2.2. Polymicrobial Sepsis (Cecal Ligation and Puncture (CLP) Model)

A modified CLP model was used to induce polymicrobial sepsis [25]. The mice were quickly anesthetized with 2.0% isoflurane, vaporized in medical oxygen (O₂), via a face mask. The abdomen was shaved, and a midline incision was performed. The cecum was isolated and ligated with 6–0 silk thread below the ileocecal valve without causing bowel obstruction. The cecum was then punctured with an 18-gauge needle to induce severe septic injury (SSI). Bowel content was gently extruded through the puncture, and the cecum was then replaced to its original position. The abdomen was then sutured. Sham-operated animals (controls) underwent the same procedures, except for cecal ligation and puncturing. To prevent dehydration, all mice received subcutaneous doses of saline (50 mL/kg of body weight) immediately and 12 hours after the surgical procedure. For pain relief, sodium dipyrone solution (10 mg/100 g body weight, i.p.) was administered at the start of the surgery and 6-12 hours after surgery. Mice were monitored daily for signs of disease, such as piloerection, hunched gait, lethargy, and eye discharge. Mice displaying severe signs of distress (labored breathing, nonresponsiveness to cage tapping, failure of grooming, and severe eye discharge) were humanely euthanized by injecting a mixture of ketamine (90–120 mg/kg) and xylazine (10 mg/kg), followed by cervical dislocation.