Label-free 3D characterization of cardiac fibrosis in muscular dystrophy using SHG imaging of cleared tissue

Abstract

Background information

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the gene encoding dystrophin. It leads to repeated cycles of muscle fiber necrosis and regeneration and progressive replacement of fibers by fibrotic and adipose tissue, with consequent muscle weakness and premature death. Fibrosis and, in particular, collagen accumulation are important pathological features of dystrophic muscle. A better understanding of the development of fibrosis is crucial to enable better management of DMD. Three-dimensional (3D) characterization of collagen organization by second harmonic generation (SHG) microscopy has already proven a highly informative means of studying the fibrotic network in tissue.

Results

Here, we combine for the first-time tissue clearing with SHG microscopy to characterize in depth the 3D cardiac fibrosis network from DMD^mdx rat model. Heart sections (1-mm-thick) from 1-year-old wild-type (WT) and DMD^mdx rats were cleared using the CUBIC protocol. SHG microscopy revealed significantly greater collagen deposition in DMD^mdx versus WT sections. Analyses revealed a specific pattern of SHG⁺ segmented objects in DMD^mdx cardiac muscle, characterized by a less elongated shape and increased density. Compared with the observed alignment of SHG⁺ collagen fibers in WT rats, profound fiber disorganization was observed in DMD^mdx rats, in which we observed two distinct SHG⁺ collagen fiber profiles, which may reflect two distinct stages of the fibrotic process in DMD.

Conclusion and significance

The current work highlights the interest to combine multiphoton SHG microscopy and tissue clearing for 3D fibrosis network characterization in label free organ. It could be a relevant tool to characterize the fibrotic tissue remodeling in relation to the disease progression and/or to evaluate the efficacy of therapeutic strategies in preclinical studies in DMD model or others fibrosis-related cardiomyopathies diseases.