Transient micro-elastography: A novel non-invasive approach to measure liver stiffness in mice

Abstract

Aim: To develop and validate a transient micro-elastography device to measure liver stiffness (LS) in mice.

Methods: A novel transient micro-elastography (TME) device, dedicated to LS measurements in mice with a range of measurement from 1-170 kPa, was developed using an optimized vibration frequency of 300 Hz and a 2 mm piston. The novel probe was validated in a classical fibrosis model (CCl(4)) and in a transgenic murine model of systemic amyloidosis.

Results: TME could be successfully performed in control mice below the xiphoid cartilage, with a mean LS of 4.4 ± 1.3 kPa, a mean success rate of 88%, and an excellent intra-observer agreement (0.98). Treatment with CCl(4) over seven weeks drastically increased LS as compared to controls (18.2 ± 3.7 kPa vs 3.6 ± 1.2 kPa). Moreover, fibrosis stage was highly correlated with LS (Spearman coefficient = 0.88, P < 0.01). In the amyloidosis model, much higher LS values were obtained, reaching maximum values of > 150 kPa. LS significantly correlated with the amyloidosis index (0.93, P < 0.0001) and the plasma concentration of mutant hapoA-II (0.62, P < 0.005).

Conclusion: Here, we have established the first non-invasive approach to measure LS in mice, and have successfully validated it in two murine models of high LS.

Keywords: Amyloidosis; Fibrosis; Liver; Liver stiffness; Mice; Micro-elastography; Transient elastography; Ultrasound.

Figure 1

Representative livers from normal control mice (A), and mice with stage 3 amyloidosis (B).

Figure 2

Amplitude of the strains induced in the liver of a mouse with systemic amyloidosis (stage 1), as a function of depth and time. The shear wave velocity (V_S) is the slope of the wave pattern. The steeper the slope, the higher the velocity of the shear wave and the higher the Young’s modulus (here E = 79.3 kPa).

Figure 3

Transducer (A) and experimental setup (B) used in transient micro-elastography.

Figure 4

Bar plot showing median liver stiffness in control (untreated vs oil-treated) and CCl₄-induced fibrosis. Liver stiffness is expressed in kPa. Whiskers indicate the extent of the data. The difference between the groups is significant (^aP ≤ 0.05, Mann-Whitney).

Figure 5

Histological characterization of amyloidosis mice. Light microscopy of a liver section from a 6-mo-old K mouse stained with Congo red (A) and the same section under polarized light showing green birefringence (B); Confocal microscopy of liver sections with immunolocalization of mutant hapoA-II using an anti hapoA-II antibody and CY2 as the secondary antibody (green fluorescence): Control mouse, 8 mo old (C); K mouse, 2 mo old (D); K mouse, 6 mo old (E); F mouse, 8 mo old (F).

Figure 6

Bar plot showing median liver stiffness as a function of amyloidosis severity. Liver stiffness is expressed in kPa, and amyloidosis severity is given according to the scoring system described in Materials and Methods. Whiskers indicate the extent of the data. The difference between index 0, 1, 2, and 3 is significant (^aP < 0.005 and ^bP < 0.01; Mann-Whitney).