| Cerebrovascular disease stands as one of the three leading threats against human being's health. In many countries, it's a major cause of death and the primary cause of adult disability. Among all the types of the disease, ischemic stroke is the most frequent one, which occupies about 70% incidence. As the survivors suffer high disability rate, there is a heavy burden on their families and society. Unfortunately, to date, there is little pharmacological treatment available for sufferers of this disorder. In order to develop clinically relevant neuroprotective agents, light has been thrown to the mechanisms in development of the infarct. Recent evidence has demonstrated an intimate relationship between astrocytes and neurons, thus dysfunction if one cell type is likely to compromise the function of the other. Due to the high content of gap junctions, astrocytes compose syncytia in brain. As gap junctions are relatively freely permeable to substances up to approximately 1.2 kDal, ions and metabolites can diffuse from regions of high concentration to regions of low concentration, which improve the spatial buffer capacity of astrocytes. The functional cell network arouses the interest of academic circles, and then several hypotheses have been put forward trying to explain the delayed neuron death in penumbra and the propagation of infarct. Under that background, the in vitro and vivo study was carried out, trying to find the alteration and regulation mechanism of astrocytes gap junction communication in forcal ischemic stroke and the influence of the alteration on propagation of infarct.Part One BackgroundAt the beginning of this paper, part of international and domestic documents in the domain had been summarized. The focus was put on three aspects: (1) the molecular composition of neuronal and glial gap junctions and the role played by junctional channels in the physiology and pathology in the CNS; (2) the culture of astrocytes; (3) the relationship between astrocytes gap junction communication and ischemic stroke.Part Two In vitro studyAstrocytes in primary culture were prepared from the brain of 1-day-old newborn Wistar rats as previously described by Manthorp. In brief, animals were decapitated and their brains immediately excised. After removing the meninges and blood vessels, the forebrains were cut into small pieces and dissociated with 0.125% trypsin. After the dissociation procedure, the cells were cultured in flasks in DMEM, supplemented with 20% (v/v) newborn calf serum. The cells were cultured in a 5%CO2 humidified incubator at 37°C. The cells were re-plated once confluent. After 3~4 replatation procedure, about 95% of the cultured cells stained positively for GFAP. Then the purified cultured astrocytes were resuspended and plated into 60mm flasks. When the cells reached 80% confluent, the cultures were transferred into ischemic incubator of N289%/CO25%/O27% for 24 hours and reoxygenation were undertaken for 6 hours. Similar to previous work, we found that a long period of hypoxia did not affect the viability of astrocytes, indicating that these cells are highly resistant to the lost of mitochondrial function. But at the beginning of reoxygenation, the cultured cells showed injure display under microscopy, and the morphological changes became more and more obvious as time went on. We could safely conclude that the hypoxia/reoxygenation model of cultured astrocytes was successful. Dye coupling method was taken to identify the alteration of the function state of gap junction. Confluent astrocyte cultures were used. 5% Lucifer yellow was microinjected into a randomly chosen cell. At lest 10 cells were microinjected in each flask. The index of dye coupling was scored by count the neighboring cells that accumulated the dye after 20min. The incidence of dye coupling was scored by count the injected cells showing dye coupling. The results were as follow: 1) under hypoxia/reoxygenation condition, the gap junctions of cultured astrocytes still remained open; 2) there was no obvious in...
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