Quantitative Evaluation of Pulmonary Fibrosis

The total sets of lung slices used for the above standard fibrosis evaluation were scanned at x20 magnification using a NanoZoomer-SQ and digital images of entire lung sections were captured using the NDP.view 2 software (both from Hamamatsu Corporation, Hamamatsu, Japan). To focus the analysis on alveolar parenchyma the walls of large bronchi located in the vicinity of the lung lobes, small bronchi and vessels (diameter >200 μm) associated with alveolar parenchyma as well as their surrounding collagen fibers were manually deleted using Gimp 2.8 software (Free Software Foundation Inc.). Digital images were then reduced from x20 to x2.5 magnification with a pixel size of 3.632 μm allowing both a high-resolution visualization of morphological structure of pulmonary tissue and a very short processing time for the software analysis (<1 sec per entire lung section). The quantification of the BLM-induced fibrotic alterations has been assessed on the basis of pulmonary tissue density. For this purpose Biocellvia has developed a proprietary software program allowing the determination of pulmonary tissue density from thousands of micro-tiles (30–56 μm²) crisscrossing the selected pulmonary tissue of entire lung sections. The density of the pulmonary tissue was evaluated for all individual micro-tiles corresponding to the ratio of the area of the lung tissue inside the micro-tile and the total area of the micro-tile. To quantify the distribution of pulmonary tissue densities they were graded in 20 classes of increasing values in increments of 0.05 (Fig 1). The frequency of tissue density was calculated dividing the number of tissue density values in a given class by the total number of density values in all 20 classes. A distribution of the frequency of tissue density according to their classification was then determined for a comparison between saline-treated control lungs and BLM-treated lungs. To visualize the distribution of density values 2D-reconstructed images of lung sections were composed by assigning pseudocolors to tissue density values according to their classification (Fig 1). These 2D-reconstructions were compared with the Masson trichrome-stained slices.
Two pulmonary tissue density indexes were defined with the aim to quantify and compare pulmonary fibrotic alterations in saline-treated control lungs and BLM-treated groups: (i) the mean tissue density (Dm) and (ii) the high tissue density frequency (HDFm). Dm corresponds to the mean density value of alveolar parenchyma determined from individual density values of the micro-tiles. Dm can be expressed per lung section, per animal or per group. HDFm was established to specifically quantify fibrotic alterations in alveolar parenchyma. HDFm corresponds to the sum of frequencies of high tissue densities limited to fibrotic alterations. The range of classes allocated to HDFm was established from the saline-treated control group wherein fibrotic alterations were missing and for which high tissue densities frequency per class were less than 1%. In the present study HDFm corresponded to the sum of frequencies ranging from class 12 to 20 (Fig 1).