| Background:Acute brain injuries like ischemia and reperfusion (I/R) can induce delayed neuronal death that often occurs in the pronumbra area around the ischemic core. The underlying molecular mechanism is yet unclear. Overactivation of NMDA receptor has been proposed as a key player for glutamate-induced neuroexcitotocixity. Notably, recent studies show that NMDA receptor is not only located in the postsynaptic density-associated synaptic membrane, but a significant proportion of the receptor is also distributed to the perisynaptic membranes, and intracellular light membranes, whereas activation of these different receptor pools may render them a distinct physiological or pathological effect on postsynaptic neurons.Interestingly, our preliminary study found that Src, a core tyrosine kinase functioning to integrate synaptic signalings to synaptic plasticity by regulating receptor trafficking, was significantly activated after I/R, and NMDA receptor subunit NR2A and NR2B were rapidly clusterd to synaptic membrane from perisynaptic and intercellular membranes after I/R. Thus, we hypothesize that Src-mediated phosphorylation signaling leads to the postsynaptic clustering of NMDA receptors after brain I/R, thereby increasing glutamate receptor number and changing their composition at postsynaptic membrane, and consequently resulting in neuronal death.Objective:To investigate the molecular mechanism of delayed neuronal death induced by brain ischemia and reperfusion and neuroprotective mechanism of PP2.Methods:In this study, rats with global cerebral ischemia-reperfusion model are used. And before30minutes rats have global cerebral ischemia, drugs are administrated into the ventricular. According to the time of reperfusion,0.5h,4h and24h. On arriving reperfusion, brain tissue of rat is fixed by formalin and stained by HE. The number of survival neuron in hippocampal CA1between the groups is observed. Under anesthesia, brain tissues from another part of the rats are rapidly frozen in liquid nitrogen. And then, brain tissues from hypothalamic dorsolateral cortex are separated in the-20℃, the various subcellular fractions are separated by differential centrifugation, the phosphorylation status and distribution of Src kinase, NMDA receptors and PSD protein in the cerebral cortex and various subcellular fractions are analyzed by biochemical means.Results:1. With cerebral ischemia and reperfusion for72h, the number of neuronal death in the hippocampal CA1from PP2treatment group with intraventricular15ug PP2given30min before ischemia was significantly less than that of the control group according to pathologial section.2. That the activation status of Src protein kinase in the postsynaptic membrane was super after cerebral ischemia and reperfusion confirmed by Western Blot. After the brain tissue homogenate, the homogenate (HOM) are separated for the different components of the postsynaptic membrane (SYN) and extrasynaptic membrane(EXTRA) by differential centrifugation and then the expression and distribution of Src are analyzed by Western Blotting. Compared to the control group,0.5h after the brain ischemia and reperfusion, SRC-P (Y416) expression in the SYN component was significantly higher (p<0.01), reached peak after reperfusion4h (p<0.01), subsequently reduced;0.5h after reperfusion SRC-P (Y416) expression in the EXTRA component was also was significantly higher (p<0.05), and there after gradually decline. In contrast,0.5h after reperfusion SRC-P (Y527)expression in the SYN component significantly decreased (p<0.01), up to4h after reperfusion, and subsequently increased (p<0.01). The Y416can enhance Src activity while the Y527inhibit Src activity. Therefore, these results suggest that Src protein kinase in postsynaptic membrane was overactivitated after cerebral ischemia and reperfusion.3. After cerebral ischemia and reperfusion, the NMDA receptor transferred from the perisynaptic membrane and intracellular vesicles to postsynaptic density. After the brain tissue homogenate, the homogenate (HOM) are separated for the different components of the postsynaptic membrane (SYN) and extrasynaptic membrane (EXTRA) by differential centrifugation and then the expression and distribution of NR2A and NR2B are analyzed by Western Blotting. Compared to the control group,0.5h after the brain ischemia and reperfusion, NR2A expression in EXTRA components significantly reduced (P<0.01),until4h (P<0.05) and24h (P<0.05) after reperfusion; NR2A expression in LM components in reperfusion at each time point were decreased significantly (P<0.01).With NR2A similar, NR2B expression and distribution in EXTRA components0.5h after reperfusion significantly decreased (P<0.01) and until24h after reperfusion (P<0.01). The difference is the NR2B expression in HOM significantly decreased4h after reperfusion (P<0.01) and until24h after reperfusion (P<0.01); the NR2B expression in LM components in reperfusion at each time point was significantly decreased (P<0.01). These results suggest that after cerebral ischemia and reperfusion, the NMDA receptor gathered from the perisynaptic membrane and intracellular vesicles to the postsynaptic membrane.4. That PP2can downregulate the phosphorylation expression of NMDAR2B, NMDAR2A, PSD93and Src after cerebral ischemia was confirmed by Western Blot.4h after cerebral ischemia and reperfusion, Tyr-P expression in the PP3control group increased, but compared with the PP3control group, Tyr-P expression in the PP2treatment group down-regulated significantly (p<0.01). PSD93-P (Y340) in the PP3control group increased significantly(p<0.01),and PP2injection did not significantly inhibit the rise.4h after cerebral ischemia and reperfusion, compared with the sham group, NR2B-P (Y1246) in PP3group and PP2up-regulated significantly (p<0.05). NR2B-P (Y1325) in PP3group significantly increased (p<0.05). However, Compared with the PP3group, the expression of NR2B-P (Y1325) in PP2group decreased significantly (p<0.01).Conclusions:1. After cerebral ischemia and reperfusion, src protein kinase in the postsynaptic density was overactivate and the phosphorylation levels of NMDA receptor in postsynaptic membrane increased significantly. 2. After cerebral ischemia and reperfusion, the NMDA receptor transferred and aggregated from the perisynaptic membrane and intracellular vesicles to postsynaptic density.3. PP2via the inhibition of Src kinase prevent the delayed death of neurons resulting from cerebral ischemia and reperfusion.In short, through this experiment, we found that the PP2played protective role in the delayed death of neurons resulting from cerebral ischemia and reperfusion.After cerebral ischemia and reperfusion,the position migration of NMDA receptors in the postsynaptic membrane was observed and its possible mechanism was analyzed,which provided a experimental and theoretical basis for clinical use of Src kinase inhibitors in the treatment of cerebral ischemia in the future. |
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