Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis

Abstract

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations.

Bleomycin Revisited: A Direct Comparison of the Intratracheal Micro-Spraying and the Oropharyngeal Aspiration Routes of Bleomycin Administration in Mice

“…OA administration exerted similar to IT administration functional impairment on respiratory mechanics compared to saline-treated controls, as assessed by significant reductions in: (1) static lung compliance ( Cst ), (2) respiratory system compliance ( Crs ), and (3) total lung capacity ( A ), as well as increases in: (4) respiratory system elastance ( Ers ), (5) tissue elastance ( H ), (6) curvature of the upper portion of the deflation limb of the PV curve ( K ), (Figure 1D ). Both static and dynamic lung compliance as well as elastance and total (inspiratory) lung capacity were found to be reliable indices of fibrotic lung injury, as recently suggested ( 18 ), well correlating with the Ashcroft score (Table S1 ). Therefore, and as the method is the most relevant to clinical measurements in human patients, not requiring additional mouse numbers, it is thus proposed as a valuable surrogate analysis of BLM-induced pulmonary fibrosis.…”

Oral Administration of Chitosan Attenuates Bleomycin-induced Pulmonary Fibrosis in Rats

“…It has been reported that BLM administered intratracheally to induce PF can reduce body weight while increasing lung weight, although these changes can be improved with suitable medication (13). Lung weight increases in proportion to the dose of intratracheally administered BLM, and the increased lung weight is associated with histopathological fibrosis scores (14).…”

CSF1R inhibition prevents radiation pulmonary fibrosis by depletion of interstitial macrophages

“…Histopathological examination of the lungs performed 20 weeks post-irradiation showed collagen deposition, as detected by the saffron (yellow) staining (figure 2e) and sirius red staining (online supplementary figure S1a) in irradiated lungs. Quantitative assessment of parenchymal fibrosis using automated histological image analysis software [18] showed a significant FIGURE 3 Inflammatory cell characterisation during thoracic radiation-induced injury. a) Fold increase of number of interstitial neutrophils within parenchyma during radiation injury in nonirradiated and irradiated C57BL/6 mice; b) gating strategy to identify neutrophils (Neutr; Gr1 + CD11b + ) and monocytes (Mono; Gr1 inter CD11b + ) within parenchyma of nonirradiated and irradiated mice 20 weeks after irradiation; c) fold increase of neutrophil number in bronchoalveolar lavage (BAL) during radiation injury in irradiated and nonirradiated mice; d) gating strategy to identify neutrophil (Gr1 high CD11b + ) and monocytes (Gr1 + CD11b + ) in BAL of nonirradiated and irradiated mice 20 weeks after irradiation; e) fold increase of number of interstitial monocytes within parenchyma during radiation injury in irradiated and nonirradiated mice; f) fold increase of monocyte number in BAL during radiation injury in irradiated and non-irradiated mice; g) fold increase in number of alveolar macrophages (AMs) during radiation injury in irradiated and nonirradiated mice; h) gating strategy to identify AMs (CD11c + CD11b − ) in nonirradiated and irradiated mice 20 weeks after radiation; i) fold increase of number of tissue-infiltrating macrophages (IMs) during radiation injury in irradiated and nonirradiated mice; j) gating strategy to identify IMs (F4/80 + Gr1 − ) in nonirradiated and irradiated mice 20 weeks after irradiation.…”