In this work we demonstrated that qualitative ultrasound analysis is sensitive and specific enough to diagnose fatty liver disease and cirrhosis in rats and, therefore, can reliably substitute histological diagnosis in experimental liver disease models. Ultrasound imaging has many advantages, namely its low cost, extensive availability and the fact that it can be easily done in murine models. Only slight sedation and abdominal shaving are required, not demanding total immobility or apnea while obtaining the images , therefore, consisting in a low risk procedure. Moreover, it is an accurate, innocuous and non-invasive technique and it is the most commonly used method for the investigation of liver diseases in humans and small animals [1, 18–21, 40, 41].
Although ultrasound is an essentially subjective diagnostic method, its validity as an imaging technique for liver evaluation has been extensively described in the literature for humans [40, 42], dogs [21–23] and cats [1, 21]. In our study, we focused our attention in a rodent model of liver disease, since these are the most commonly used models in pre-clinical studies [37, 43–46]. The rat model was chosen because they are larger than mice, facilitating US imaging procedure.
Healthy rats showed homogeneous liver parenchyma, with medium level echogenicity and regular liver surface, as previously described in humans , dogs [21, 23, 48] and cats [2, 21]. Liver echogenicity was lower in comparison to the right renal cortex, a finding that contrasts with humans , but is in accordance to what has been described in dogs . In cats, renal cortical echogenicity varies in normal animals . Fatty liver disease in rats was characterized by homogeneous and diffusely increased echogenicity ("bright liver"), equal or greater to the right renal cortex, reduced visualization of the diaphragm and of small peripheral vessels, and by no changes in liver surface, as described in humans , dogs [21, 23], and cats [1, 21].
Liver echogenicity increases due to the presence of fatty infiltration and/or fibrosis [21, 23], changing the relation between liver and right renal cortex echogenicity [21, 22]. This finding had high sensitivity (90%), specificity (100%), positive and negative predictive values (100% and 76.9%), and accuracy (92.5%) for the detection of fatty liver disease, values that were compatible with the ones previously described in the literature. The increased liver echogenicity has been reported to have sensitivity of 60 to 82% [1, 23, 52], specificity of 97 to 100% [1, 52] and positive predictive value of 96 to 100% [1, 52] for detection of fatty liver disease. However, it is important to exclude kidney diseases when utilizing this approach .
Three rats with mild hepatic steatosis appeared to be normal in US examination, which is also comparable to previous reports in the literature [23, 52]. US has been reported to have sensitivity of 90 - 91% for the detection of moderate to marked steatosis, but its sensitivity decreases to 30 - 64% when steatosis is mild [23, 52].
The finding of an extensive coarse and heterogeneous parenchyma associated to irregular or nodular hepatic surface proved to be relevant in the detection of cirrhosis with sensitivity of 70.6%, specificity of 100%, positive predictive value of 100%, negative predictive value of 82.1% and accuracy of 87.5%, as seen in humans [40, 53] and in dogs . Its presence implied a definitive diagnosis of cirrhosis since there were no false negative results. Therefore, US imaging can consistently substitute histology to diagnose cirrhosis in rats, constituting a useful method to avoid unnecessary animal sacrifice or repeated liver biopsies when following long experimental protocols.
The evaluation of portal hypertension is also important to assess the severity of liver diseases [20, 21, 40] and one of its most important signs is the widening of the portal vein [20, 21, 47]. PVD was statistically different when comparing cirrhotic to non-cirrhotic rats (p < 0.001). An increase in its caliber when equal or superior to 2.1 mm can be considered a relevant ultrasound finding with sensitivity of 100%, specificity of 90.5%, positive predictive value of 89.5% and negative predictive value of 100% for the detection of cirrhosis. There was one false positive, a rat bearing liver steatosis with fibrosis. There were no false negatives since none of the rats with PVD lower than 2.1 mm were cirrhotic. Additionally, PVD measurements were unable to distinguish normal from steatotic livers because of the considerable overlap between PVD values encountered in normal rats compared to rats carrying steatosis and fibrosis. This fact is, nonetheless, expected since fatty liver disease does not lead to portal hypertension and, consequently, to the widening of the portal vein. Doppler evaluation of the portal vein was also tried, yet it was not possible due to technical difficulties. The caliper could not be stably positioned inside such a small vessel, we could not induce apnea and whirl flow was found even in normal rats, causing an oscillation between hepatopetal and hepatofugal flow.
The presence of ascites, which is also an important indicator of the severity of liver disease, could be detected even when the amount of fluid present in the peritoneal cavity was small, as previously reported by Vörös et al . Among the imaging diagnostic methods, ultrasound is considered ideal for the study of ascites [6, 21, 23]. In our study, its presence invariably corresponded to cirrhosis in histology, although only 5 animals presented with ascites out of 17 cirrhotic rats. This was, however, expected since ascites is only observed when cirrhosis is decompensated. In addition, although splenomegaly should be expected in severe cirrhosis with portal hypertension, there were no statistical differences in splenic area among the three groups analyzed, indicating that splenomegaly was not a prominent feature in our study of cirrhotic rats.