Supersonic shear imaging (SSI) is a relatively new technique to measure the elasticity of target tissues based on the shear wave propagation. The aim of this study was to evaluate the value of SSI in discriminating nonfibrotic nonalcoholic steatohepatitis (NASH) from the less severe nonalcoholic fatty liver disease (NAFLD), NASH with fibrosis, and the normal liver, as well as the relationship between various NAFLD pathologic or biochemical findings and SSI liver elasticity. Rabbits with NAFLD of different degrees were subjected to SSI for liver elasticity measurement. Plasma was collected for biochemical examinations, and liver tissues were harvested for pathologic assessment. Results showed that liver elasticity of rabbits with nonfibrotic NASH was significantly different from that of rabbits with simple steatosis, borderline, NASH with fibrosis, and normal liver (
Nonalcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome and characterized by excessive accumulation of fat in hepatocytes. In recent years, the prevalence of NAFLD is increasing worldwide and it has become the leading cause of chronic liver disease [
Liver biopsy is the gold standard for the diagnosis of NAFLD, but it is invasive and has potentially serious complications [
Ultrasonic shear wave elastography techniques developed in recent years (such as transient elastography (TE), acoustic radiation force impulse (ARFI) elastography, and supersonic shear imaging (SSI)) can be used to detect the liver elasticity for the evaluation of NAFLD and have shown great potential in assisting fibrosis staging [
Available studies have confirmed that fibrosis can affect liver elasticity in the case of NAFLD, but there is no consensus about the effect of other NAFLD pathological changes (such as steatosis, ballooning degeneration, and inflammation) on the liver elasticity [
A total of 40 New Zealand male rabbits (Shanghai Slack Laboratory Animal Co. Ltd.) weighing 1.80-2.23 kg were housed in isolated cages in an environment with 12 h/12 h light/dark cycle at room temperature of 22°C and relative humidity of 30%-50%, and animals were given ad libitum access to food (provided by Fujian Medical Research Institute) and water. Rabbits were housed for 1 week for accommodation to the environment and then randomly divided into four groups. Ten rabbits in the control group were fed with a standard general feed (150 g/d), and the remaining rabbits were fed with high-fat diet (HFD) (1% cholesterol, 0.5% bile salt, 5% egg yolk powder, 10% lard, and 83.5% general feed, 150 g/d, provided by Fujian Medical Research Institute) for 4 weeks (
SSI was performed at the predesigned time points. Before examinations, the rabbits were fasted for 12 h and then intravenously anesthetized with 3% sodium pentobarbital (30 ml/kg). The animals were fixed on the experimental table and hair on the chest and abdomen was removed. An Aixplorer ultrasound imaging system (SuperSonic Imagine, Aix-en-Provence, France) equipped with a line array probe (SL10-4) with a frequency of 4-10 MHz was used for elasticity measurements which were performed by a sonographer who had a two-year experience on elastography and were blind to the experimental design. First, the liver was observed on two-dimensional grayscale images, and then the probe was fixed lightly on the skin with the right lobe of the liver as the detection site before share wave elastography. The mean liver elasticity (Emean) was expressed in kilopascal (kPa) with a range of 0-100 kPa. The ROI of elastography was localized in the right liver lobe about 1.0-2.5 cm away from the body surface, and the gallbladder, interlobular fissure, biliary tract, and blood vessels were avoided. When the color signals filling in the ROI were over 90% and stable, the image was frozen. Then the Emean was measured in the ROI using a circular Q-Box with the diameter of 5 mm. Measurement was repeated 10 times for each rabbit, and the average was calculated as the final elasticity for further statistical analysis. Figure
Share wave elastography of the rabbit liver. (a) After overall observation of the two-dimensional grayscale images of the rabbit liver, the line array probe (SL10-4) of an Aixplorer ultrasound imaging system was fixed lightly on the rabbit skin with the target liver lobe as the detection site. (b) Before the SWE test, the measurement section can be preselected by the two-dimensional grayscale image. (c) When the SWE mode is started, the screen displays a double frame. The two-dimensional grayscale diagram is used to guide the placement of ROI (square), and the gallbladder, biliary tract, and blood vessels shall be avoided. In the ROI is a real-time color map showing liver elasticity. After the color signal in the ROI is filled by more than 90% and stabilized, the Q-Box (circle) is activated to measure the elasticity value and the result is displayed on the screen. SWE: share wave elastography; ROI: region of interest.
