The Relationship Of Occludin Expression With Brain Edema In Liver Cirrhosis Rat Models Induced By High Ammonia

ObjectiveHepatic encephalopathy is one of the severe complications of cirrhosis. It is the common cause of cirrhosis due to its poor prognosis and high incidence. The exact pathogenesis of hepatic encephalopathy is not fully understood by now. Several different hypotheses were put forward, among them the most important was ammonia poisoning theory. Varying degrees of cerebral edema exist in patients with hepatic encephalopathy, elevated level of blood ammonia can promote the occurrence of hepatic encephalopathy, then cerebral edema appeared. Current studies in cerebral edema shows the two main pathogenesis of brain edema is vasogenic and cytotoxic cerebral edema,which participate in the formation of cerebral edema simultaneously. The major cause of vasogenic cerebral edema is the change of blood-brain barrier (BBB) permeability. The integrity of tight junction (TJ), the most important structure in BBB, directly affects the function of BBB. In the TJ complexes, occludin protein is the main regulatory proteins. Its high level expression in the brain vascular endothelial cells enables it to work as the most sensitive and reliable sign of TJ. In recent years,the study of TJ related protein in chronic liver disease is rare. In this study, we made researches on the Relationship of occludin expression with Brain Edema in Liver Cirrhosis Rat models induced by high ammonia.Material and methodExperimental animals:healthy male (SD) rats, the rats were randomly divided into four groups 1.normal group:no intervention factors; 2.normal ammonia load group: give ammonia load at the end of experiment;3. cirrhosis group:Preparation of liver cirrhosis model, no intervention factors; cirrhosis 4.ammonia load group:Preparation of liver cirrhosis model, give ammonia load at the end of experiment. This cirrhosis models was prepared by injection of carbon tetrachloride in olive oil solution intraperitoneally.1.1ml of boold samples were collected before and 30 minutes after ammonia load respectively, detecting the level of blood ammmonia within 30 minutes.2.Preparation and judgement of model:rats were observed 2 hours after administration, corneal reflex and flip reflection were done to determine the existance of accompanied hepatic encephalopathy3.Blood-brain barrier permeability test:Rats were killed two hours after the injection of Evans blue solution. Brain tissue was dissolved in formamide to obtain supernatant after centrifugation, using 632nm light for Colorimetric. The brain tissue EB content was calculated according to the standard curve to detect the permeability changes of BBB.4.Brain tissue water content test:The brain tissue was taken out wholy after killing rats, and brain tissue water content was tested by dry wet weight method.5.Microscopic observation of liver and brain tissue after HE stain:Fix brain tissue and the right lobe of liver with 40% paraformaldehyde. Observe the its histological changes under the microscope after HE stain.6.Detect the distribution and expression of occludin in the brain tissue with immunohistochemical technique:the vascular endothelial cells of which cytoplasm was stained brown, or the cytoplasm along the membrane was stained brown linearly are positive cells.7.Semi-quantitative analysis of the expression of occludin protein was carried out ursing Western blot tests.8.Statical analysis:Quantitative date were expressed as means±SE, and were analyzed with one-way analysis of variance (ANOVA). Statistical analysis was performed using the SPSS 11.5statistical software. The results were considered statistically different at p＜0.05.Results1.the model preparation:The normal group, and normal ammonia load group and cirrhotic group with the flip reflex and corneal reflex exists, hepatic encephalopathy does not occur. In the cirrhotic ammonia load group, five rats was proven with hepatic encephalopathy, whose flip reflex and corneal reflex disappeared. Four generate ascites in the cirrhotic group, while six in the cirrhotic ammonia load group. Under the microscope observation:the liver lobule structure in the two cirrhotic group was destructed, fibrous tissue hyperplasia, with a clear sign of pseudolobuli formation. While in the normal group and normal ammonia load group such manifestations were not observed. The brain tissue of all four groups were normal both gross and under microscope.2.Ammonia test results:compared with the normal and normal ammonia load group, the blood ammonia levels in the cirrhotic group and cirrhotic ammonia load group elevated markedly. And the blood ammonia levels elevated further more, and was significantly different with other three groups.3.The results of EB contents:Compared to the other three groups,there is a higher level of EB contents in experimental group, the maxlmum arrived 1.9890 ug/g. there is a significant difference compared to the other three groups, P＜0.05.4.The results of brain water content:Compared to the other three groups,there is a higher level of brain water contents in experimental group, the maxlmum arrived 86%, there is a significant difference compared to the other three groups, P＜0.05.5.occludin protein expression:Occludin protein expression could be seen in the four groups of rats by immunohistochemical staining. The Cirrhotic and the cirrhotic ammonia load group have a weak positive staining (P＜0.05) compared with the normal and normal ammonia load group. Ammonia load group compared with cirrhosis group have a weak positive staining (P＜0.05); there was no significant difference between the normal group and the normal ammonia load group. In the Western blot analysis, the normal group (normal ammonia load group), the cirrhotic group, the cirrhotic ammonia load group expressed less occludin protein gradually. There was no significant difference between the normal group and the normal ammonia load group in occludin protein expression.Conclusions1.Animal model of liver cirrhosis can be successfully prepared by intraperitoneal injection of carbon tetrachloride in olive oil solution. The metabolic disorder of ammonia exists in animal model of liver cirrhosis. After the induction of high ammonia, blood ammonia level was elevated further, brain water content increased, BBB permeability-increased which shows that blood ammonia play an important role in hepatic encephalopathy.2.occludin(TJ-associated protein) expression was significantly decreased, suggesting that low expression of occludin in TJ may be the molecular basis of its structural damage, and it plays an important role in the regulation of BBB permeability.