Central slices of right lung tissue were cut, collected in cryotubes, quick-frozen by immersion in liquid nitrogen and stored at -80��C. Total RNA was extracted from Tofacitinib Citrate CP-690550 frozen tissue using the SV Total RNA Isolation System (Promega, Madison, WI, USA), following the manufacturer��s recommendations. RNA concentration was measured by spectrophotometry in a NanoDrop ND-1000 System (NanoDrop Products, Wilmington, DE, USA). First-strand cDNA was synthesized from total RNA using the GoTaq 2-Step RT-qPCR System (Promega). Relative mRNA levels were measured by SYBR Green�Cbased detection using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Samples were measured in triplicate.

For each sample, the expression of each gene was normalized to housekeeping gene expression (acidic ribosomal phosphoprotein P0, 36B4) using the 2-����Ct method, where ��Ct = Ct (reference gene) – Ct (target gene).Table 1Target gene primersaStatistical analysisData were tested for normal distribution (Kolmogorov-Smirnov test with Lilliefors correction) and homogeneity of variances (Levene median test). If both conditions were met, the effects of different ventilatory strategies on ALI groups were analyzed by means of one-way analysis of variance (ANOVA) followed by Tukey��s test. Otherwise, one-way ANOVA on ranks followed by Dunn��s post hoc test was employed. The significance level was always set at 5%. Parametric data were expressed as means �� SD, and nonparametric data were expressed as medians (IQR). All tests were performed using SigmaStat 3.1 software (Jandel Corp, San Raphael, CA, USA).

ResultsMean arterial pressure was higher than 70 mmHg throughout the experiments in both ALI groups, but was significantly lower during PCV than during BIVENT (Figure 4). In the ALIexp group, animals required additional fluid administration to keep MAP higher than 70 mmHg (P < 0.05).Figure 4Mean arterial pressure of animals with pulmonary and extrapulmonary acute respiratory distress syndrome. ALIexp: extrapulmonary acute lung injury; ALIp: pulmonary acute lung injury; BIVENT: biphasic positive airway pressure; MAP: mean arterial pressure; ...At baseline, the overall respiratory mechanics parameters were comparable among groups, except for PTP, which was lower for BIVENT-100 in the ALIp group (Table 2).

At the end of the experiment, minutes on ventilation, mean Vt and peak airway pressure were comparable in the PCV and BIVENT groups, regardless of ALI etiology. The RR of spontaneous breaths, minutes on ventilation, PTP/min and P0.1 during Plow were significantly increased for BIVENT-50 compared to BIVENT-75 and BIVENT-100. Inspiratory pressure and mean airway pressure were similar in all assisted ventilation modes (Table 3). However, with regard to spontaneous breaths Brefeldin_A during Plow, P0.1 was higher for BIVENT-50 than for BIVENT-75 and BIVENT-100.