After 12 h fasting, blood was collected and then centrifuged to obtain plasma. The plasma samples were processed for the detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).
Paraffin-embedded tissue blocks from the right lobe of the liver where SSI was performed were sliced (7
Statistical analysis was performed using IBM SPSS Statistics Version 22.0 (IBM Corp., Armonk, NY, USA) and MedCalc software (MedCalc Software, Mariakerke, Belgium), and the measurement data are expressed as
Three rabbits from HFD groups died during the feeding period (one at 7th, 9th, and 10th week). The death was ascribed to the intolerance to HFD and relevant diarrhea. Finally, 37 rabbits were analyzed including 10 in the control group, 10 in the HFD-4w group, 9 in the HFD-8w group, and 8 in the HFD-12w group. Table
Pathologic characteristics of rabbit models.
Control group |
HFD-4w group |
HFD-8w group |
HFD-12w group |
|
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Pathologic findings | ||||
Steatosis grade | 8/2/0/0 | 0/6/4/0 | 0/1/3/5 | 0/2/5/1 |
Ballooning grade | 9/1/0 | 2/8/0 | 1/4/4 | 0/2/6 |
Inflammation grade | 9/1/0/0 | 4/5/1/0 | 0/1/6/2 | 0/0/5/3 |
Fibrosis stage | 10/0/0/0/0 | 10/0/0/0/0 | 6/3/0/0/0 | 0/3/3/2/0 |
NAS | 8/2/0/0 | 0/4/6/0 | 0/0/1/8 | 0/0/0/8 |
HFD: high-fat diet; NAS: nonalcoholic fatty liver disease activity score.
SSI measurements in the NASH (
The SSI measurements of the rabbit liver with NAFLD in various severe degrees. Emean: mean elasticity value; SS: simple steatosis; NASH: nonalcoholic steatohepatitis; NAFLD: nonalcoholic fatty liver disease; SSI: supersonic shear imaging. Asterisks indicate pairs having statistically significant differences in Bonferroni’s post hoc test after ANOVA test.
The SSI measurements of nonfibrotic NASH and NASH with fibrosis in rabbits. Emean: mean elasticity value; SS: simple steatosis; NASH: nonalcoholic steatohepatitis; SSI: supersonic shear imaging. Asterisks indicate pairs having statistically significant differences in Bonferroni’s post hoc test after ANOVA test.
Supersonic share wave elasticity images and HE- and MT-stained liver sections in rabbit models of NAFLD. (a) From a normal rabbit liver. Share wave elasticity image shows that the Emean value was 5.7 kPa. Liver sections show normal histologic performances without steatosis, ballooning degeneration, inflammation, and fibrosis. (b) From a rabbit liver with SS. The Emean value was 8.2 kPa. Liver sections show hepatic steatosis without ballooning degeneration, inflammation, and fibrosis. (c) From a borderline rabbit liver. The Emean value was 9.5 kPa. Liver sections show dispersive hepatic steatosis with sporadic ballooning degeneration, and no significant inflammation and fibrosis can be found. (d) From a rabbit liver with NASH. The Emean value was 12.9 kPa. Liver sections show diffuse hepatic steatosis with sporadic ballooning degeneration and inflammation, without fibrosis. (e) From a rabbit liver with NASH. The Emean value was 15.1 kPa. Liver sections show dispersive hepatic steatosis with significant ballooning degeneration and hyperplasia of perisinusoidal fibrous tissue. HE: hematoxylin-eosin; MT: Masson’s trichrome; NAFLD: nonalcoholic fatty liver disease; Emean: mean elasticity value; SS: simple steatosis; NASH: nonalcoholic steatohepatitis.
The area under ROC curve (AUROC) of SSI for distinguishing nonfibrotic NASH from less severe NAFLD or normal liver was 0.997 (95% CI, 0.899–1.000), and the optimal cutoff value was 10.17 kPa, with the sensitivity of 100% and the specificity of 95.24%. In the differentiation of nonfibrotic NASH and NASH with fibrosis, the AUROC of SSI was 0.967 (95% CI, 0.849–0.998) and the optimal cutoff value was 12.82 kPa with the sensitivity of 90.00% and the specificity of 92.59%.
