Observations And Analysis Of The Endothelial Vacuolation In The Building Of EAN Cell Model From SD Rat Cerebral Cortex

Objective: With the increase of population ageing and the change of the masseslifestyles, the incidence of brain disease is rising significantly. Many drugs have beendeveloped to treat these diseases, but they can not be effective to control the diseasesbecause most of them can not get through the blood brain barrier (BBB). As a result, thetreatment and prevention of the brain diseases become large challenge in medical field.In our experiment, we set up EAN tricellular model system using the primary culturesof Sprague Dawley (SD) rat cerebral endothelial cells (E)、astrogcytes (A) and neuron(N) to provide a new and effective tool for research on brain diseases therapy. On thebasis of setting up the successful EAN model system, we observed and analyzed theinstability factors during the model building, improves the conditions and methods ofmodel building and make up a better and more stable system model in vitro to providemore reliable and ideal new method for research on brain diseases mechanism and newsystem for new drugs research and screening.Methods:1. We culture primary three cells successfully, then GFAP, vWF, MAP2were detected by immunofluorescence staining with to identify the purity of three cells.2. We set up the EAN model system by planting these cells in transwell system withcollagen IV，according to the different time and space.3. After the establishment ofEAN model system, we check the TEER every day and check the Pe NaF on the5th dayof the EAN co-culture model system, and identify endothelial cell tight junctionstructure and observe morphology.4. After the EAN model system building, accordingto the instable problems, we summarize the original datas and pictures of7times, anduse relevant statistic methods and Image-Pro-Plus to check and analyze the instablefactors.Results:1.We successfully isolate and culture astrocytes, capillary endothelial cells and neurons from SD rat cerebral cortex and established EAN tricellular networkmodel system preliminarily.2.We measure the TEER and sodium fluoresceinpermeability coefficient of the different models of co-culture after5days.The TEER ofEAN, EA, EN and E are22.77±1.71×cm2,19.47±1.4×cm2,11.55±0.93×cm2and8.42±1.63×cm2. The Pe NaF of EAN, EA, EN and E are45.05×106cm/s,76.08×106cm/s,52.1×106cm/s and167.1×106cm/s.3. We summarized7times ofexperimental results. Occording to the results, we find cytoplasmic vacuolation4times,and measure the TEER of the model3times and the Pe NaF of the model4times. Bythe IPP software measuring on the3rd,5th,6th and7th time original pictures. The4times results of the ratio of the sum of vacuoles to the number of cells are0.7734,9.2202,16.9858and0.455. The ratios of the sum of vacuoles to the area of the cells are0.00629,0.0397,0.09577and0.002004. The TEER of EAN model system in the4th,5th and7th experiments are measured, and the experimental results obtained are that thehighest resistance TEER are49.25×cm2,22.77×cm2and47.28×cm2. The Pe NaFof EAN model system in the3rd,4th,5th and7th experiments are measured, and theexperimental results are44.4×106cm/s,39.7×106cm/s,42.5×106cm/s and40.76×106cm/s.Conclusion:1.The TEER of the SD rat cerebral cortex EAN cell network modelmeasured is higher than that of the other cell culture models; the Pe NaF of the SD ratcerebral cortex EAN cell network model measured is lower than that of other cellculture models, so the EAN three cells model may be closer to the brain environmentthan other models.2. In the process of building the model, we found that sometimes theprimary culture endothelial cells show the cytoplasmic vacuoles phenomenon; theTEER and Pe NaF result show numerical instability phenomenon.3. Endothelialcytoplasm vacuolization phenomenon maybe influence the TEER and the Pe NaFmeasurement. In the further experiments we should try to improve the endothelial cellculture methods, ensure the stable growth of capillary endothelial cells, and correctthese destabilizing factors, in order to establish a more complete and stable system invitro model to provide a new evaluation of new drug development and screening system and an effective tool for the study of brain diseases.