| ObjectiveTo investigate the expression of aquaporin-4(AQP4)mRNA in the early stage of cerebral edema following ischemia in rats and elucidate the relationship between the dynamic changes of brain water content and expression of AQP4 mRNA.The results may reveal the function and effect of AQP4 in early stage of ischemic brain edema and be beneficial to find a new approach for the therapy of cerebral edema.Methods1.Materials1.1 Animal and grouping:66 healthy Wistar rats were randomly assigned to pseudo-operation group,operation group,TTC staining group.Each group was divided into six sub-group according to different time points(1h,3h,6h,12h, 18h,24h).Every subgroup contained five rats.1.2 Main agents and apparatus:RNA PCR Kit,PCR amplification system,gel imaging analysis system,ultraviolet spectrometer.1.3 Animal model:A revised model of Line hitch law according to Zea Longa.1.4 TTC staining:It demonstrates the model is succeed.2.Index detection2.1 Brain water content:determined by wet-dry weight.2.2 Detecting of AQP4mRNA in brain tissue:The expression of AQP4mRNA was assessed by RT-PCR.RNA1uL was extracted from the brain tissue,adding related reagents.The cDNA was synthesized by mRNA through reverse transcription,mixed the cDNA with AQP4 specific primers and related reagents,during 40 seconds with 94℃,1 minute with 50℃,1 minute with 72℃,after 35 cycles,extension 7 minutes with 72℃,the amplified products of AQP4 was mixed with equalβ-actin amplified products,then brought 5 ul mixed products in 2%agarose gel bed,electrophoresis,the ratio(Gray Of the ratio)of AQP4 mRNA to the optical density integral ofβ-actin electrophoresis products were observed and calculated by gel imaging analysis system observation and calculation,which determined the expression level AQP4 mRNA.3.Statistical processingAll data were analyzed by SPSS 13.0 statistical soft.The data from every group were presented by(?)±s,the difference of mean value among every group was performed with single factor ANOVA.Significant difference was considered if P＜0.05.Results①The brain water content(%)in operation group were 78.64±1.00,78.93±0.57,79.88±0.24,80.60±0.84,80.95±1.03,81.09±0.69;in pseudo-operation group were 77.63±0.40,77.73±0.87,77.24±0.86,77.51±0.78,77.49±0.30,77.67±0.47 for 1h,3h,6h,12h,18h,24h respectively.The comparison was significant difference (P＜0.05)②The expression level of AQP4 mRNA in operation group were 0.89±0.02, 0.93±0.07,1.06±0.06,1.08±0.07,1.10±0.03,1.11±0.23;in pseudo-operation group were 0.77±0.01,0.75±0.08,0.75±0.01,0.76±0.01,0.76±0.02,0.77±0.02 respectively. The difference between the two groups was significant(P＜0.05)③The changes of brain water content were positively correlated with the dynamic expression of AQP4 mRNA in different time-point(r=0.871,P＜0.01).DiscussionIschemic cerebrovascular disease is characterized by high morbidity,high morbidity and high mortality rates.Brain edema following ischemic stroke play a important role in the process of pathophysiological changes,leading to clinical condition deteriorated.It is one of the key factors affecting the the prognosis of ischemic stroke.The method of middle cerebral artery occlusion was used to establish the model of brain edema induced by focal cerebral ischemia,which was similar to the process of cerebral edematization.Through the dynamic changes of the study,we found that brain water content in the operation group were significantly higher than that in the pseudo-operation group,and furthermore,brain water content began to increase in operation group at 1h and gradually increased within 24 hours. Ischemia produced an increase in water content in operation group as compared with pseudo-operation group.Significant difference was shown between two groups at each time point(P＜0.05).Aquaporin(aquaporins,AQPs)is the main molecular basis of transmembrane water transport.It plays an important role in maintenance of water,electrolyte balance in brain tissue,which is involved in the formation and elimination of cerebral edema.Recent study found that the expression of AQP4 in the ischemic penumbra began to increase at 15 minutes after middle cerebral artery occlusion in rats.Then a rapid increase within the first hour and a slow increase until 24h were also shown, which corresponded well with the generation and resolution of brain edema monitored by MRI.The results showed that pseudo-operation group also had AQP4 mRNA expression.The expression of AQP4 mRNA in operation group began to increase at 1h and gradually increased in 24h.The result was significant different(P＜0.05)compared with the pseudo-operation group at different time points.Experiments also showed that there was a positive correlation between the brain water content and expression of AQP4 mRNA(r=0.871,P＜0.01),suggesting that ischemia prompted up-regulation of expression of AQP4 mRNA and increased water channel permeability,leading to the aggravation of ischemic cerebral edema.Conclusion1.The results suggested that brain water content was increased in rats after middle cerebral artery occlusion.2.The results suggested that expression of AQP4 mRNA was increased in rats after middle cerebral artery occlusion.3.The results suggested that there was a positive correlation between the high level expression of AQP4 mRNA and cerebral edema.Up-regulation of AQP4 mRNA expression could increase water channel permeability,and aggravate ischemic cerebral edema. |
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