Morphological Characteristics Of Astrocytes In EAN–a Tricellular System For Study On Neuroprotection And Neuroregeneration

Objective:During the research of the neuroprotection and the neuroregeneration, that often uses two different methods, which are in vitro experiment and in vivo experiment. The cell still retained similar original body characteristics in vivo experiment, but, its environment is complicated, which cannot control a several of factors, and it also cause the barrier of analysis to the interference of single factor. However, monoculture has neglected the interaction of different cells in the brain (such as, brain capillary endothelial cells, astrocytes and neurons). As a result, the co-culture technique has appeared. Generally, the primary culture cells introduce from the different species of animal to using the co-culture. According to several scientific references, those two methods have ineluctable discrepancy that usually affects an experiment result. In order to gain more objective and effective experiment platform, the Transwell technique of this experiment usage creates a three kind of the Sprague-Dawley (SD) rat's primary culture cells (brain capillary endothelial cells, astrocytes and neurons--EAN) in the co–culture model system. From different previous studies, the primary culture cell still retained original partial characteristics in vivo and it also realizes the simulation to be similar under the blood brain barrier structure and the objective micro-environment survival, the growth, the differentiation, the function, the damage, the repair and the interaction. Therefore, it can enhance experiment's accuracy and the reliability, has provided a new scientific credible platform for the future neuroprotection and neuroregeneration research.Methods:According to several scientific references and the laboratory work experience, the author chooses the SD rat brain capillary endothelial cells, astrocytes and neurons that can be primary cultured, through the GLUT-1, GFAP and MAP-2 to mark brain capillary endothelial cell, astrocyte and neuron separately to measure the purity of cells. These cells are planted at a regular time and space via the transwell in sequence to establish EAN three cell co–culture model system. When the system became stable, using the confocal microscopy system, photographs astrocyte's GFAP immunity fluorescence ICON to intuitively describe its change of morphological character in the monoculture and the EAN model system separately. By using Image-pro Plus6.0 software, it can measure the peripheral projection length and area of astrocyte's GFAP positive structure and the central complex area of astrocyte's GFAP positive structure. Basic on the statistics method, analysis its change of morphological character objectively. Simultaneously using the three dimensional reconstruction technology, analysis the astrocyte's GFAP positive structure that projects on the Transwell semi-permeable membrane pore growth status.Results:1. The central complex area of astrocyte's GFAP positive structure is 940.00±178.15μm2 in the EAN model, while in monoculture is 1946±173.01μm2. Both difference has statistics significance (P<0.05).2. The peripheral projection length of astrocyte's GFAP positive structure is 156.33±17.64μm, and the area is 2196.00±241.50μm2 in the EAN model. The peripheral projection length of astrocyte's GFAP positive structure is 108.48±13.33μm, and the area is 515.50±156.84μm2 in monoculture. Both the peripheral projection length of GFAP positive structure difference has statistics significance (P<0.05), and also both the peripheral projection area of GFAP positive structure difference has statistics significance (P<0.05).3. During Transwell the semi-permeable membrane pore that discover the existence of the astrocyte's GFAP positive structure.Conclusions:1. Using the primary culture the brain capillary endothelial cell, the astrocyte and the neuron established in the EAN three cell co–culture model system, which the GFAP positive structure shape of astrocyte compare with the condition of the sole culture existing difference. Compare to monoculture of astrocyte, the astrocyte's GFAP positive structure center of complex area reduces in the EAN model, but the periphery of the GFAP positive structure projection the length and the area increase.2. In the EAN model the astrocyte's GFAP positive structure can grow along Transwell in the semi-permeable membrane pore,which cultivates the direction of the brain capillary endothelial cell to growth.