| Ischemic stroke causes different degrees of brain damages,repairing these damages are critical in diagnosis,prognosis and treatment of brain ischemia.In the model of global cerebral ischemia in mice,transient ischemia leads to structural impairment of the dendrites and dendritic spines in cortical neurons,which could be reversibly recovered after timely reperfusion.This reversible change provides hope for the treatment of neuronal injury in the brain.The change of neuronal dendritic spine structures must be accompanied by the change of synaptic structures.As an important part of synaptic structures,the changes of synaptic molecules in the process of reversible recovery of neuronal structure are not clear.In this study,the structural changes of dendrites and dendritic spines during transient cerebral ischemia-reperfusion were observed by confocal microscopy and two-photon in vivo imaging.The density of synapses was counted,and their related proteins were marked by immunofluorescence staining.Furthermore,the expression of synaptic molecules was explored by means of molecular biology.Finally,the behavioral test was used to observe and evaluate the effects of transient cerebral ischemia on the behavior and activity of mice.The results showed that the dendrites of pyramidal neurons in the fifth layer of the cerebral cortex of Thy1-YFP H-line transgenic mice were beaded after 20 min of bilateral cortical artery ligation(BCAL).Interestingly,confocal microscopic imaging showed that after transient cerebral ischemia,this structural disruption was mainly observed on the apical branches of the dendrites but not on the main shafts of the dendrites.Results of in vivo two-photon imaging showed that some of the dendritic spines were lost after ischemia but then reappeared in their original position during reperfusion.We also utilized immunofluorescence co-localization staining to quantitate the number of synapse connections and the density of excitatory postsynaptic scaffold protein PSD-95,and found that they decreased significantly after 20 min-BCAL,but gradually recovered during reperfusion,which is consistent with the changes of dendritic spines at different time points following ischemia-reperfusion.Through the methods and techniques of molecular biology,it was found that the change of PSD-95 protein expression level was consistent with the staining results.At the same time,the results of staining and WB also showed that the phosphorylation level of Synapsin1 increased significantly after 1 hour of reperfusion.At the transcriptional level,the m RNA expression of PSD-95,Synapsin 1,Synaptophysin,AMPAR 1 and NMDAR 1decreased significantly after ischemia 20 min,increased significantly after 1 hour of reperfusion,and then gradually returned to the expression level of Control group after3 hours of reperfusion.At the behavioral level,the grip strength of the forelimb and time of walking on rotarod of ischemic mice were significantly decreased at 1 day after stroke,and gradually recovered after 3 and 7 days of reperfusion.Intriguingly,it was found that the total distance and time of spontaneous activity increased in the ischemic mice,respectively shown as the increased activity in the central area and decreased in the peripheral area of the testing box.These results suggest that reversible changes of neuronal dendrites and dendritic spines occurred during transient cerebral ischemia-reperfusion,and this reversible change also occurred at the protein expression level of PSD-95 and the m RNA expression level of synaptic-related molecules.The behavior of mice also recovered after cerebral ischemia-reperfusion,and the activity ability was even enhanced.The results of this study provide a certain reference basis for the repair of neuronal dendrites and dendritic spines after stroke. |
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