Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice

Abstract

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.

Quantification of Lung Fibrosis in IPF-Like Mouse Model and Pharmacological Response to Treatment by Micro-Computed Tomography

“…The division in a frequency distribution of Ashcroft score classified as mild (0–3), moderate (=4) and severe (≥5) fibrotic lesions was applied at each time point ( Figure 1C ). This method of quantification allows to weight the contribution of each category within the final score ( Stellari et al., 2017 ).…”

“…For Ashcroft score analysis the whole lung parenchyma was considered for the analysis at the magnification of 20× after slide digitalization. To improve the Ashcroft score informativeness, the point score was subdivided into three major categories: mild (0–3), moderate (4) and severe (5–8); their frequency distribution was then analyzed ( Stellari et al., 2017 ). In the pharmacological experiment with Nintedanib, the left lobe was fixed and used for histological analysis and right lobes for the hydroxyproline quantification.…”

“…In our laboratory a mouse model of lung fibrosis induced by double BLM intratracheal instillation was recently used for primary screening showing some limitations such as a patchy fibrosis distribution through the lobes ( Ruscitti et al., 2017 ; Stellari et al., 2017 ).…”

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

“…Recently, non-invasive imaging technologies have been applied to monitor airway remodeling, inflammation, and fibrosis progression in murine models: Magnetic Resonance Imaging (MRI), Micro Computer Tomography (Micro-CT), Fluorescence Molecular Tomography (FMT) and Bioluminescent (BLI) 11,12,13,14,15,16,17,18,19,20,21 . We propose a non-invasive imaging approach to monitor longitudinally lung fibrosis progression by FMT and Micro-CT at different time-points after a bleomycin challenge 22 .…”

“…The role of MMPs has been investigated by injecting either vehicle or bleomycin treated mice with MMP activable fluorescent probes at selected time points. Twenty-four hours after injection, the mice were imaged by FMT revealing that fibrotic mice can activate the specific MMP fluorescent probe in vivo (Figures 6A-6D) 18 and ex vivo ( Figure 6E) 18 .…”