Molecular MR imaging of liver fibrosis: a feasibility study using rat and mouse models

Abstract

Background & aims: Liver biopsy, the current clinical gold standard for fibrosis assessment, is invasive and has sampling errors, and is not optimal for screening, monitoring, or clinical decision-making. Fibrosis is characterized by excessive accumulation of extracellular matrix proteins including type I collagen. We hypothesize that molecular magnetic resonance imaging (MRI) with a probe targeted to type I collagen could provide a direct and non-invasive method of fibrosis assessment.

Methods: Liver fibrosis was induced in rats with diethylnitrosamine and in mice with carbon tetrachloride. Animals were imaged prior to and immediately following i.v. administration of either collagen-targeted probe EP-3533 or non-targeted control Gd-DTPA. Magnetic resonance (MR) signal washout characteristics were evaluated from T1 maps and T1-weighted images. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline.

Results: EP-3533-enhanced MR showed greater signal intensity on delayed imaging (normalized signal enhancement mice: control=0.39 ± 0.04, fibrotic=0.55 ± 0.03, p<0.01) and slower signal washout in the fibrotic liver compared to controls (liver t(1/2)=51.3 ± 3.6 vs. 42.0 ± 2.5 min, p<0.05 and 54.5 ± 1.9 vs. 44.1 ± 2.9 min, p<0.01 for fibrotic vs. controls in rat and mouse models, respectively). Gd-DTPA-enhanced MR could not distinguish fibrotic from control animals. EP-3533 gadolinium concentration in the liver showed strong positive correlations with hydroxyproline levels (r=0.74 (rats), r=0.77 (mice)) and with Ishak scoring (r=0.84 (rats), r=0.79 (mice)).

Conclusions: Molecular MRI of liver fibrosis with a collagen-specific probe identifies fibrotic tissue in two rodent models of disease.

Fig. 1. Characterization of fibrosis and collagen deposition in the rat DEN model

(A) Masson’s trichrome staining reveals numerous portal fibrotic expansions in the DEN sample corresponding to advanced fibrosis, Ishak 5 in this example. There is no detectable fibrosis in the control sample. (B) Immunostaining for type I collagen in liver shows rich staining in the DEN treated animals but is absent in the controls. (C) Expression of liver α1(I) procollagen mRNA was 15-fold higher (* p <0.05) in the DEN treated group compared to controls. (D) Total liver collagen (as hydroxyproline) is 2.5-fold higher in DEN treated animals compared to controls (* p <0.05). Box plots in Panels (C) and (D) illustrate median (line inside box), interquartile range (box), and minimal and maximal (lines extending above and below box) values.

Fig. 2. MR imaging of liver fibrosis in the rat DEN model

(A) Inversion recovery MRI (TR/TE/TI=3200ms/4ms/507ms) of rat liver before and 40 min after injection of 20 μmol/kg EP-3533; S = stomach, L = liver. The inversion time was chosen to minimize liver signal prior to probe injection. Post injection there is higher signal in the liver of the DEN treated animal compared to the control. Differences in baseline stomach signal are due to the absence/presence of chow which shortens stomach T1 [42]. (B) EP-3533 was retained longer in fibrotic livers than in controls (* p <0.05), while (C) the half-life of Gd-DTPA in the liver did not statistically differ between DEN and control groups. (D) Ex vivo analysis of EP-3533 (as gadolinium) at 120 min post injection showed 70% higher gadolinium levels in the DEN group compared to controls (** p <0.01).

Fig. 3. Dynamic MR enhancement of the liver in CCl4 treated and control mice

Dynamic MRI (TR/TE/flip angle=50ms/1.92ms/35°) after injection of either collagen-binding EP-3533 or non-specific Gd-DTPA probe normalized to peak signal enhancement. (A, C) Expansion of first 4.5 min after injection showing that with EP-3533 (A), the peak enhancement appears later and the wash-out is slower in the CCl4 group compared to control, but Gd-DTPA (C) shows no noticeable difference between the control and fibrotic groups. (B, D) Complete signal enhancement profiles out to 60 min post injection. EP-3533 (B) shows slower liver wash-out from CCl4 animals compared to controls resulting into higher relative signal enhancement at later time points. Gd-DTPA (D) does not show significant differences between the two groups.

Fig. 4. Correlations between MRI and ex vivo analyses

Open squares: controls, filled squares: CCl4 treated animals. (A) Gadolinium in liver at 80 min after EP-3533 injection correlates linearly with relaxation rate enhancement (ΔR1) observed at 60 min. (B) Gadolinium (EP-3533) liver:blood ratios correlate with hydroxyproline (total collagen), showing an association of the probe with increased collagen levels. (C) Half-life of EP-3533 in the liver as determined by MRI correlates with total collagen, showing the feasibility of molecular MR imaging to detect fibrosis. (D) Increasing stage of fibrosis (Ishak score) is well reflected in the increase of hydroxyproline concentration in tissue. (E) Probe uptake into liver (gadolinium liver:blood) increases with increasing stage of fibrosis.