The Correlation Between The Free-radical Production And Apoptosis After Intracerebral Hemorrhage In Rats

Background and ObjectiveIt has been proved that apoptosis is the major form of secondary brain damage following an intracerebral hemorrhage(ICH). Its hypothetic mechanism includes secondary ischemia around the haematoma, toxic effect of thrombin and the breakdown product of haemoglobin(HB), free-radical production and mediation of various inflammatory factors. The brain which contains a large portion of lipid is extremely sensitive to hypoxia and oxidative damage. Moreover, the serumal Fe3+ resulting from an ICH may lead to more free-radical production through the Fenton/Haber-Weiss reaction which triggers the H2O2 to produce more OH. Accordingly, comparing with ischemic stroke, the free-radical production may produce greater damage when an ICH occurs. Previous studies has proved that the gene therapy which inhibits free-radical productions may alleviate post hemorrhagic secondary damage, reducing apoptosis around the haematoma. The current study intervened rats'ICH model by using the hydroxy radical scavenger and observed the apoptosis process of neuron and gliacyte under different brain free-radical levels. Also, our study paid particular attention to the free-radical's effect on the expression and activation of TNF-α,caspase-3,8 which shed light on the mechanism of free-radical's act on post cerebral hemorrhagic apoptosis.Part One: The correlation of free-radical level and apoptosis after intracerebral hemorrhage in ratsI Objective: to find out the effect on apoptosis under different free-radical levels following an ICH around the haematoma.II Method: Animals were randomly divided into 4 groups: sham operated group, model group, Edaravone group 1(1mg/Kg) and Edaravone group2(3mg/kg). Respectively, each group was allocated into 7 subgroups which were 6h, 12h, 24h, 48h, 72h, 7d, 14d. By Horseley-Clarke technique(HCT), SD rats'left caudate putamen were administered 80ul of autoblood in a double administration and withdrawal way. The ICH model then was evaluated by Bederson's scale. Around the haematoma, the level of MDA and hydroxyl radical was tested by spectrophotometer and so did the process of apoptosis being tested by immunohistochemistry techniques.III Result:1.significant increase of MDA and hydroxyl radical production has been observed in the model group comparing with the other two. Significant difference of MDA and hydroxyl radical production was also observed in all other groups.2.In sham operated group, a small amount of TUNEL positive cells were found. They slightly increased as time proceeded and hit its peak at the time of 3d, then they fell after that. For the other 3 groups, TUNEL positive cells were observed at the time of 6h. They increased significantly at 24h and reached peak level at 3d; then they fell profoundly at 7d though a few still could be seen even at 14d. There was significant difference between the groups.3.There is correlation between apoptosis and brain free-radical production(r=0.2003) and MDA level(r=0.6563).IV Conclusion:Post hemorrhagic apoptosis goes hand in hand with the changes of free-radical level and the two are correlated. This indicates that free-radical acts on cell apoptosis of post ICH and free-radical may induce the apoptosis of neuron and glial cells. Part Two: The correlated pathway of free-radical induced apoptosis after intracerebral hemorrhage in ratsI Objective: To investigate the effect of post ICH free-radical on apoptosis pathway.II Method: Through immunohistochemical semi-quantitation measure, post ICH TNF-α's expression was tested, and so did brain Caspase correlating with 3 pathways by Western blot, including the expression and activation of Caspase-3, Caspase-8,Caspase-9,Caspase-12. As a result, the signal transduction pathway of post ICH free-radical related apoptosis was examined.III Result:1.The expression and secretion of TNF-αin cerebral cortex, hippocampus and parahematoma tissue increased significantly comparing with sham operated group.The different expression level of model group and drug treatment group can be found.So we could concluded that free-radical had relationship with the level of TNF-α,that free-radical could induce the expression of TNF-α.2.From half-quantitating of the protein level of Caspase-3 and Caspase-8,we found that both Caspase-3 and Caspase-8 were activated after ICH.The total level of them were higher in model group and the two-dosage drug treatment group than the sham operated group,and the level of model group were higher than the drug treatment group.IV Conclusion:After ICH,free-radical may induce the expression and secretion of TNF-α, and the expression level of Caspase-3 and Caspase-8 is upregulated.It is considered that Death Receptor Pathways may participate in apoptosis after ICH, and that is correlated with the level of free-radical.