 days of sepsis.

BMDMs were collected from the femur and tibia of 6–8-week-old mice and cultured for 4–5 days in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, USA, BE12-60fF) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, 16000-044) and penicillin streptomycin amphotericin mixture (Lonza, 17-745E) containing 25 ng/ml macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) at 37°C in 5% CO₂. For the isolation of PMs from Atg7-floxed and Atg7-lacking mice (8-week-old), intraperitoneal injection of mice were performed using 1 ml of 3% Brewer thioglycollate (BD Biosciences, 211716). After 3 days from injection, the isolation of cells were conducted by flushing out the peritoneal cavity with 10 ml of Dulbecco's PBS (DPBS; Cytiva Hyclone^TM, Marlborough, MA, SH30028.02) containing 10% fetal bovine serum. The appropriate number of cells were seeded and incubated for 1 day in DMEM supplemented with 10% fetal bovine serum and penicillin streptomycin amphotericin mixture.