Mouse models of sepsis

Lipopolysaccharide (LPS)-induced endotoxemia and Cecal Ligation and Puncture (CLP) were performed according to Animal Resource Protocol approved by the Committee at the University of Rochester (UCAR 2008-039R). For endotoxemia assays, 8–12-week-old C57BL/6J, Ly6G^Cre/+:C1qa^+/+ (WT), or Ly6G^Cre/+:C1qa^fl/fl (C1q cKO) male mice were weighed in order to deliver equal 12.5 mg/kg dose of LPS (E. coli O127:B8, Sigma-Aldrich) diluted in PBS via intraperitoneal (IP) injection. Animals were subsequently weighed daily for up to 7 days and closely monitored where in the event that percentage body weight loss exceeded 25% mice were euthanized via IACUC small rodent euthanasia protocols. For CLP, procedures were administrated following Rittirsch et al. (51). Briefly, mice were anesthetized with an IP injection of xylazine/ketamine cocktail (dose range of 80-100 mg/kg). Secondary anesthesia was maintained with Isoflurane/0₂ gas mixture throughout the entire surgical procedure. Standard aseptic techniques were used to prepare the incision site. A vertical incision in the abdomen left lower quadrant was used to cut skin and peritoneum in order to access the peritoneal cavity. The cecum was ligated with silk sutures and punctured through with a 21-gauge needle, with sham mice receiving no ligation or puncture. Peritoneum and skin were approximated and closed using standard small animal surgical metal clips. Lidocaine was used topically on the wound site after stapling. Mice were resuscitated with 1 ml Ringers lactate injected subcutaneously. Animals were subsequently weighed daily for up to 7 days and given daily topical doses of Lidocaine until sacrifice to monitor disease progression and recovery. For antibody-mediated C1q neutralization in vivo, mice were pretreated with 10 μg of anti-C1q (clone JL-1, Hycult Biotech) or Rat IgG2b Isotype control (Abcam) in 100 μl of PBS via IP injection 1h prior to sepsis induction. For C1q treatment, mice were previously weighed and received single IP injection of LPS with or without treatment of 20 μg Native Human C1q protein (Abcam) administered as follows: 1 mg of C1q was reconstituted in 1 ml of distilled water with each animal receiving a single IP injection of 100 μl C1q/PBS (20 μl C1q + 80 μl PBS) at three separate time points. Dose 1 was given 30 min prior to LPS injection, dose 2 was given 6h after LPS injection and dose 3 was given 24h after LPS injection. Similar experimental processes were used for C3a (CompTech) and C5a (Sino Biological).