Tidal volume (VT) was 7 mL/kg and respiratory frequency (f) was twelve breaths per minute. A five-centimeter H2O PEEP was maintained and 10,000 U of Heparin i.v. (Sanofi-Aventis, Ploërmel, France) was administered. The pigs were killed using pentobarbital i.v. (Chemische Fabrik, Berg, Germany) (25 mg/kg) and potassium chloride i.v. (5 g). Pneumoplegia was performed by infusing 1 L of the preservation fluid

Perfadex® (Vitrolife AB, Gothenburg, Sweden) at 4°C in the right ventricule. Perfadex® was buffered with Trometamol (Addex-THAM, Kabi, Sweden). Finally, the lungs were extracted and stored in a cold room at 4°C for 30 minutes. The usefulness of EVLP Cobimetinib mw is well known and described in literature [7, 12, 17, 43]. Many parameters of our ex vivo preparation was performed in a “state of the art” EVLP setting and published by research teams that are experts in the field [36]. In our experiments, our purpose was not to demonstrate or suggest an evolution of the EVLP technique, but rather to use such experimental preparations to evaluate the benefit of the CsA to reduce IRI. The ex CP-673451 mouse vivo lung function assessment system was primed with 2.8 L of Perfadex® added with 5% of bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), and 2 mg/L of Trinitrine (Sanofi-Aventis). The pulmonary artery was cannulated with a 20-F cannula (Turemo,

Ann Arbor, MI, USA) connected to the extracorporeal circuit. A pressure probe (Baxter, Uden, Holland) was first placed into the pulmonary artery, then a temperature probe (Sorin Group, Arvada, CO, USA) was connected to the membrane oxygenator; finally, a second temperature probe (Integral Process, Conflans Sainte Honorine, France) was placed at the pulmonary vein exit. During the rewarming

phase, 2 L/min of oxygen and 2 L/min of nitrogen (93%) mixed with carbon dioxide (7%) were carried to the membrane oxygenator. Isotonic trometamol was used to obtain a physiologic pH in the mixed solution. The rewarming of the lung preparations was initiated by a slow infusion (100 mL/min) at 25°C. The peristaltic pump flow was gradually increased along with the temperature of the perfusion fluid. At 32°C, ventilation was started (VT = 50 mL, f = 12/min, find more PEEP = 5 cmH2O, FiO2 = 50%) and then gradually increased by increments of 20 mL up to a maximal VT of 7 mL/kg. During this rewarming phase, the pump flow was progressively increased up to 1.3 L/min (normal cardiac output for a 20 kg pig). However, PAP was never allowed to exceed 25 mmHg. The pump flow was fixed with a lower pressure less than 25 mmHg in order to preserve the integrity of the capillary-alveolar membrane. The rewarming phase was considered complete when the temperature of the solution from the pulmonary veins reached 36°C, while full cardiac output and ventilation were also obtained.