The Role And Mechanism Of Bile Duct Hyperplasia And Angiogenesis In Bile Duct Ischemic Injury

Establishment of rat Liver Cirrhosis by Carbon Tetrachloride Subcutaneous InjectionAim:To establish a rat model of liver cirrhosis for the study of bile duct ischemic injury in rats.Methods:Fifty-six male SD rats were randomly divided into two groups:(1) cirrhosis model group (n=50) and (2) control group (n=6). The rats in cirrhosis model group were induced by subcutaneous injection of 50% carbon tetrachloride olive oil at 0.2 mL/100 g twice weekly for 12 weeks, while the rat in control group were injected with olive oil at 0.2 mL/100 g twice weekly for 12 weeks subcutaneously. Two rats were killed during fourth week, eighth week and twelfth week, respectively.Results:In cirrhosis model group, periportal steatosis was observed in rat liver after 4-week injection of carbon tetrachloride, and portal fibrosis was obvious after 8-week injection. Rat model with liver cirrhosis was established using carbon tetrachloride after 12-week injection. Obvious nodular cirrhosis was visible and pseudolobule formation was observed under an optical microscope in all rats accepted 12-week injection of carbon tetrachloride. Two rats in cirrhosis model group died during the establishment of liver cirrhosis model, and no rats died in control group. The model success ratio was 82%.Conclusions:1. The method of subcutaneous injection of 50% carbon tetrachloride olive oil to induce rat liver cirrhosis model was safe and effective, with high model success ratio.2. The method was simple and suitable for the preparation of large quantities of rats with liver cirrhosis.The role and mechanism of bile duct proliferation and angiogenesis in the ischemic injury of bile ductAim:To investigate whether the bile duct proliferation and angiogenesis play a protective role in the ischemic injury of bile duct and the corresponding mechanism, through the study on the bile duct density and microvessel density of the liver before and after bile duct ischemia in normal rats and cirrhotic rats.Mathods:Sixty-four male SD rats were divided into two groups (n=32):(1) normal group (rats with normal liver); (2) cirrhosis group (rats with cirrhotic liver). Each group was further divided into Oh,6h,3d and 14d subgroups according to ischemia time (n=8). A cirrhosis model was induced by subcutaneously injection of carbon tetrachloride for 12 weeks. All rats were underwent separation of liver ligaments, and ligation of the main hepatic artery and the extrahepatic peribiliary vascular plexus, to prepare the model of bile duct ischemia. Blood samples were centrifuged and supernatants were collected. After that, total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using an automatic biochemical analyzer. Biloma information was examined. Liver tissues were cut as 4-μm tissue sections, stained with HE and Masson. Bile duct ischemia injury was assessed using the bile duct injury score. Immunohistochemistry for CD34, CK19 and Ki67 was performed. CD34 was stained to assess the microvessel density (MVD) and angiogenesis, and staining of CK19 and Ki67 was used to evaluate the bile duct density and the proliferation of cholangiocyte, respectively. SPSS 19.0 statistical software was used for data processing.Results:In all animals, a total of three biloma cases were found in normal rats with bile duct ischemia for 14d. In the normal rats with bile duct ischemia, TBIL and DBIL levels increased significantly after 6-hour and 3-day surgery, respectively (P<0.01), and the levels both increased to the peaks after 14-day surgery (P<0.01). In the cirrhotic rats with bile duct ischemia, TBIL and DBIL levels both increased significantly after 6-hour (P<0.01), began to decline after 3-day surgery (P<0.01), and after 14-day surgery the levels declined to the levels as before the surgery (P>0.05). The peak levels of TBIL and DBIL in the cirrhotic rats were also significantly lower than that in the normal rats after bile duct ischemia, respectively (P<0.01). Bile duct injury score showed that the bile duct injury was the most severe in normal rats and cirrhotic rats with bile duct ischemia both for 6h (P<0.01). With the prolongation of ischemic time, the bile duct injury scores decreased gradually, and restored to the level before ischemia returned to the level before the ischemia in cirrhotic rats after bile duct ischemia for 14d(P>0.05). The microvessel density, bile duct density and activity of cholangiocyte proliferation were all higher in cirrhotic rats than those in normal rats before ischemia and after ischemia for 6 hours, 3 days and 14days, respectively(P<0.01).Conclusions:1.The degree of bile duct injury gradually reduced, accompanied by bile duct proliferation and angiogenesis, after bile duct ischemia in both normal and cirrhotic rats.2. The bile duct density and microvessel density were both higher in cirrhotic liver than that in normal liver, and the degree of bile duct injury was less in cirrhotic rats.3. The bile duct proliferation may compensate for ischemia injury of bile duct and the angiogenesis may reconstruct the bile duct blood supply, thus exerting protective effect on ischemic injury of bile duct, which provide a new idea and target for treatment and prevention of bile duct ischemic injury.