Experimental Study On The Diagnostic Value Of Shear Waveelastrography And Contrast-enhanced Ultrasound In Nonalcoholic Fatty Liver Disease

ObjectiveTo investigate the diagnostic value of shear wave elastrograph(SWE)and contrast-enhanced ultrasound(CEUS)in different stages of nonalcoholic fatty liver disease(NAFLD),and to analyze its mechanism.Methods(1)39 healthy New Zealand white rabbits were randomly divided into normal diet group(A1 group,A2 group,A3 group),3 in each group,and high-fat diet group(B group,C group,and D group),10 in each group.The A1 group,A2 group,and A3 group were fed an ordinary diet for 4 weeks,8 weeks,12 weeks,respectively.The high-fat diets of group B,C,and D were fed for 4 weeks,8 weeks,and 12 weeks,respectively.(2)Before the feeding of normal diet group and high-fat diet group rabbits,conventional two-dimensional ultrasound and SWE examination were performed to obtain the liver Emean value.(3)After the feeding period,rabbits in each group were subjected to conventional two-dimensional ultrasound,SWE,and contrast-enhanced ultrasound.The changes in Emean values and related imaging parameters(PI,TP,MTT,and AUC)were analyzed.After the end of the ultrasound examination,the rabbits were taken 5 ml of blood from the vena cava,and the alanine aminotransferase(ALT)? aspartate aminotransferase(AST)?total cholesterol(TC)?triglyeride(TG)?low-density lipoprotein cholesterol(LDL-C)values were measured in the laboratory.At the same time,the liver tissue of rabbits was subjected to HE staining,oil red O staining,Masson staining,TUNEL and electron microscopy.The histopathological and ultrastructural changes of normal control group,nonalcoholic simple fatty liver(NAFL)group,nonalcoholic steatohepatitis(NASH)group and fibrosis group were observed and compared.Results1.With the increase of feeding time 4W,8W,and 12 W,the average weight,liver weight,and liver index of the normal diet group and the high-fat diet group increased,and the high-fat diet group was significantly higher than the normal diet group(B>A1,C>A2,D>A3),the differences were statistically significant(P<0.01).2.Emean values of rabbit liver elastography with different pathological stages of NAFLD,the performance was normal control group <NAFL group <NASH group <fibrosis group,that is,with the progression of the NAFLD disease spectrum,Emean values are increasing,and the two are highly positively correlated,the Spearman's correlation coefficient was 0.948(P=0.000<0.01),and the difference in the Emean values between the groups was statistically significant(P=0.000<0.01).3.Contrast-enhanced Ultrasonography of liver in rabbits with different pathological stages of NAFLD,the peak Intensity(PI)and AUC of NAFL group,NASH group and fibrosis group were significantly lower than those of normal control group,and the differences were statistically significant(P<0.05).The difference between PI and mean transmit time(MTT)between NAFL group and NASH group was also statistically significant(P<0.05).4.The Emean value of Elastography and the combined parameters of contrast-enhanced ultrasound(PI+TP+MTT+AUC)had higher diagnostic efficacy for NAFLD with different pathological staging,and the area under the ROC curve was similar.5.Liver function tests in rabbits with different pathological stages of NAFLDshowed thatthe ALT levels in fibrosis group and NASH group were significantly higher than those in other groups(P<0.01).AST increased with the progress of NAFL group,NASH group,and fibrosis group,and only fibrosis group had statistically significant difference compared with other groups(P<0.01).6.Changes in blood lipids in rabbits with different pathological stages of NAFLD,the levels of TC,TG,and LDL-C in NAFL group,NASH group,and fibrosis group were higher than those in normal control group,but only TC and LDL-C in NASH group and fibrosis group were statistically different from those in normal control group(P<0.05).7.HE staining of liver tissue in rabbits with different pathological stages of NAFLD.In the normal control group,normal liver tissue was observed under the microscope,and there was no or occasional hepatocyte fat accumulation in the liver lobule;In the NAFL group,> 33% hepatocyte steatosis in the liver lobules was observed under low magnification,but no histological changes such as inflammation,ballooning,necrosis and fibrosis were observed;In the NASH group,there was a large accumulation of fat droplets in liver cells,ballooning or necrosis of hepatocytes,and infiltration of inflammatory cells,and mild peri-sinusoidal fibrosis(S1 phase);In the fibrosis group,more extensive fibrosis around the sinus peri-tal and portal veins was seen,and bridging fibrosis was seen(S2,S3phase).8.Oil Red O Staining of liver tissue in rabbits with different pathological stages of NAFLD.Hepatocytes in NAFL group,NASH group and fibrosis group were swollen.A lot of red lipid droplets were found in cytoplasm.9.Masson staining of liver tissue in rabbits with different pathological stages of NAFLD.In the NASH group,a small amount of blue collagen fibers were seen around the sinus in the liver tissue.In the fibrosis group,extensive fibrous tissue hyperplasia in the sinus and portal areas was seen,and bridging fibrosis was seen in some of them.10.TUNEL assay of liver tissue in rabbits with different pathological stages of NAFLD showed that brown apoptotic cells were observed in NAFL group,NASH group and fibrosis group.11.The ultrastructural changes of liver tissue in rabbits with different pathological stages ofNAFLD,there were a lot of lipid droplets in hepatocytes of NAFL group,and mitochondria and rough endoplasmic reticulum were swollen.In the NASH group,hepatocytes were filled with lipid droplets of different sizes,and the nucleus and organelle structure changed more significantly,and apoptotic cells and eosinophils were also seen.The structure of the fibrosis group was more disordered and collagen fibers were seen.Conclusions1.With the progress of NAFLD disease spectrum,the Emean values of elastography showed an increasing trend,and the two were highly positively correlated.The difference in Emean values between the groups was statistically significant(P=0.000<0.01),suggesting that SWE can be used for the staging diagnosis of NAFL,NASH and advanced liver fibrosis inexperimentalrabbit fatty liver,and hashigh diagnostic efficiency.To provide experimental basis for clinical application research,Emean value is expected to become a quantitative parameter for clinical diagnosis of fatty liver.2.The results of this study show that the increased Emean value may be related to histological changes such as cell swelling,tissue tension,inflammatory reaction and fibrosis caused by hepatocyte steatosis.3.Contrast-enhanced ultrasound parameter PI and AUC are valuable for the differential diagnosis between NAFLD and normal controls,while PI and MTT may be helpful for differential diagnosis of NAFL and NASH,and their joint parameters also have higher diagnostic efficacy in different stages of NAFLD progression.4.The liver function index ALT has certain reference significance for the diagnosis of NASH and fibrosis group,while AST has certain reference significance for the diagnosis of fibrosis group.Serum lipids TC,TG,and LDL-C have little value in differential diagnosis of NAFL,NASH,and NASH-related hepatic fibrosis.5.In this experiment,multimodality tests such as liver function,histopathology,and ultrastructure were performed to confirm that the use of high-fat diets for feeding New Zealand white rabbits can replicate series lesion models of the NAFL,NASH and NASH-related liver fibrosis in a short period of time.