Study On Cell-cell Communication Based On Calcium Dispassive Structure In Multi-BV-2Microglial Cells

The dissipative structure theory is a widely applicable theory and provides very effective support for various disciplines, especially in life science. Molecular oscillations and concentration waves are two important dissipative structures at molecular level, which are referred to calcium oscillations and calcium waves specific for calcium. Intracellular calcium is an important second messenger in cells, as the signal encoding from its temporal and spatial information could direct various metabolic activity of the cells. Thus, the temporal and spatial information of calcium signaling, especially its two important dissipative structures, has been wildly studied. Cell-cell communication is the most important prerequisite for the existence and maintenance of multi-cellular organism, especially for microglia-the first immune defense of the nervous system. The cell-cell communication in microglia is particularly important for its immune function, but the specific mechanism is still not clear. Therefore, under the guidance of the dissipation theory, we use the fluorescence imaging and microscopic local stimulation techniques to study the cell-cell communication based on calcium dissipative structure in microglial cells.In order to obtain real-time images of calcium oscillations and calcium waves, we constructed weak light micro-imaging system with high temporal and spatial resolution, based on the wide-field fluorescence microscopy and electron multiplying charge-coupled device (EMCCD). In addition, we also constructed a microscopic local stimulation system by combining a three-dimensional microscopic operating system and a microinjection control system.The spontaneous oscillations of the cytosolic calcium concentration in multi-BV-2microglial cells were studied using the high-spatiotemporal-resolution imaging system. First, cross-correlation analysis of the temporal dependence of the oscillations indicated the existence of cell-cell communication. Then, short-time imaging analysis showed that spontaneous calcium oscillations resulted from calcium waves generated by other cells as well as from calcium elevation inside the cell. Thus, cell-cell communications could induce random spikes of spontaneous calcium oscillations. At last, numerical simulations based on the minimal model for calcium oscillation suggested that there existed threshold effect in calcium oscillations and the cell-cell communications could induce random spikes of spontaneous calcium oscillations in the multi-cell system.Intercellular calcium communication plays an extremely important role in the process of immune function of microglia. In order to study the propagations and mechanisms of intercellular calcium waves, we used the microscopic local stimulation system to apply mechanical stimulus in BV-2microglial cells with different planting densities. Results showed that calcium signaling responded to mechanical stimulus and propagated to neighboring cells in isolated BV-2microglial cells, which suggested that the paracrine pathway mediated intercellular calcium waves. And the paracrine message may be ATP. We also found similar intercellular calcium waves in the densely planted BV-2microglia cells, and both paracrine and gap junction pathways were involved in the mechanically-induced intercellular calcium waves. More importantly, TNT-like structure between BV-2microglial cells may also mediated the propagation of intercellular calcium wave. Thus, our studies demonstrated that three distinct pathways could mediate the intercellular calcium waves in BV-2microglial cells.In summary, the calcium oscillations and intercellular calcium waves in multi-BV-2microglial cells were imaged to study the mechanism of intercellular calcium communications, and three distinct pathways were demonstrated to be involved in the communications. The results are able to provide a new perspective and experimental support for microglia related immune diseases, and a new experimental model to study the dissipative patterns of calcium signals.