Non-invasive imaging evaluation of liver fibrosis and liver function is of great importance at present, where great efforts have been made to develop new drug for liver fibrosis and chronic liver disease. In this regard, we aim to validate SWE and gadoxetate-enhanced DCE-MRI in an animal model of liver fibrosis. Liver fibrosis was induced in SD rats by intraperitoneal administration of thioacetamide for 8 weeks: 200 mg/kg for high dose group and 150 mg/kg for low dose group.
In the ROC analysis to diagnose liver fibrosis (i.e., high-dose group and low-dose group), the diagnostic accuracy of RLE-3, iAUC-3, iAUC-15, and Emax was 100% (AUROC 1,000) with complete differentiation. between the liver fibrosis groups and the control group. Theoretically, the ultrasound SWE reflects liver fibrosis and the gadoxetate-enhanced DCE-MRI reflects liver function. In our study using the animal liver fibrosis model, the ultrasonographic SWE and gadoxetate-enhanced DCE-MRI are quite feasible to non-invasively evaluate both histopathological liver fibrosis and physiological liver function in preclinical research.
There have been major efforts to develop a new drug for liver fibrosis and chronic hepatitis in recent decades. These liver fibrosis and liver function imaging are mainly studied in clinical research. In this regard, the use of gadexetate-enhanced SWE and DCE-MRI technique in preclinical trial may be a very powerful tool, as long as technical feasibility is proven and repeatability is ensured.
From this perspective, we conducted an animal study to evaluate biomarker validation in the rat liver fibrosis model.
Materials and Methods
Measurements were acquired by a single operator (Y.C.C), a radiographer with 5 years of experience in animal CT and ultrasound imaging. Under isoflurane anesthesia, the rats were placed in a supine position with the two forelimbs abducted. The operator measured SWE using a corresponding depth of 1 cm from the liver surface in all animals.
Among the measurements, we chose four measurements in the middle, according to the WUFUMB guidelines. In all rats, SWE examinations were performed in two measurement sessions with 3-day intervals to assess test-retest reproducibility of SWE. Regions of interest (ROIs) are placed in the region with homogeneous color in the elasticity map (a) and the velocity map (b). a) Liver stiffness map showing the distribution of liver stiffness in kPa in the liver.
An en-bloc liver resection was performed and the liver was cut in cross-section at 5-mm intervals. To evaluate the extent of liver fibrosis, Masson's trichrome staining was performed with a commercially available kit (Sigma-Aldrich Korea, Seoul, Korea). In Masson's trichrome staining, cytoplasm and muscle fibers are stained red, while collagen is stained blue (16, 17).
To evaluate the liver function, the ICG retention rate at 15 minutes (ICG-R15) test was performed, which is the most used method in clinical practice (18). The ICG (Daiichi Sankyo, Tokyo, JP) was dissolved in normal saline to a final concentration of 2.5 mg/ml. The ICG solution with a concentration of 2.5 mg/Kg body weight was injected through the tail vein.
Blood sample was obtained 15 minutes after ICG injection and mixed with ethylenediaminetetraacetic acid (EDTA) 20 μl. In addition, the test-retest repeatability was also evaluated using Bland-Altman plots with the mean relative difference and 95% limit of agreement (LOA) (23). The 95% LOA is the range of the mean relative difference ± 1.96SDs of the mean relative difference.
Results
The SI of the liver in the gadoxetate-enhanced MRI is generally the highest in the control group, followed by low-dose group and high-dose group (Figure 6). Indeed, all the MRI indices of liver function differed significantly between groups (p<0.001, One-way ANOVA), indicating that the low value of MRI indices indicates liver fibrosis and low liver function. Of these, iAUC-15 may be the best semi-quantitative parameter to evaluate both liver function and histopathological liver fibrosis.
In our preclinical study using liver fibrosis animal model, we showed that the ultrasound SWE and gadoxetate-enhanced DCE-MRI are quite feasible to evaluate histopathological liver fibrosis and physiological liver function non-invasively. Among the imaging parameters of SWE and DCE-MRI, iAUC-15 may be the best index to evaluate both histopathological liver fibrosis and physiological liver function based on the highest correlation coefficients (r = -0.81 and -0.65, respectively). The need to monitor the degree of liver fibrosis in a non-invasive and repeatable way is increasing in the preclinical trial.
On the contrary, MRI has advantages that the accurate functional evaluation of liver function as well as detailed anatomical evaluation is possible. Therefore, the quantitative liver function evaluation is based on the degree of reduction of liver parenchymal enhancement on gadoxetate-enhanced MRI (12). The liver function estimation using gadoxetate-enhanced MR can be categorized into three methods: (1) measurement of liver parenchymal SI on hepatobiliary phase (aka RLE method), (2) MR relaxometry such as T1 map or T2* map, and ( 3 ) DCE-MRI for use in pharmacokinetic modeling (32-35).
Since DCE-MRI techniques have greatly advanced in the last decade, DCE-MRI methods have been increasingly used for the evaluation of liver function (35, 37). Of the five MRI indices, iAUC-15 may be the best index to assess liver function based on the highest correlation with ICG-R15 (r=-0.65). Assessment of liver function with gadoxetate-enhanced MR imaging has several advantages over the traditional ICG test (12).
The MRI can evaluate liver anatomy and liver function in localized liver abnormalities, which is more clinically relevant than a global assessment (38). To include gadoxetate-enhanced MRI in the preclinical trial and research for liver function estimation, standardization is a very important prerequisite. In preclinical trial with animal liver fibrosis model, the ultrasonographic SWE and gadoxetate-enhanced DCE-MRI are quite feasible to non-invasively evaluate histopathological liver fibrosis and physiological liver function.
Indocyanine green kinetics to assess liver function: Ready for a clinical dynamic assessment in major liver surgery. Gd-EOB-DTPA-enhanced MRI for assessment of liver function and volume in liver cirrhosis. Liver parenchymal enhancement of hepatocyte phase images in Gd-EOB-DTPA-enhanced MR imaging: which biological markers of liver function influence the enhancement.
Dynamic gadoxetate-enhanced MRI for the assessment of total and segmental liver function and volume in primary sclerosing cholangitis.
