Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy.
The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.
Pena
Ana-Maria
AM
Laboratory for Optics and Biosciences, Ecole Polytechnique, 91128 Palaiseau, France.
Fabre
Aurélie
A
Débarre
Delphine
D
Marchal-Somme
Joëlle
J
Crestani
Bruno
B
Martin
Jean-Louis
JL
Beaurepaire
Emmanuel
E
Schanne-Klein
Marie-Claire
MC
D000818:Animals; D001761:Bleomycin; D003094:Collagen / Q000648:ultrastructure*; D005109:Extracellular Matrix / Q000648:ultrastructure*; D006801:Humans; D008168:Lung / Q000473:pathology*; D016676:Macrophages, Alveolar / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D010994:Pleura / Q000473:pathology; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000473:pathology*
D001761:Bleomycin; D003094:Collagen