Longitudinal micro-computed tomography-derived biomarkers quantify non-resolving lung fibrosis in a silicosis mouse model

Abstract

In spite of many compounds identified as antifibrotic in preclinical studies, pulmonary fibrosis remains a life-threatening condition for which highly effective treatment is still lacking. Towards improving the success-rate of bench-to-bedside translation, we investigated in vivo µCT-derived biomarkers to repeatedly quantify experimental silica-induced pulmonary fibrosis and assessed clinically relevant readouts up to several months after silicosis induction. Mice were oropharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. At weeks 1, 5, 9 and 15, we assessed lung function, inflammation and fibrosis in subsets of mice in a cross-sectional manner. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. The volume of healthy, aerated lung first dropped then increased, corresponding to an acute inflammatory response followed by recovery into lower elevated aerated lung volume. Imaging results were confirmed by a significantly decreased Tiffeneau index, increased neutrophilic inflammation, increased IL-13, MCP-1, MIP-2 and TNF-α concentration in bronchoalveolar lavage fluid, increased collagen content and fibrotic nodules. µCT-derived biomarkers enable longitudinal evaluation of early onset inflammation and non-resolving pulmonary fibrosis as well as lung volumes in a sensitive and non-invasive manner. This approach and model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.

Innate Lymphoid Cells Are Required to Induce Airway Hyperreactivity in a Murine Neutrophilic Asthma Model

“…Twenty-four hours after the last LPS challenge, micro-computed tomography (µCT) was performed to obtain in vivo lung scans of free-breathing mice (SkyScan 1278, Bruker µCT, Kontich, Belgium). Therefore, the mice were anesthetized with 1.5%-2% isoflurane in 100% oxygen and scanned according to a previously validated scan protocol (25). µCT scans were analysed with the software provided by the manufacturer (TSort, NRecon, DataViewer, and CTan) to respectively gate, reconstruct, visualize, and process µCT data as described previously (25)(26)(27).…”

“…Therefore, the mice were anesthetized with 1.5%-2% isoflurane in 100% oxygen and scanned according to a previously validated scan protocol (25). µCT scans were analysed with the software provided by the manufacturer (TSort, NRecon, DataViewer, and CTan) to respectively gate, reconstruct, visualize, and process µCT data as described previously (25)(26)(27).…”

Regional Quantification of Bleomycin-Induced Pulmonary Fibrosis in Mice by Microcomputed Tomography for the Assessment of the Therapeutic Responses of Antifibrotic Drugs

“…So, there must be a distribution pattern of in ammation and brosis in BLM model. In most of the studies which exhibited their representative images of BLM lungs in the papers, the micro-CT images from different labs showed that the pattern of distribution of the upper middle lung, peritrachea, and dorsal segment existed and was very common, and was repeated in many laboratories no matter how they administrated BLM buffer [13,14,[23][24][25][26][27][28]. The ATS documents also showed that the same lung should be consistently used for this assay to allow comparison between mice and groups if the brotic stimulus was deposited with excellent reproducibility [10].…”

The importance of routine quality control for reproducible pulmonary measurements by in vivo micro-CT

“…Histogram-based analyses are used to discriminate between aeration levels, identifying variations in lung density to allow accurate longitudinal assessment of lung disease progression 8 , 9 in compliance with the 3R rules (Replacement, Reduction, Refinement). Moreover, the integration of micro-CT technology in antifibrotic drug discovery can be useful for investigating drug efficacy in preclinical settings 10 – 13 .…”