Quantitative Assessment With Contrast-enhanced Ultrasonography In A Rat Model Of Liver Fibrosis

Liver fibrosis is an early reversible process in the development of chronic liver diseases.It's necessary to identify the grade of liver fibrosis in clinical diagnosis, management and whereafter follow-up.The animal model is the foundation of studies on the pathogenesis and the pathophysiological process of liver fibrosis,and it's also an important means to screen clinical medicine and evaluate the curative effectiveness. Thioacetamide(TAA) is one of commonly used chemistry medicines to induce liver fibrosis.However,how many will the success ratio of this modeling have? Whether the model can meet the needs of further studies? These need to be proven.At present liver biopsy is the golden standard to identify the grade of liver fibrosis,but shortcomings such as invasive wound have limited the application clinically.In recent years the domestic and foreign scholars have sought non-invasive diagnostic methods to evaluate the liver fibrosis grade,and contrast-enhanced ultrasonography(CEUS) is also one of them.Whether the combination of CEUS and the new quantitative analysis software reflects the characteristics of blood perfusion in normal rat's liver? Are there any quantitative parameters suitable to assess the liver fibrosis grade? In order to answer the above questions,we have undertaken the following researches.Part one:The establishment and assessment of liver fibrosis model in ratsObjective To evaluate the effect of modeling of liver fibrosis in rats with the use of thioacetamide(TAA).Methods One hundred male Sprague-Dawley rats,SPF (specified-pathogens free) level,were divided into the model group(90) and the control group(10) stochastically.The rats in model group were administered 10% TAA solution via intra-peritoneal injection according to the 200mg/kg dosage,three times in the first week,and afterward twice a week till the 12th week.The rats in control group were administered physiological saline via intra-peritoneal injection according to the 2ml/100g dosage with the same frequency as model group.From the third week,we draw ten rats each week at random to perform CEUS,then collected 2.5ml blood sample to do serologic examination reflecting liver function and the liver fibrosis grade,and the liver specimens were fixed with formalin solution for pathological assessment at last.All the results of serology,morphology and pathology were analyzed to evaluate the effect of liver fibrosis model.Results During the modeling process,8 rats died,7 were in the model group,and 1 was in the control group.Among the rest 83 rats in model group,there were 81 rats with various grades of liver fibrosis,which was confirmed by pathology.Therefore,the success ratio of making the model was 90%(81/90).The changes of this rat model were similar with the changes occurs in human liver fibrosis,including serology,morphology,and pathology.Conclusions The method of inducing liver fibrosis with TAA in rats has a high success ratio and it can meet the need of further studies.Part two:Contrast-enhanced ultrasonography with GAMMA curve fitting technique in the quantitative assessment of liver blood perfusion in normal ratsObjective To investigate the value of the new quantitative software(SW-UCS-1) in the quantitative assessment of liver blood perfusion in normal rats.Methods CEUS were performed on ten health male SD rats.The new quantitative software with GAMMA fitting technique was used to analyze the dynamic images with DICOM format.The software could draw the time-intensity curve(TIC) from the region of interest(ROI),and fit with gamma:technique to acquire the fitting curves and the quantitative parameters.The validity of the fitting curves was evaluated by the goodness of fitting index(GOF).①We selected 200 different ROIs stochastically, and observed the distribution of GOF values;②We selected 60 different ROIs stochastically,which were located at the superficial part(30 ROIs) and deep part(30 ROIs) of rat liver separately(the ROIs' size were similar),and observed whether there were differences of quantitative parameters between them.Results The GOF value bigger than 0.90 accounted for 56%(112/200),and GOF bigger than 0.85 accounted for 94%(188/200).The majority of fitting curves were close to time-intensity curves. Some quantitative parameters between liver superficial part group and the deep part group had statistical differences(P＜0.05),such as peak intensity(PI),increased intensity(a1),rate of half contrast material washout(a2) and area under the curve (AREA).Conclusions SW-UCS-1 quantitative analysis software can reflect the liver blood perfusion in normal rats objectively.GOF is useful for observers to evaluate the accuracy of fitting curves and the quantitative parameters.The influence of the depth to partial quantitative parameters should be considered when choosing the ROI. Part three:Quantitative assessment of liver fibrosis grade in rat model with contrast-enhanced ultrasonographyObjective Contrast-enhanced ultrasonography(CEUS) was used in a rat model with liver fibrosis in order to investigate quantitative parameters correlated with fibrosis grades.Methods Ninety-two male SD rats were induced as liver fibrosis models by TAA.CEUS was performed on these rats randomly since the third week and blood samples were collected for serologic examination.The new quantitative software (SW-UCS-1) was used to analyze the dynamic images and acquire the quantitative parameters.The Spearman rank correlation examination was used to analyze the correlations of the serological indexes,the quantitative parameters and the liver fibrosis grades.The rats were divided into five groups according to pathological fibrosis grade(S0-S4),and t-test was used to seek the group differences further.The fibrosis grade S≥S2,≥S3 and＞S4 were taken as the different research end point,we draw the receive operating characteristic curves(ROC) to evaluate the diagnostic value of quantitative parameters.Results The fibrosis grade on pathology was as the following:S0=11,S1=14,S2=30,S3=27and S4=10.There were statistical correlations between fibrosis grade and some serological indexes as ALT,γ-GT,total bile acid,and total bilirubin.Similarly there were statistical correlations between fibrosis grade and some quantitative parameters as a1,a2,PI,AREA,and HA-HVTT. In the above serological indexes,only the S0 and the S4 group had statistical differences.However,there were statistical differences of quantitative parameters in almost all the groups.According to the ROC curve,the quantitative parameter AREA was most valuable to predict the liver fibrosis grade(S≥S2,AUC=0.899;S≥S3 AUC=0.902;S=S4,AUC=0.951).Conclusions The quantitative analysis technology with CEUS may reflect the liver fibrosis grade in rat model.Quantitative parameters, such as a2,PI,AREA and HA-HVTT are valuable to predict the liver fibrosis grade.