Jean-Claude Gilhodes1, Yvon JuleÂ1*, Sebastian Kreuz2, Birgit Stierstorfer2, Detlef Stiller2,
Lutz Wollin2
1 Biocellvia, Marseille, France, 2 Immunology and Respiratory, Boehringer Ingelheim Pharma GmbH & Co.
KG, Biberach, Germany
* yvon.jule@biocellvia.com
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.
