Changes of expression of FADD and Daxx following focal cerebral ischemia in rats / 中华神经科杂志

ABSTRACT

Objective To investigate the changes of expression of Fas-associated proteins named Fas-associated death domain protein(FADD)and death-associated protein(Daxx)in the ischemic penumbra following transient focal cerebra ischemia in rats.Methods ①Adult male Sprague-Dawley rats were randomly divided into the sham-operated group and the cerebral ischemia model group.Rats underwent right middle cerebral artery occlusion(MCAO)for 2 h and reperfusion for 1,3,6,12 and 24 h using an intraluminal suture technique.The expression of FADD and Daxx mRNA and protein were measured with methods of immunohistochemistry.Western blot and reverse transcription-polymerase chain reaction(RT- PCR)respectively were used in the ischemic penumbra of rats.②Double-label fluorescence confocal laser scanning microscopy(CLSM)was performed to monitor FADD and Daxx intracellular location before and after ischemia.Results RT-PCR,Immunohistochemistry,Western blot experiments indicated that a very low level of FADD mRNA and protein were detected in the cerebral cortex of sham rats.The expression level both of FADD mRNA and protein increased significantly at 3 h after reperfusion,peaked at 12 h,then declined markedly at 24 h in the ischemic penumbra of model rats.RT-PCR,Immunohistochemistry indicated that a relatively high level of Daxx mRNA was detected in the cerebral cotex of sham rats.The expression level of Daxx mRNA increased significantly at 3 h after reperfusion and persisted to 24 h at a high level,whose protein had a same change of expression level in the ischemic penumbra of model rats. Immunofluorescence double-staining laser scanning by CLSM showed that the immunoreactivity of FADD was located in cytoplasm,and the intracellular translocation of the immunoreactivity of Daxx from nucleus to cytoplasm was monitored by measuring the green fluorescence after ischemia.Conclusion The transient upregulation of FADD and the persistant high level of expression of Daxx may contribute to neuronal apoptosis following cerebral ischemia/reperfusion.