Our project aims at:
- Quantifying collagen fibers as a predictive marker of the evolution and reversibility of liver fibrosis,
- Determining the organization (orientation / crosslinking) of fibrotic collagen in order to define structural signatures, predictive of pathological and tumor developments.
The method chosen is second harmonic generation (SHG) microscopy, which we developed in collaboration with the physicists (IPR, UMR 6226 CNRS-University of Rennes1). It is a reliable, standardized and reproducible method for the precise quantification of fibrillar collagen characteristic of pathological liver fibrosis (SHG Fibrosis Index). The main advantage of this method is that the SHG signal is intrinsically emitted by the endogenous collagen fibers and does not require any addition of extrinsic dyes. The principle of SHG microscopy is based on the use and development of infrared (IR) lasers that excite molecules through non-linear processes. In addition, analyzes of the collagen network can be performed with polarized light (P-SHG), to precisely determine the 3D organization (orientation / crosslinking) of collagen fibers.
We have demonstrated the interest and power of SHG microscopy for the histopathological evaluation of hepatic fibrosis in humans (SHG Fibrosis Index) and validated this method with respect to the Metavir classification and Ultrasound Elastometry (Gailhouste et al 2010 ).
Collagen detection by second-harmonic generation (SHG) microscopy
The quantification of fibrillar collagen and its scoring by SHG makes it possible to precisely follow the evolution of liver fibrosis (regression or aggravation) and thus be an aid to the diagnosis and validation of experimental therapeutic protocols, in addition to the Metavir score.
We also studied, by P-SHG microscopy, the remodeling of the collagen of the extracellular matrix during hepatic fibrosis through the experimental model of CCl4-treated mice (Rouède et al 2017). Our study shows:
- an expansion of collagen fibers around intrahepatic vessels, which are the site of the initiation of liver fibrosis,
- that this perivascular fibrosis is characterized by remodeling of the fibers resulting in reduction of their disorder.
Measurement of Collagen structural parameters by Polarization dependence second harmonic generation (P-SHG) microscopy
We conclude that the analysis of P-SHG images is a powerful method for estimating the remodeling and organization of fibrillar collagen in situ in physiological and pathological tissues.