INTRODUCTION

Despite the development of novel treatment options for chronic liver diseases, mortality rates of liver disease patients are still high in Korea. A lot of research efforts are invested in developing better regimes to cure those diseases; however, failures of new drug development still occur during each step.¹ The evaluation system for drug efficacy with the imaging modality is not well established in preclinical research areas. Although many biomarkers have been emerged as the indicator for subject assignments, it is still difficult to distinguish the disease severity of the subjects that would assign into the control and experimental groups.² Therefore, we aim to develop the bioimaging database and assess the feasibility for preclinical liver fibrosis severity index establishment with MRI.

MATERIALS & METHODS

Six-week-old male Sprague-Dawley (SD) rats were used and randomly divided into two groups as follows: Control (n = 7) and thioacetamide (TAA; n = 6) groups. To induce liver fibrosis, TAA was intraperitoneally injected with 200 mg/kg three times weekly for eight weeks (Fig.1). MR imaging was assessed every two weeks using a 9.4-T magnet. The MR images were obtained as follow: (1) T₁-weighted dynamic contrast-enhanced (DCE)-MRI with TR/TE = 70/2.47 ms, FOV = 60 × 35 mm², 400 images, total scan time = about 46 min. 25 μmol/kg Gadoxetic acid (Gd-EOB-DTPA; Primovist®) was intravenously injected after 120 sec. (2) T₂* map with TR/TE = 800/2.56 ms, FOV = 60 × 35 mm². The relative enhancement rate (RER) was calculated using the RER(t) = [SI(t)-SI(0)/SI(0)]×100(%) equation.³ The area under the curve to maximum time (AUC_tmax) was calculated by the integration of areas from 0 to 6 min 72 sec (SI(t); SI of the liver after Gd-EOB-DTPA injection, SI(0); average of pre-contrast SI). The blood samples were collected from the tail vein every two weeks. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL) and albumin (ALB) were measured with Hitachi 7180 Autoanalyzer. The 4% paraformaldehyde-fixed, and paraffin-embedded liver tissue samples were sectioned at 4 µm and stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT). Images were obtained under a Zeiss-AxioscopeⅡ microscope. The lobular inflammation was scored as follow: no foci (score-0), <2 foci / 200× (score-1), 2-4 foci / 200× (score-2), >4 foci / 200× (score-3). The hepatocyte ballooning was scored as follow: none (score-0), few ballooned cells (score-1), many ballooned cells (score-2). The fibrosis stage by Brunt⁴ was graded as follow: none (stage-0), portal fibrosis (stage-1), portal fibrosis with few septa (stage-2), septal or bridging fibrosis (stage-3), cirrhosis (stage-4). Statistical analysis was performed using SPSS 18.0. One-way ANOVA, and independent t-tests were performed to compare the mean between different values and p < 0.05 was considered significant.

RESULTS AND DISCUSSION

The TAA-induced group showed significantly decreased body weights at four weeks (Fig.2A). The AST, ALT, and TBIL were significantly increased in the TAA-induced group at 8-weeks (Fig.2B). In the histological analyses, inflammatory cells and scarring were observed near the portal triad following TAA injection (Fig.3). TAA-induced group showed increased lobular inflammation (0 vs. 2), hepatocyte ballooning (0 vs. 2), and fibrosis stage (0 vs. 3.3) (Table 1). With the analyses of the serum levels and histology, we confirmed that TAA injection generated the animal models that bear the liver fibrosis. We next evaluated the degree of hepatic fibrosis with MRI. T₁-weighted DCE and T₂*map images was acquired (Fig.4A and C). In comparison analyses of RER, TAA-induced group showed decrease of RER compared to the control from the second weeks (Fig.4B). The AUC_tmax and T₂* map showed significantly decreased in the TAA-induced group, especially at 6^th and 8^th weeks compared to the control (Fig.4D). All the MRI data also implied that liver fibrosis was progressed under the TAA injection. After all, Pearson correlation were calculated between MR data and several hepatic enzymes. T₂* map with AST or with ALT have shown significant negative correlations. Between AST and ALT, AUC_tmax and T₂* map showed significant positive correlations (Table 2). Our data demonstrated that correlations between blood tests and MR images were existed as the fibrosis progressed.

CONCLUSION

The current report is the part of the ongoing project for the preclinical liver fibrosis imaging severity index establishment. Due to the small numbers of subjects, we haven’t generated the severity index standard in this report. However, as MRI data with serum and histological data showed correlative relations, we had preliminary severity index guideline (data not shown) with our current database. Because our study will be carried out until the sufficient analyzed data are gathered, we expect that the imaging-based severity index will be established soon.

Acknowledgements

This study was supported by grants of Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education [2018R1A2B2007694]