| ObjectiveIschemic brain edema is one of the important complications of cerebral stroke, as well as main cause of death in early brain infarction, and now it is the focus of neurology study.Aquaporin-4 (AQP4) is a intrinsic membrane protein having an elective water-permeation ,which can increase permeability to water ,so it plays a very important role in regulation of water in brain tissue.Calcium ion is the most active ion in organism , its concentration plays a key role in regulation of cell's activity. This experiment studies the brain water content of ischemic brain edema ,calcium concentration , the change regularity of AQP4mRNA expression according to the model of middle cerebral artery occlusion(MCAO),and uses calcium antagonist Nimotop to protect it in order to discuss the effect of AQP4 and calcium antagonist in the formation of ischemic brain edema and the protect mechanism of calcium antagonist, by this way can find a new method for clinical treatment of brain edema.Materials and Methods1. Materials1.1 Animal and grouping: 80 healthy Wistar rats were randomly assigned to pseudo-operation group(n=25),operation group(n=25), Nimotop group(n=25) and TTC group(n=5) . Each of pseudo-operation group, operation group and Nimotop group was divided into five sub-group according to 6h,ld,3d,5d,7d after treatment ,every group contains five rats. 1.2 Main agents: Nimotop, RNAiso Reagent, RNA PCR Kit(AMV), DNA Maker, 6xLoadin Buffer, Erasol, agarose, Fura-2/AM, DMEM, Triton X-100, EGTA.1.3 Main apparatus: Constant temperature water cabinet, pyro-drying cabinet, Supercold refrigerator of -80℃, Low temperature freezing centrifuge, Low temperature supercentrifuge, tissue homogenater, PCR amplification system, gel imaging analysis system, ultraviolet spectrometer, dual wavelength spectrofluorophotometer.1.4 animal model: We could make them by improved way of Line hitch law according to Zea Longa.1.5 TTC staining: It donmonstrates the model is succeed.2. Index detection2.1 brain water content: determined by wet-dry weight.2.2 Calcium concentration in brain cell: determined by dual wavelength spectrofluorophotometer with Fura-2/AM as fluorescence indicator.2.3 AQP4mRNA in brain tissue: The expression of AQP4mRNA was assessed by RT-PCR.3. statistical processingAll data were analyzed by SPSS13.0 soft ware package. The data from every group were presented by x|-±s, the difference of mean vale among every group was done with single factor ANOVA. Significant difference was considered if P<0.05.ResultThe brain water content,the expression of AQP4mRNA and the concentration of calcium ion in brain cell increased after 6 hour of MCAO rats, reached peak at the 3rd day, then the level was decreased slowly ,it was still more than normal at the 7th day .They were all positively correlated with each other (P<0.01).Calcium ion antagonist Nimotop could decrease the expression of AQP4mRNA and the concentration of calcium ion.Discussion Aquaporin(AQP) discovered at the beginning of 1990th was a high elected water molecule specified protein, which regulated the water transportation and water balance. AQP4 mainly existed in astrocyte and ependymal cell, it was important constructive basis of water regulation and transportation between glial cell and brain cerebral spinal fluid, as well as intervascular transportation. We could see that there was AQP4mRNA in brain tissue of pseudo-operation group rats. In operation group, AQP4mRNA in brain tissue began to increase after 6 hour, reached peak at 3rd day. The expression of AQP4mRNA in operation group was all higher than pseudo-operation group with significant difference(P<0.05). The brain water content and the expression of AQP4mRNA had the relevance through related analysis(r=0.768, p<0.01). So AQP4 and brain edema had close correlation, the expression of AQP4mRNA and brain edema have direct ratio relations.Calcium ion looked as second messenger in cell, could synthesize and release neutotrasmitter, maintain excitability of nerve and activity of enzyme. This experiment showed that the concentration of calcium ion in operation group were dominant higher than pseudo-operation group with significant difference(P<0.05). Calcium ion concentration of operation group in ischemic brain tissue began to be higher than pseudo-operation group after 6 hours, reached peak at 3rd day. The brain water content and the calcium ion concentration had the relevance through related analysis(r=0.814, p<0.01). So ischemic brain edema and calcium ion overload had close correlation. Calcium ion concentration and AQP4mRNA also had direct ratio relations(r=0.817, p<0.01). Both of them took part in the formation of brain edema.Nimotop was calcium ion antagonist of dihydropyridine (DHPs), which can go through blood-brain barrier and had high affinity to brain tissue, it had protective effect to nerve cell in some degree. In this experiment the brain water content,calcium ion concentration in brain cell and the expression of AQP4mRNA in Nimotop group were lower than operation group, which also presented that Nimotop as calcium antagonist could reduce the inflow of calcium ion, decline the expression of AQP4mRNA in edema tissue, which lead to decrease the degree of brain edema and protect brain tissue.Conclusion1. The result show that brain water content, the expression of AQP4mRNA as well as calcium concentration in cell were all increase after MCAO. The high expression of AQP4mRNA and the increase of calcium ion can lead to brain edema.2. The result show that there existed relavence among brain water content, the expression of AQP4mRNA and calcium ion concentration in cell.3. Calcium antagonist Nimotop can reduce the expression of AQP4mRNA and the concentration of calcium in brain cell, in this way it can alleviate brain edema.
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