The lungs were then kept in 100% ethanol for 24 h at 4 °C (Nagase et al., 1996). After fixation, tissue blocks were embedded in paraffin and 4-μm thick slices were cut and mounted. Slides were stained with hematoxylin–eosin. Morphometric analysis was done with an integrating eyepiece with a coherent system made of a 100-point grid consisting of 50 lines,
coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany). The volume fraction of collapsed and normal pulmonary areas and the fraction of the lung occupied by large-volume gas-exchanging air spaces (wider than 120 μm) were determined by the point-counting technique (Gundersen et al., 1988 and Weibel, 1990) at a magnification of 200× across 10 OSI-906 cost random, non-coincident microscopic fields. Points falling on collapsed, normal or hyperinflated alveoli were counted and divided by the total number of points hitting alveoli in each microscopic field. Polymorpho- (PMN) and mononuclear (MN) cells were counted at 1000× magnification, and divided by the total number of points falling on tissue area in each microscopic field. Thus, data are reported as the fractional area of pulmonary tissue. Lung parenchyma strips (3 mm × 3 mm × 10 mm) were longitudinally cut from right lungs. Pleural tissue was removed, and the strips were stored in liquid nitrogen for analysis of type-III procollagen (PCIII)
mRNA expression. Total RNA was isolated from the
frozen lung tissue (Chomczynsky and Sacchi, 1987). The relative expression of type-III procollagen mRNA (PCIII mRNA) was Sorafenib ic50 obtained by semi-quantitative reverse-transcription and polymerase chain reaction (RT-PCR). In the PCIII mRNA detection by RT-PCR, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as internal positive control. The semi-quantitative method Pyruvate dehydrogenase lipoamide kinase isozyme 1 of RT-PCR, used to quantify the PCIII mRNA expression in the experimental rat lung, was validated in preliminary experiments (Garcia et al., 2004 and Farias et al., 2005). All reactions included a negative control RT (-). The identity of the amplification was confirmed by determination of the molecular size on agarose gel electrophoresis with 100 bp DNA molecular markers (Gibco BRL, Grand Island, NY, USA). SigmaPlot 11 software package (SYSTAT, Chicago, IL, USA) was used. To evaluate the consequences of mechanical ventilation, ventilated groups were compared to Non-Vent. In order to analyze the effects of PEEP during OLV with low VT, comparisons between V5P2 and V5P5 were done, while the effects of high VT during OLV with physiological PEEP were assessed by comparisons between V5P2 and V10P2. The normality of the data (Kolmogorov–Smirnov test with Lilliefors’ correction) and the homogeneity of variances (Levene median test) were tested. When both conditions were satisfied one-way ANOVA test followed by Dunnett’s test and Student t-test were used.