Cecal ligation and puncture (CLP)

Sepsis was induced using CLP, a murine model of polymicrobial sepsis. Injury was titrated to achieve a ~50% 14-day mortality to mimic the clinical scenario of sepsis [31]. In brief, C57BL/6 mice were anesthetized using isoflurane and underwent laparotomy, the cecum was exteriorized, ligated distal to the ileocecal valve, and punctured twice with a 25-gauge needle. Sham-operated animals underwent laparotomy and exteriorization of the cecum only. Personnel conducting sham and CLP surgeries received training and competency testing from Emory University Division of Animal Resources veterinary staff. All animals received buprenorphine (0.1mg/kg) preoperatively for pain relief and 1mL of normal saline for intraoperative fluid losses as well as antibiotics (ceftriaxone 25mg/kg and metronidazole 12.5mg/kg) subcutaneously postoperatively. Antibiotics were continued on a q12hr dosing schedule for 48 hours postoperatively. For experiments evaluating CXCR4 blockade, mice designated to the CLP+ Plerixafor group received a 100μl SQ injection of 5mg/kg Plerixafor (AMD3100, Sigma-Aldrich) one hour after abdominal closure. Throughout this manuscript, mice that underwent CLP and were given normal saline as a control injection are referred to as “septic control mice”. Mice were sacrificed by CO₂ asphyxiation. For survival studies, mice were observed daily for 7-days after surgery. Animals were observed every 12 hours during this 7-day period. The following criteria were used as humane endpoints; animals meeting any one of these criteria were considered moribund, counted as deceased in the enumeration of surviving animals, and sacrificed by CO₂ asphyxiation. 1) Loss of 25% of body weight from baseline weight. 2) Major organ failure or medical conditions unresponsive to treatment such as severe respiratory distress, icterus, uremia, intractable diarrhea, or self-mutilation. 3) Surgical complications unresponsive to immediate intervention (bleeding, infection, wound dehiscence). 4) Clinical or behavioral signs unresponsive to appropriate intervention persisting for 24 hours including significant inactivity, labored breathing, sunken eyes, hunched posture, piloerection/matted fur, and abnormal vocalization when handled. Once any animal reached endpoint criteria, the amount of time elapsed before euthanasia was <12 hours. Some animals died before meeting the criteria for euthanasia. A total of 42 animals were used in the survival study, and 26 of them died or met endpoint criteria. The cause of death for all animals was septic shock.