Animal Treatment and cecal ligation and puncture (CLP)
Mice were anesthetized with isofluorane and a midline abdominal incision
was made. The cecum was mobilized, ligated below the ileocecal valve,
and punctured twice with a 22 gauge needle to induce polymicrobial
peritonitis. Sham-operated mice underwent the same procedure, including
opening the peritoneum and exposing the bowel, but without ligation and
needle perforation of the cecum. After surgery, the mice were injected
with 1 mL physiologic saline solution for fluid resuscitation and 20ug
butorphanol for post-operative analgesia. All mice had unlimited access
to food and water both pre- and post-operatively, allowed to die
spontaneously from sepsis. According to the literature and our previous
research, 5 mg/kg CD28 functional antibody was administered
intraperitoneally at the same time[21]. Mice were scored with murine
sepsis severity scale and survival rate by two independent, blinded
researchers.