Morphological Characteristics Of Brain Capillary Endothelial Cells And Neurons In A Ticellular System For Study On Neuroprotection And Neuroregeneration In Vitro

Objective:The brain capillary endothelial cells and neurons of the brain play an important role in the central nervous system. In vivo testing is often preferred over in vitro since it is better suited for observing the overall effects of an experiment on a living subject. However, it is difficult to study the properties of brain capillary endothelial cells in vivo because they represent a small fraction of the total cell population in the brain. Further more; the functions of neurons and capillary endothelium cells in vivo are influenced by a number of factors which are beyond our control. Adding to that, in vivo study could not neglect the individual differences between laboratory animals. In vitro Experiment of neurons or the capillary endothelium can observe a simple environment, having no uncontrollable factors. However these isolated cells which are cultured alone can't maintain a reasonable semblance of their in vivo phenotype. To provide an in vitro system for studies of brain capillary endothelial cells and neurons morphologic characteristics, we developed a new in vitro model which uses primary cultures of brain capillary endothelial cells, astrocytes and neurons to establish a tricellular co-culture model named EAN system.Methods:We chose the Sprague-Dawley (SD) rat brain capillary endothelial cells, astrocytes and neurons that can be primary cultured, and purified those cells by special ways. Consequently, these cells were planted at a certain time and space in the transwell, in order to establish EAN three cell co-culture model systems. In the same time, we cultured brain capillary endothelial cells and neurons alone in a Flask. When the system became stable, morphologic characteristics of brain capillary endothelial cells and neurons were indicated by immunofluorescence staining of microtubule-associated protein 2 (MAP-2) and glucose transport 1 (Glut-1) in the EAN system. Meanwhile, Image-pro Plus6.0 software was used to measure length of MAP-2 positive area in neurons dendrites and the ratio between major axis and minor axis of Glut-1 positive area of brain capillary endothelial cells.Results:1. In EAN system the length of MAP-2 positive area in neurons dendrites were significantly bigger than those neurons whose cultured alone (n=10, P<0.01).2. In EAN system the ratio between major axis and minor axis of Glut-1 positive area of brain capillary endothelial cells cultured was significantly bigger than those minor axis whose cultured alone(n=5, P<0.01).Conclusion:The morphologic characteristics of brain capillary endothelial cells and neurons cultured in the EAN system were significantly different from those cells cultured alone.