Study On The Alteration Of Microvasculature In Liver Fibrosis And Its Influence On Portal Hypertension Based On X-ray Phase-contrast CT

X-ray phase-contrast imaging（PCI）,which utilizes the phase shifts of X-rays passing through the object to generate image contrast,can detect subtle differences in the biological soft tissues without the aid of contrast agent,and it has approximately1000-fold higher sensitivity than conventional absorption-based X-ray methods in imaging soft tissues.As a combination of PCI and computed tomography（CT）,X-ray phase-contrast CT（PCCT）could achieve high resolution three-dimensional（3D）imaging of biological soft tissues on the micron scale,which provides a solid technical foundation for its extensive application in the biomedical field.Liver fibrosis is a reversible repair response to chronic liver injury that may eventually develop into liver cirrhosis if not cured for a long time,and can lead to portal hypertension,esophagogastric varices,ascites and other complications.The main pathological features in the development of liver fibrosis and cirrhosis are the damage of the liver microcirculation and the disorder of the microvascular structure.At present,the morphological studies of intrahepatic microstructure are mainly based on two-dimensional（2D）histopathology,while PCCT technology can realize high-resolution 3D visualization of intrahepatic microstructure,and can further explore the pathogenesis of diseases through accurate 3D quantitative analysis.By PCCT combined with histopathology,the dynamic evolution of the microvascular system in the liver explants of patients with post-necrotic and biliary cirrhosis and the corresponding rat liver cirrhosis models induced by carbon tetrachloride（CCl₄）and bile duct ligation（BDL）was observed with the development of the disease,and the pathogenesis of cirrhotic portal hypertension was further explored.The main research contents are as follows:（1）.Investigation of the dynamic adaptation of the biliary system during liver fibrosisA total of 40 rats with liver fibrosis induced by BDL were randomly divided into five groups:control group,2-week,4-week,6-week and 8-week group post-BDL（each group with 8 rats）.According to different pathological characteristics,the biliary system was divided into three domains:downstream,midstream,and upstream.Combined with histopathology and PCCT,the entire biliary and intrahepatic vascular system was observed from macroscopy to microscopy based on 2D and 3D perspective.Additionally,liver explant tissues from 28 biliary atresia（BA）patients were used to investigate the impact of biliary tract reconstruction by Kasai portoenterostomy.To offset the increased biliary pressure within the biliary system,the ductular reaction in the downstream,midstream and upstream domains was manifested by dilation,"spider web-like"looping and interconnected"honeycomb-like"patterns,respectively.Moreover,the"connection channel"between the original bile duct and the proliferative bile duct was traced by PCCT-based 3D endoscopic technique.In BA patients,both fibrosis and proliferative ductules regressed after successful biliary tract reconstruction following Kasai portoenterostomy.Furthermore,histological analysis of HE,SMA,CK19,and CD34showed that the ductular reaction was accompanied by a progressive increase in the arterial supply,but a loss of portal blood supply.（2）.Dynamic evaluation of the vascular circulation in liver fibrosis and its subsequent regressionThe rats with liver fibrosis induced by CCl₄were divided into normal group,moderate group,severe group and regressive group after spontaneous recovery（each group with 6 rats）,and the 3D vascular network of the whole liver was reconstructed by PCCT.In this study,a novel approach for calculating Murray’s deviation based on the vascular volume was proposed instead of the traditional method for evaluating vascular branch circulation based on diameter.Detailed comparative analysis of the volume-based Murray’s deviation and the traditional diameter-based method was performed to verify accuracy of the volume-based method.Moreover,the volume-based method was explored to assess the vascular circulation of major（diameter≥200μm）and small vascular branches（diameter<200μm）in different periods of liver fibrosis.The results showed that the Murray’s deviation increased with the progression of fibrosis,and the value rapidly decreased after the regression,but did not return to the normal condition.Compared to the Murray’s deviation in the small branches,the value in major branches was significantly smaller,which indicates that the influence on the branch circulation of small branches is greater than that of major branches in the process of fibrosis.（3）.Dynamic evolution of portal vein structure and its mechanism in cirrhotic portal hypertensionIn this study,63 cases of BA,18 cases of primary biliary cirrhosis（PBC）and 35cases of hepatitis B-related cirrhosis（Hep-B-cirrhosis）patients were retrospectively screened.Six histological parameters,including obliteration of portal venules（OPV）,fibrosis proportionate area,septal width,nodule size,cholestasis and ductular reaction,were assessed between different types of cirrhosis by histopathology.Then,the effects of OPV and other deformations of the portal system on portal pressure in BDL and CCl₄-induced biliary cirrhosis and post-necrotic cirrhosis rat models were further investigated in a 3D manner using PCCT.In addition,the deep cellular mechanism of OPV formation based on the toxicity of bile acids was also explored.The results showed that the degree of OPV and fibrosis were significantly higher in biliary cirrhosis than in post-necrotic cirrhosis,and OPV could not be reversed by Kasai portoenterostomy for patients with BA.The 3D visualization of OPV was achieved by PCCT and the further 3D quantitative evaluation showed that the OPV rate of BDL model was significantly higher than that of CCl₄ model,and there was a significant correlation between the OPV rate and portal pressure in both models.Moreover,the lumen deformation of portal vein and the structural changes of peripheral microvessels cooperate with OPV to influence the development of portal pressure.Finally,cell experiments showed that the toxic effects of bile acids could damage endothelial cells and lead to their apoptosis,which may be an important factor for the formation of OPV.