Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Abstract

Background: Domestic dogs and cats are very well known to develop chronic hepatic diseases, including hepatic lipidosis and cirrhosis. Ultrasonographic examination is extensively used to detect them. However, there are still few reports on the use of the ultrasound B-mode scan in correlation with histological findings to evaluate diffuse hepatic changes in rodents, which represent the most important animal group used in experimental models of liver diseases. The purpose of this study was to determine the reliability of ultrasound findings in the assessment of fatty liver disease and cirrhosis when compared to histological results in Wistar rats by following up a murine model of chronic hepatic disease.

Results: Forty Wistar rats (30 treated, 10 controls) were included. Liver injury was induced by dual exposure to CCl4 and ethanol for 4, 8 and 15 weeks. Liver echogenicity, its correlation to the right renal cortex echogenicity, measurement of portal vein diameter (PVD) and the presence of ascites were evaluated and compared to histological findings of hepatic steatosis and cirrhosis. Liver echogenicity correlated to hepatic steatosis when it was greater or equal to the right renal cortex echogenicity, with a sensitivity of 90%, specificity of 100%, positive and negative predictive values of 100% and 76.9% respectively, and accuracy of 92.5%. Findings of heterogeneous liver echogenicity and irregular surface correlated to liver cirrhosis with a sensitivity of 70.6%, specificity of 100%, positive and negative predictive values of 100% and 82.1% respectively, and accuracy of 87.5%. PVD was significantly increased in both steatotic and cirrhotic rats; however, the later had greater diameters. PVD cut-off point separating steatosis from cirrhosis was 2.1 mm (sensitivity of 100% and specificity of 90.5%). One third of cirrhotic rats presented with ascites.

Conclusion: The use of ultrasound imaging in the follow-up of murine diffuse liver disease models is feasible and efficient, especially when the studied parameters are used in combination. The potential implication of this study is to provide a non-invasive method that allows follow-up studies of fatty liver disease and cirrhosis of individual rats for pre-clinical drug or cell based therapies.

Figure 1

Histological findings observed over the experiment. (a) Microscopy of Control group (normal liver): Hematoxilin/Eosin (H&E) section (magnification 100×) shows hepatocytes radially arranged in plates aligned to sinusoids (arrowheads). Sirius red (magnification 100×) staining shows only vessels stained in red (portal triad - PT), which is a normal place of collagen deposit. (b) After 4 weeks of experiment: H&E section (magnification 100×) shows moderate steatosis (arrows). Sirius red (magnification 100×) staining shows focal perivenular and pericellular fibrosis (arrowheads). (c) After 8 weeks of experiment H&E section (magnification 100×) shows moderate steatosis (arrows) with some fibrosis (arrowheads) but preserved liver parenchymal architecture. Sirius red (magnification 100×) staining some fibrous septa linking centrolobular veins with portal tracts (arrowheads). (d) After 15 weeks of protocol: H&E section (magnification 100×) shows macronodular cirrhosis, characterized by thick collagen septa, forming regenerative nodules, global architectural distortion, some inflammatory infiltrate (arrow) and steatosis. Sirius red (magnification 100×) staining shows thick collagen septa (arrowheads) linking portal tracts, centrolobular veins, and portal tracts to centrolobular veins, forming regenerative nodules (RN).

Figure 2

Correlation between ultrasound and histological findings. (a) Sonograms and macroscopy of Control group (normal liver): Transversal sonogram demonstrates homogeneous liver parenchyma, with medium level echogenicity and a regular hepatic surface (arrowheads). Parasagittal sonogram presents right renal cortex more echogenic than liver. Macroscopy shows smooth surface, brownish-red color and friable consistency. (b) Sonograms and macroscopy of fatty liver: Transversal sonogram presents diffusely increased parenchymal echogenicity. Parasagittal sonogram demonstrates hepatic echogenicity greater than that of right renal cortex. Macroscopy exhibits wrinkled surface, yellowish color and friable consistency. (c) Sonograms and macroscopy of liver carrying steatosis and fibrosis: Transversal sonogram shows discrete coarse and heterogeneous parenchymal echogenicity and the liver surface as a dotted or slightly irregular line (arrowheads). Parasagittal sonogram presents hepatic echogenicity equal to the right renal cortex echogenicity and a slightly irregular liver surface (arrowhead). Macroscopy shows a slightly irregular surface, pale red color and more rigid consistency. (d) Sonograms and macroscopy of cirrhotic liver: Transversal sonogram presents extensive coarse and heterogeneous parenchymal echogenicity, extremely irregular hepatic surface (arrowheads), a hypoechoic regenerative nodule (arrow) and mild ascites (*). Parasagittal sonogram shows hepatic echogenicity slightly greater than that of the right renal cortex, extremely irregular liver surface (arrowhead) and mild ascites (*). Macroscopy reveals extremely irregular surface, reflecting the presence of underling regenerative nodules, reddish color and rigid consistency.

Figure 3

Portal vein widening over the experiment. (a) Parasagittal sonogram shows a straight and thin portal vein (diameter = 1.5 mm) in a Control group rat (arrowheads). (b) Parasagittal sonogram demonstrates a slightly tortuous and thin portal vein (diameter = 2.0 mm) in a rat with hepatic steatosis and fibrosis (arrowheads). (c) Parasagittal sonogram presents a tortuous and wide portal vein (diameter = 2.4 mm) in a cirrhotic rat (arrowheads).

Figure 4

Comparison between portal vein diameter and histological findings. Quantification of portal vein diameter shows increase in this value in the group of cirrhotic rats (mean = 2.2 mm) and in the group of rodents with steatosis and fibrosis (mean = 1.8 mm) compared to the Control group (mean = 1.5 mm). Data are shown as mean ± SD and the test utilized was ANOVA with the Tukey's post-test for multiple comparisons.

Figure 5

ROC (Receiver Operating Characteristic) analysis of Portal Vein Diameter (PVD) values.