Univariate analysis showed that the liver elasticity on SSI had a positive correlation with all NAFLD pathologic findings, including steatosis, ballooning degeneration, inflammation, and fibrosis (
Correlation of liver elasticity values with histopathology findings in all rabbit models.
Univariate analysis | Multivariate analysis | ||||
---|---|---|---|---|---|
|
|
|
95% CI |
|
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Steatosis grade | 0.695 | ≤0.001 | 0.527 | -0.194-1.249 | 0.146 |
Ballooning grade | 0.806 | ≤0.001 | 0.784 | -0.151-1.718 | 0.097 |
Inflammation grade | 0.870 | ≤0.001 | 1.248 | 0.519-1.977 | 0.001 |
Fibrosis stage | 0.739 | ≤0.001 | 2.892 | 2.167-3.617 | ≤0.001 |
Correlation of liver elasticity values with histopathology findings in rabbit models without fibrosis.
Univariate analysis | Multivariate analysis | ||||
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95% CI |
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Steatosis grade | 0.857 | ≤0.001 | 0.926 | 0.254-1.598 | 0.009 |
Ballooning grade | 0.708 | ≤0.001 | 1.186 | 0.358-2.013 | 0.007 |
Inflammation grade | 0.833 | ≤0.001 | 1.089 | 0.426-1.751 | 0.003 |
Biochemical findings of rabbits with various degrees of NAFLD are shown in Table
Biochemical examination results of blood plasma from rabbit models.
Pathologic diagnoses | ALT (U/l) | AST (U/l) | TC (mmol/l) | TG (mmol/l) | HDL-C (mmol/l) | LDL-C (mmol/l) |
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Normal |
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Simple steatosis |
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Borderline |
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Nonfibrotic NASH |
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NASH with fibrosis |
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ALT: alanine aminotransferase; AST: aspartate aminotransferase; TC: total cholesterol; TG: triglyceride; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; NASH: nonalcoholic steatohepatitis. Data are
The correlation of liver elasticity with various biochemical indicators is shown in Table
Correlation of liver elasticity values with biochemical findings in rabbit models.
Univariate analysis | Multivariate analysis | ||||
---|---|---|---|---|---|
|
|
|
95% CI |
|
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ALT | 0.909 | ≤0.001 | 0.169 | 0.125-0.213 | ≤0.001 |
AST | 0.799 | ≤0.001 | 0.008 | -0.052-0.068 | 0.796 |
TC | 0.568 | ≤0.001 | -0.009 | -0.121-0.103 | 0.874 |
TG | 0.015 | 0.929 | — | — | — |
HDL-C | 0.354 | 0.032 | -0.045 | -0.632-0.542 | 0.877 |
LDL-C | 0.577 | ≤0.001 | 0.088 | -0.012-0.188 | 0.083 |
ALT: alanine aminotransferase; AST: aspartate aminotransferase; TC: total cholesterol; TG: triglyceride; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol;
In available studies, mice, rats, and rabbits are frequently used to establish animal models of NAFLD [
In the disease spectrum of NAFLD, NASH has the greatest potential risk of developing into cirrhosis and even HCC. The severity of NASH and the accompanying fibrosis may directly affect the prognosis of NAFLD [
Our study showed that liver fibrosis was a major factor that had positive influence on the liver elasticity of rabbits with NAFLD (
Serum biochemical examination is another noninvasive method in evaluating NAFLD [
In this experiment, a high-fat diet was used to establish the rabbit NAFLD model and NAFLD in this model is similar to human NAFLD in terms of etiology and pathology. After 12-week high-fat diet, cirrhosis was not found in these rabbits. Different from previous studies [
In conclusion, nonfibrotic NASH can be effectively identified from NAFLD of other degrees and normal liver in rabbits by SSI, based on the difference in liver elasticity. The difference in liver elasticity might be related to not only the degree of fibrosis but also the degrees of inflammation, steatosis, and ballooning degeneration in the liver with NAFLD.
The data used to support the findings of this study are available from the corresponding author upon request.
We declare that we have no financial or personal relationships with other people or organizations that can inappropriately influence our work and that there is no professional or other personal interest of any nature in any product, service, and/or company that could be construed as influencing the manuscript.
This work was supported by the Key Clinical Specialty Fund of Fujian Province (no. 2011724) and Joint Funds for the Innovation of Science and Technology of Fujian Province (no. 2017Y9028).