Cerebral Ischemic Injury In The Dynamic Process Of Research And Its Application

Cerebrovascular diseases are one of the causes of death and disability in the world, and they are closely related to disturbances of blood flow and ischemia. Recent studies found that ischemic brain damage is resulted from many reasons such as functional rearrangement of glia cells, an ion balance disorder, lipide peroxidation induced by free radicals, and changes of neurotransmitters. However, the mechanisms underlying ischemia-induced cell death are currently unclar.In the present study, microdialysis-HPLC, confocal laser scanning microscopy, flow cytometry, and RT-PCR techniques were used to investigate the dynamics of ischemic brain demage and its mechanisms, further to study the protection of puerarin against cerebral ischemia.the time-course of infarect size was first observed after middle cerebral artery occlusion (MCAo) for 50 min followed by reperfusion in rats. The results showed that there is a course of development of infarct induced by MCAO. No obvious infarction was appeared until 4 hr after reperfusion. Infarction development was earlier in striatum than in the cortex. A near total infarct was appeared in striatum 24 h after MCAo. However, a delayed infarct was evident in the cortex. By day 3 post MCAO, the infarct had progressively expanded to the nearly whole area of the frontparietal cortex.To clarify the effects of neurotransmitters on ischemic brain damage, microdialysis and allied techniques were used to continuous sampling and analysis time course changes of amino acid transmitters in the extracellular fluid of striatum following MCAO/reperfusion. It was found that the level of amino acids largely increased in striatum during MCAO, and peaked on 60 min of MCAO. The levels of amino acids began to return following reperfusion and to the normal on 24 hr after reperfusion. Efflux of excitatory amino acid ischemia-evoked is stronger than inhibitatory amino acid, and it results in a higher ratio of Glu/GABA under ischemia. The ratio of Glu/GABA was returned to normal value 24 hr after reperfusion.Furthermore, the changes in intracellular Ca2+ and generation of NO in cultured hippocampal neurons were measured during oxygen-glucose deprivation (OGD) using confocal laser scanning microscopy with Fluo-3, a Ca2+ probe, and diaminofluorescein diacetate (DAF-2 DA), a NO probe respectively. Marked changes were seen during 30 min exposure to OGD, with [Ca2+]i and level of NO increasing rapidly about 2-15 min after the beginning of OGD and gradually during the later time course of OGD. The influx of Ca2+was appeared to be associated with the activated NMDA receptor on cell membrane during OGD, because MK801, the inhibitor of NMDA receptor, blocked the influx of Ca2+. These results indicated that ischemic brain damage was associated with the activation of glutamate release/Ca2+ influx/NO signaling pathway.In addition, flow cytometric analysis of Annexin-V and PI labeling cells showed that cells died after reperfusion, but not during 50 min MCAO, and necrosis appeared earlier than apoptosis after MCAO. Two patterns of cell death were co-existed in both ischemic core and penumbra. However, MCAO dominantly induced necrosis in the striatum (ischemic core), and necrosis rate in this region peaked 8 h after MCAO. The percentage of necrotic cells at this time point was significantly promoted by 1,009% as compared to sham rats. In contrast, delayed apoptosis was appeared in the dorsolateral cortex. At early time points after MCAO, no obvious apoptosis occurred in this region. However, a rapid increase of apoptosis was appeared 24 hr after MCAO, and the peak (433% of the level in sham rats) was on day 3 post-MCAO.Furthermore RT-PCR experiments indicated that there was a biphasic regulation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA expression in time- and region-dependent manner after ischemia. Following a transient increase during early time course post-MCAO, expression of XIAP mRNA decreased. A negative correlation was observed between cell apoptosis and regulation of XIAP gene in these regions.