The Study Of The Brain Neurovascular Unit Model In Vitro

BackgroundThe brain neurovascular unit is an overall concept which including neurons, glial, microvascular and so on, and is of great significance in the research and treatment of brain vascular diseases. Combined with transwell, our research team had established the brain neurovascular unit model with neurons, astrocytes and microvascular endothelial cells of rat. Under the anoxia-reoxygenation, the BBB function change, cell damage and protection reaction was studied in the triple co-culture model. This work was supported by the Ministry of Education Doctoral Program special fund (No.20110182110012) and the Key Projects of Chinese Medicine Research of Chongqing Municipal Health Bureau(No.2010[60]2010-1-4).Objective1. Stabilizing and improving the methods for isolation and culturing the three types cells, and the method of establishment of in vitro NVU model.2. Studying the cell-specific protein changes at different time points and the dynamic interaction between the three types of cells. Verifying the relationship of NVU between in vitro and in vivo under hypoxia eoxygenation injury.Methods1. To improve the isolating, culturing and identification of three types cellsImprovement research group pre method of using DMEM/F12medium containing10%FBS, inhibited the growth of non-neuronal cells purified with cytarabine and identified the neurons with NSE. In this study, the DMEM/F12medium supplemented with B27serum-free culture was used, neuron-specific microtubule-associated protein2(MAP-2) were identified. With the early method, the astrocytes were isolated, cultured, purified and identified. Distinguished from "Twice Enzyme Digestion Combine gradient centrifugation Method", in this study, we added BSA directly to cerebral cortex and homogenized, microvessel fragments were digested after gradient centrifugation.2. Stabled and improved the methods of establishment the neurovascular unit vitro model and its anoxia-reoxygenation damageThe neurons were directly plate the bottom of the hole and purified. After culturing for10days, replaced the translucent transwell with transparent transwell, lmL astrocytes suspension were planted in the outside of the transwell membrane, and1d after, the purified brain microvascular endothelial cells were planted in the inside of the transwell membrane for3d. Replaced with serum-free medium, the co-culture system was put into a sealed box which continuous feeding the mixed gas at37℃. After4h, the co-culture system was replaced by reoxygenation liquid and incubated at normal condition. By inverted phase contrast microscope, the three cell growth state was observed. The4-hour leak test and transendothelial cell resistance value detection were carry out.3. Studied the dynamic changes of specific proteins of the brain vascular unit fater hypoxia reoxygenation injuryAt the six time points of. before hypoxia. reoxygenation0h.2h,6h.12h and24h. the proteins of three types of cells under different culture conditions were extracted. After pre-processing, the GAP-43of neurons, Claudin-5of microvascular endothelial cells and AQP-4of astrocytes were detected with western blooting.Results1. The three type cells had morphology uniform and showed their typical growth characteristics. The purity of neurons was more than95%. the astrocytes more than96%and microvascular endothelial cell more than99%.2. In the co-culture system, the three types cells grew well, the astrocytes and microvascular endothelial cells form a monolayer after3d. The liquid level had no significant decline in the4-hour leak test. The value of TEER was reached268.3±6.5(Ω cm2). After hypoxia reoxygenation injury, the three types cells growth state changed in varying degrees and the cell gap was significantly,4hour leak test was positive and the value of TEER was significantly decreased to227.1=3.3(Ωcm2).3. The expressions of GAP-43and Claudin-5in the triple co-culture were more than the double-culture and single-culture. The expression of AQP-4in astrocytes single-cultured was more than double co-culture and triple co-culture. After hypoxia reoxygenation injury, co-culture could restraine the decrease of GAP-43and Claudin-5, and inhibited the expression of AQP-4, especially in triple co-culture.Conclusions1. Through adding BSA directly in the cortex fragments, homogenizing and gradient centrifugation, the microvascular segments were successfully separated. After passaging one, the purity of microvascular endothelial cells was more than99%. By serum-free culture method containing B27, the purity of neurons culture were more than95%. Combined with subculture shaking, the purity of astrocytes culture was more than96%.2. In this study, by using transparent transwell, the triple co-culture model was established. In the model, the state of cell growth state was in line with in vivo, and has the basic function of BBB.3. After hypoxia reoxygenation, the expression changes of GAP-43, Claudin-5and AQP-4of triple co-culture model were in line with in vivo characteristics. The triple co-culture model could be used as a in vitro model to investigate the cerebral diseases and drug screening.