Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Abstract

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Dynamics of compartment-specific proteomic landscapes of hepatotoxic and cholestatic models of liver fibrosis

“…As variations in ECM composition and content also substantially affect the biomechanics of liver tissue, we decided to examine correlation between our proteomic results and mapping of dynamic changes in the biomechanical properties of CCl 4 -treated livers. We used atomic force microscopy (AFM) combined with polarized microscopy 17 to precisely locate and probe the following compartments: 1) regions of collagen-rich scar tissue in close vicinity of the central vein, 2) injured hepatocyte regions next to the collagen scar, and 3) regions of hepatocytes on the interface between injured and not visibly damaged hepatocytes (so-called interface hepatocytes 36 ) (Figure 7B). In parallel, we also measured stiffness of corresponding regions in nontreated control liver sections (Figure 7C).…”

Regulators, functions, and mechanotransduction pathways of matrix stiffness in hepatic disease

“…There is an equation between elastic modulus and shear modulus, and the elastic modulus is approximately three times the shear modulus for isotropic and incompressible materials. Several mechanical test methods have been used to measure the matrix stiffness, including elastography and atomic force microscopy (AFM) methodology ( Sigrist et al, 2017 ; Ojha et al, 2022 ).…”

“…One of the major drawbacks of common liver stiffness measurements including USE and MRE is that they do not provide cellular-level resolution of stiffness heterogeneity in the liver ( Ojha et al, 2022 ). During the liver disease development, areas containing rich collagens show higher stiffness compared to the surrounding parenchyma ( Calò et al, 2020 ).…”

Analysis of biomechanical properties of mouse skin dermis through atomic force microscopy: Application to demonstrate a sexual dimorphism

“…29 We then performed nanomechanical measurements on tissue slices cut parallel to the skin surface at different depths, for three male mice (Figure 2B). For these horizontal sections, each In pathological conditions involving ECM remodelling, such as in tumour microenvironment 30 or tissue fibrosis, 31 the stiffness of the stroma has often been correlated with collagen density and matrix deposition. To determine whether a higher Young's modulus in male dermis was associated with increased matrix synthesis, we first performed a quantitative analysis of the transcripts encoding (i) the main collagenous components of the dermis, that is types I (Col1a1), III (Col3a1) and V (Col5a1) collagens, (ii) the major components of the elastic fibres, that is Elastin (Eln) and Fibrillin-1 (Fbn1), and (iii) some regulators of collagen fibril spacing, that is the Decorin (Dcn)…”