An Experimental Study On The Apoptotic Mechanism Of Neurocytes In Bilirubin Encephalopathy

Objective : To explore the feasibility of animal model of bilirubin encephalopathy,the method is that injecting bilirubin solution into newborn cavies' cerebellomedullary cistern.To investigate the dynamic changes of hippocampus neurocytes' apoptosis and necrosis,the expressions of Bcl-2 and Bax proteins in the animal model of bilirubin encephalopathy,to analyse the correlations among the TUNEL positive rate of neurocytes ,the positive rates of Bcl-2 and Bax proteins,and the ratio of Bcl-2 to Bax protein.Methods : Forty-two cavies within 5-day-old,they were randomly divided into 7 groups:control group,model groupl,2,3,4,5,6.Bilirubin solution was injected into the cerebellomedullary cistern of model groups (10 g bilirubin/g weight) to set up the animal model of bilirubin encephalopathy. The cavies of model groups were sacrificed at the time of 2,6,12,24,48,72h after injection,respectively.We injected normal saline into the cerebellomedullary cistern of cavies in contrl group,then killed them at 24h after injection.The rates of neurocytes' apoptosis and necrosis,the expressions of Bcl-2 and Bax proteins were detected by Annexin V-FITC / PI label,HE stain,immunohisto-chemical method and TUNEL in the hippocampus. The correlations among the TUNEL positive rate of neurocytes ,the positive rates of Bcl-2 and Bax proteins,and the ratio of Bcl-2 to Bax were analysed.Result: (1) The cavies of control group came round soon and respired calmly with normal behaviors. The cavies of model groups appeared abnormalbehaviors in different degree.(2)Under light microscope,the neurocytes' modalities and structures were normal in control group.The brain tissues were stained with yellow bilirubin in model groups.Some neurocytes shrinked, chromatin condensated,aggregated and fragmented in the hippocampus.These changes accorded with morphologic changes of cell apoptosis.At the same time,some neurocytes swelled, cytoplasm cavitated and nucleus dissolved, even bcame"red neurons",which indicated necrotic neurons.(3) Using Annexin V-FITC / PI label,we observed the dynamic changes of neurocytes1 apoptotic and necrotic rates.The obvious increase of apoptotic rates began at 6h after injection,and reached the peak at 24h in the hippocampus,then decreased gradually.The evident increase of necrotic rates began at 6h after injection,and reached the peak at 12~24h in the hippocampus, then decreased quickly. The dynamic changes of neurocytes' apoptotic rates detected by Annexin V-FITC / PI label and TUNEL were consistent.(4) The expressions of Bcl-2 and Bax proteins were detected by immunohistochemical method in the hippocampus. There was a significantly negative correlation between the TUNEL positive rate and the positive rate of Bcl-2 protein expression, while a significantly positive correlation existed between the TUNEL positive rate and the positive rate of Bax protein expression.Conclusion: The method that injecting bilirubin solution into newborn cavies' cerebellomedullary cistern to set up the animal model of bilirubin encephalopathy is feasible. The phenomena of neurocytes' apoptosis and necrosis exist in the model cavies' hippocampus of bilirubin encephalopathy. It's crucial to prevent bilirubin encephalopathy.