| OBJECTIVE:To observe the ultrastructural changes of selectively vulnerable hippocampal CA1 neurons to investigate the influence and neuroprotective effect of ischemic postconditioning after ischemic injury,explore molecular mechanisms of protective effect of ischemic postconditioning after cerebal ischemia,and,to inquire what biological role of PI-3K/Akt signal transduction pathway play after ischemic postconditioning and cerebral ischemia in tree shrews.METHODS:Cerebral ischemia was induced by photochemical reaction in tree shrew,and then alternated employed occlusion right common carotid artery 5 min and reperfused 5min at once,carried out 3 circles to establish experimental model of ischemic postconditioning.Using terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling staining(TUNEL)to label apoptosis of pyramid neuron in hippocampus CA1 subfied after cerebral ischemia and ischemic postconditioning,the animal's brian stained by 2,3,5-triphenyltertrazolium chloride (TTC)to show the infarct size of brain.The ultrastractural impairment changes in different groups were observed under the electronmicroscope.The level of Akt-Ser-473 and Akt-Thr-308 phosphorylation which are the downstream kinase of phosphatidylinositol 3-kinase(PI-3K)were detected quantitatively by Enzyme Linked-Immuno-Sorbent Assay(ELISA)during diferrent time of cerebral ischemia and ischemic postconditioning.The activated expression and distribution of Akt[pS473]and Akt[pT308]in pyramid neuron in hippocampus CA1 in different groups and different phase were surveyed by immunohistochemistry RESULTS:The counts of TUNEL positive neuron of ipsilateral in hippocampus CA1 subfied in cerebral ischemia group and postconditioning group were more than control group significantly(P＜0.01),but the counts of TUNEL positive neuron have no significant difference among all of contralateral groups(P＞0.05).After cerebral ischemia 4 hours,the counts of TUNEL positive neuron in postconditioning group and cerebral ischemia group still.have no statistical difference(P＞0.05),but the counts of TUNEL positive neuron have shown slightly decreased in postconditioning group.After cerebral ischemia 24 hours and 72 hours,the counts of TUNEL positive neuron were significant decreased in ischemic postconditioning groups than ischemic groups(P＜0.01).TTC stained shown that the infarct size of brain was diminished with ischemic postconditioning treatment.The observation with electronmicroscope displayed that the amendment of structure of mitochondria and endoplasmic reticulum in pathological condition of neuron in hippocampus CA1 subfied after ischemic postconditioning.After cerebral ischemia 4 hours,the level of phosphorylation of the Akt protein at threonine residue 308 was significant increased than control group(P＜0.05),however,in the group of after cerebral ischemia 24 hours,the level of phosphorylation of the Akt protein at threonine residue 308 was significant decreased than the group of after cerebral ischemia 4 hours(P＜0.05).In the groups of after ischemic postconditioning 24 hours and 72 hours,the level of phosphorylation of the Akt protein at threonine residue 308 were significant increased than control group(P＜0.05),and the level of phosphorylation of the Akt protein at threonine residue 308 was significant increased in the group of after ischemic postconditioning 72 hours than the group of after cerebral ischemia 72 hours.The level of phosphorylation of the Akt protein at serine residue 473 had not significant difference among all groups(P＞0.05).The result of immunohistochemistry shown that the activated expression of Akt[pS473]and Akt[pT308]were clear in endochylema and membrane in pyramid neuron on hippocampus CA1 subfield.CONCLUSION:①Ichemic postconditioning generate neuroprotective effect after cerebral ischemia by decreased neuron apoptosis in hippocampus CA1 subfied and disminished the infarct size of brain.②Akt protein would be phosphorylated at serine residue 473 ever after cerebal ischemia or ischemic postconditioning,the Akt protein at threonine residue 308 only took place transient phosphorylation during cerebral ischemia but showed activate persistently and increasingly in the group of after ischemic postconditioning.So far,this is first report about the finding.Since the complete activation of Akt needs both serine residue 473 and threonine residue 308 are phosphorylation,so the phosphorylation threonine residue 308 may determine the Akt avtiation.③The activation of PI-3K/Akt pathway may play an important role in neuroprotective effect after cerebral ischemia postcondining.The threonine residue 308 of Akt protein as an important target point of neuroprotective drug after cerebral ischemia may has potential values,deserved to be further explored.
|
PDF Full Text Request