All the animal protocols were approved by the Animal Care Committee of University of Washington and the VA Puget Sound Healthcare System. Male C57BL/6 mice weighing 25 to 30 g received intraperitoneal injections of either PBS or 1 μg/g of
E. coli LPS, O111:B6 (Sigma Chemical Co, St Louis, MO, USA). Immediately afterwards, the mice were treated with 1 ml subcutaneous of lactated Ringer's solution for fluid replacement. The mice were returned to their cages with free access to water and food. After 14 hours, the mice were anesthetized with inhaled isoflurane. The larynx was revealed and the trachea was intubated orally with an 18-gauge Vialon
® angiocath (BD, Franklin Lakes, NJ, USA). Placement of the catheter in the trachea was verified by detecting the movement of a 100 μl bubble of water located inside a syringe connected to the catheter, and by measurement of the mixed expired CO
2 (MECO
2) with a capnograph (Novametrics Medical Systems Inc, Wallingford, CT, USA). Once intratracheal intubation had been confirmed, the animal was mechanically ventilated with a rodent ventilator Type 845 (Mini-Vent, Cambridge, MA, USA) with the following settings: tidal volume, 10 ml/kg; respiratory rate, 150 breaths/minute; fraction of inspired oxygen, 0.21; and positive end-expiratory pressure, 0. Airway pressures, rectal temperature, and MECO
2 were monitored continuously. In preliminary studies this ventilation strategy produced normal arterial blood pH values (7.36 ± 0.08,
n = 4). The respiratory rate was adjusted to maintain the MECO
2 between 10 and 15 Torr. The body temperature was maintained between 37 and 38°C with external heating. At one hour after the onset of mechanical ventilation, the mice received an initial subcutaneous fluid bolus of 0.15 ml of a 1:1 mixture of 5% dextrose and lactated Ringer's. Additional subcutaneous boluses of 0.15 ml were administered every 30 minutes. The mice were ventilated for six hours, and then killed with pentobarbital (120 mg/kg intraperitoneally). The mice were exsanguinated by direct cardiac puncture, the thorax was opened, and the left lung was removed and placed in 1 ml of protease inhibitor solution (Complete™; Roche Applied Science, Indianapolis, IN, USA). The right lung was lavaged with PBS, removed from the thorax, suspended from the fixation apparatus, and fixed with 4% paraformaldehyde at a constant pressure of 15 cmH
2O [
17]. The abdomen was incised, and one lobe of the liver and the right kidney were removed. The capsule of the kidney was pierced several times and the tissues were placed in 4% paraformaldehyde.