Male Sprague-Dawley rats (n=80, weight: 200 ± 20g) were provided by the Experimental Animal Center of our University, Guangdong, China (Animal permit number: SCXK2014-0035). Rats were housed at 18-20°C, 60-70% humidity, and on a 12 hour light/dark cycle. During the experimental period, animals were fed with distilled water and standard rat feed. All experimental procedures were approved by the Institutional Animal Ethics Committee and were performed according to the Laboratory Animal Care and Usage Manual.
At the beginning of the study, all rats were randomly assigned to two experimental groups. Group A animals underwent a carbon tetrachloride (CCl4) injury and subsequent withdrawal for recovery according to a previous protocol . In short, rats were injected with 50% CCl4 (CCl4:olive oil in a 1:1 ratio) (0.3 ml/100 g bodyweight) subcutaneously twice per week until day 40.
Group B animals underwent bile duct ligation (BDL) and recanalization. Rats were initially anesthetized with 3% pentobarbital (0.2 ml/100 g bodyweight) by intraperitoneal injection . Then, after an abdominal incision, the common bile duct was exposed aseptically, separated, and ligated near the liver below the bifurcation. The bile duct was then recanalized on day 10.
The time points for MRI scanning and histological examination for both study groups (n=8) are shown in Figure 1. The MRI time points were decided based on our previous studies .
Magnetic resonance imaging:
MRIs were acquired with an INGENIA 3.0T scanner (Philips Healthcare, Best, Netherlands) with a 4-channel animal coil (ShuZhou ZONGZHI Medical Technologies Co.). Rats were sedated using 3% sodium pentobarbital (0.2 ml/100 g bodyweight) via intraperitoneal injection and placed in the prone position. The standard sequences used for scanning include the following: (A) axial and coronal T2-weighted fast field echo (2D-FFE), repetition time/echo time (TR/TE): 203/9.2 ms, field of view (FOV): 60×60 mm, slice thickness: 3 mm, matrix: 100×100; (B) axial T1 weighted turbo spin-echo (TSE), TR/TE: 400/10ms, FOV: 60×60 mm; matrix: 120×93; slice thickness: 3 mm.
The T1rho was determined using an adiabatic RF pulse with the following parameters: TR/TE: 4.9/2.4 ms; FOV: 60×60 mm; flip angle: 40°; Matrix: 100×100; slice thickness: 3 mm; number of slices: 5; spin lock frequency: 500 Hz; rotation lock times: 0, 27, and 54 milliseconds.
MRI data analysis:
The T1rho images were fitted to an exponentially decaying T1rho function on a pixel-by-pixel basis to generate a T1rho relaxation map with the IDL software (Research Systems, Inc., USA). T1rho values were computed using a mono-exponential decay model, as seen in Eq. 1, where MTSL is the magnetization and TSL is the time of the spin-lock pulse.
The data was analyzed using the ImageJ software (NIH, Bethesda, MD, USA). Five regions of interest (ROIs) of approximately 3–4 mm2 were drawn manually to avoid the inclusion of the liver margin, vessels, and bile ducts. The mean values obtained for the five ROIs were considered as the T1rho values
Blood serum parameters of liver function
After centrifugation for 10 min at 4°C (300 × g; Centrifuge 5804R), the activity of alanine transaminase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) in the serum was determined using an automatic biochemical blood analyzer (cat. no. 7600-210; Hitachi High-Technologies Corporation).
After the MRI scan, the rats were sacrificed via excessive anesthesia. The liver tissue was removed, fixed with a formaldehyde solution and embedded in paraffin. The tissue was then cut into 5-μm sections and stained with hematoxylin and eosin (HE) and picrosirius red. Here, picrosirius staining reflects collagen content and indicates progressive changes in fibrosis after the reversal of bile duct obstruction. Two independent pathologists blinded to the MRI results evaluated the samples using a dedicated microscope (Leica DM2000).
The collagen content of the liver was measured using a semi-quantitative analysis with picrosirius staining. Briefly, ten parenchymal images of the liver (magnification of 200×) were randomly selected and the percentage of positively stained areas relative to the entire area was calculated using the Image-Pro Plus v6.0 image analysis software (Media Cybernetics, Rockville, MD) [8, 9]. Inflammation, ballooning, and steatosis in the liver were evaluated according to the nonalcoholic steatohepatitis (NASH) standards . Lobular inflammation was graded on a 0-3 scale: 0: no lesions; 1: <2 lesions; 2: 2-4 lesions; 3: >4 lesions. Portal inflammation was graded on a 0-1 scale: 0: none to minimal; 1: greater than minimal. Ballooning was graded on a 0-2 scale: 0: none; 1: few balloon cells; 2: many cells/prominent ballooning. The assessment of steatosis was based on the proportion of the steatosis area within the visual field .
The statistical package for social sciences software version 20.0 (SPSS, Chicago, IL) was used for all statistical analysis. A p-value <0.05 was considered statistically significant. The data are expressed as mean ± standard deviation. Normality was assessed using a Kolmogorov-Smirnov test. A one-way analyses of variance for multiple groups with least significant difference post hoc tests was performed on the outcome parameters. Pearson’s rank correlation coefficients, (r values) were calculated to assess the correlation between the T1rho and the pathophysiological parameters.