| Glucocorticoids (GCs) are hormones released during stress, which are very important to maintain both normal growth and development of bodies and internal homeostasis in physiological conditions and stress. But in plasma sustained GCs of high concentrations from oversecretion during stress can make their access to brain through blood-brain barrier and endanger central nervous system(CNS). In CNS, hippocampus, a structure possessing high concentrations of receptors for GCs, is likely to be victims of excessive circulating GCs, that put forward a question of how to use GCs such as dexamethasone and methlprednisolone to alleviate cerebral edema. Many evidence support GCs can induce damages of hippocampal neurons, but the complexity of in vivo environment makes it difficult for the identification of the exact mechanism of effects of GCs, and unsystematic in vitro experiments aiming at different destinations are impossible to present enough evidence. At this point, the knowledge about effects of GCs on hippocampal neurons is still at the preliminary stage. There has been virtually no enough proofs and systematic reports. If the proccess and mechanism of insults of high-concentration GCs to hippocampal neurons can be expounded, it can present theoretical reference to proper application of GCs to cerebral edema.ObjectiveThe objectives of our experiments were to observe insults from dexamethsones of different concentrations on hippocampal neurons. Employing analysis of celluar morphology, celluar emzymology and celluar apoptosis-related proteins, these studies can make the insulting proccess and mechanism of GCs on hippocampal neurons understood more deeply. At the same time, these studies were to explore the protective mechanism of bFGF to insults of hippocampalneurons induced by GCs. Our experiments will provide a reasonable reference to GCs on cerebral edema.MethodsIn these studies, astrocyte(AS)-conditioned medium was employed to optimize primary culture of rat hippocampal neurons to obtain relatively pure culture(the ratio of neurons could reach 95% and single neural network could be observed). Microscopy, typan blue staining and MTT were used to evaluate changes of celluar morphology and celluar enzymology induced by dexamethasone of defferent concentrations. TUNEL was used to examine celluar apoptosis of hippocampal neurons induced by dexamethasone of different concentrations. By immunohistochemistry staining, the changes of apoptosis-related protein expression of Bcl-2 and Bax were examined. And at last, the effects of bFGF on apoptosis induced by dexamethasones were compared among groups.Results1. We obtained relatively pure culture through optimizing primary culture of rat hippocampal neurons with AS-conditioned medium. Compared with control, glial cells decreased significantly, the physiological apoptosis was inhibited markedly, and the quantities of cells increased, the bodies enlarged, the processes lengthened and widen. All these phenomena supported the viability of neurons were enhanced. The positive ratio of NSE in cells on day 7 in culture was about 95%, and that of GFAP was about 3%. By day 12-14, the cells reached their best states to exhibit strong refraction and third dimension and the wide neural processes formed dense network. This optimal method is a perfect one to observe various indexes of single hippocampal neuron.2. The injuries of dexamethasone to hippocampal neurons(1) After incubation with dexamethasone of different concentrations for 24 hours, the cell morphology of 10~9,10~8M groups did not change compared with control by microscopy. InlO"7, lO^lO^M groups, the processes shorten, the membranes shrinked, the refraction weakened, the bodies became smaller, and some cells presented karyopyknosis and karyorrhexis.(2) Incubation with dexamethasone of different concentrations for 24 hours, OD values from MTT of 10~9,10~*M groups did not vary markedly compared with control. In 10~7,10"*,10~5M groups, OD values varied significantly, while OD values between thr... |
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