The animals were then paralyzed (vecuronium bromide 2 mg/kg, intravenously) and mechanically ventilated (Servo i, MAQUET, Switzerland) with the following parameters: VT = 6 ml/kg, respiratory rate (RR) = 80 breaths/min, inspiratory to expiratory ratio = 1:2, fraction of inspired oxygen (FiO2) = 1.0, and PEEP equal selleckchem to 0 cmH2O (zero end-expiratory pressure (ZEEP)). Blood (300 ��l) was drawn into a heparinized syringe for measurement of arterial oxygen partial pressure (PaO2), arterial carbon dioxide partial pressure (PaCO2) and arterial pH (pHa) (i-STAT, Abbott Laboratories, North Chicago, IL, USA) (BASELINE-ZEEP). Afterwards, mechanical ventilation was set according to the following parameters: VT = 6 ml/kg, RR = 80 bpm, PEEP = 5 cmH2O, and FiO2 = 0.3 (Figure (Figure1).1).

Est,L was then measured (BASELINE) and the animals were randomly assigned to one of the following groups: 1) hypovolemia (HYPO); 2) normovolemia (NORMO), and 3) hypervolemia (HYPER). Hypovolemia was induced by blood drainage in order to achieve a MAP of about 70 mmHg. Normovolemia was maintained at a MAP of about 100 mmHg. Hypervolemia was obtained with colloid administration (Gelafundin?; B. Braun, Melsungen, Germany) at an infusion rate of 2 ml/kg/min to achieve a MAP of about 130 mmHg. Following that, the colloid infusion rate was reduced to 1 ml/kg/min in order to maintain a constant MAP. Depth of anesthesia was similar in all animals and a comparable amount of sedative and anesthetic drugs were given in all groups.

After achieving volemic status, animals were further randomized to be recruited, with a single RM consisting of continuous positive airway pressure (CPAP) of 40 cmH2O for 40 seconds (RM-CPAP), or not (NR) (n = 6 per group; Figure Figure1).1). After one hour of mechanical ventilation (END), Est,L was measured. FiO2 was then increased to 1.0, and after five minutes arterial blood gases were analyzed (END). Finally, the animals were euthanized and lungs, kidney, liver Brefeldin_A and small intestine were prepared for histology. IL-6, IL-1��, caspase-3, and PCIII mRNA expressions were measured in lung tissue. The experiments took no longer than 80 minutes.Figure 1Timeline representation of the experimental protocol. CLP, cecal ligation and puncture; I:E, inspiratory-to-expiratory ratio; PEEP, positive end-expiratory pressure; RR, respiratory rate; RT-PCR, real time-polymerase chain reaction; VT, tidal volume; …Respiratory parametersAirflow, airway and esophageal pressures were measured [9,21]. Changes in esophageal pressure, which reflect chest wall pressure, were measured with a water-filled catheter (PE205) with side holes at the tip connected to a SCIREQ differential pressure transducer (SC-24, Montreal, Canada).