Cecal Ligation and Puncture (CLP)-Induced Polymicrobial Sepsis

The CLP procedure was performed as previously described and according to the general guidelines.^11–13 Briefly, under isoflurane anesthesia, mice were laid supine and a midline incision was made in the lower abdomen after shaving and sterilizing the abdominal skin. The cecum was identified and carefully extracted. Stool in the cecum was squeezed distally and a 3–0 suture was tied around the proximal portion of the cecum and secured tightly, interrupting continuity with the bowel and cutting off blood flow. A 21 g needle was used to make a single puncture in the proximal, antimesenteric aspect of the cecum and a small amount of stool was expressed. The cecum was placed back in the abdominal cavity and the abdominal wall closed with 6–0 suture. Skin was approximated and closed with Gluture tissue adhesive. Immediately post op, mice were given 20 mL/kg normal saline solution and buprenorphine 0.05 mg/kg. Two hours later, mice were given imipenem 25 mg/kg. At 12 hours mice were dosed with normal saline solution 10 mL/kg, and the same doses of imipenem and buprenorphine. Mice were monitored using a clinical severity score every 12 hours, as previously reported.¹² Tissue samples were collected at the specified time points. Anesthesia was induced again with isoflurane and blood collected via cardiac puncture. The chest was opened and heart, and lungs collected rapidly into cryovials which were immediately snap frozen in liquid nitrogen. Abdominal organs were collected in a similar fashion immediately thereafter.