Study On Growth And Development Of Spiral Ganglion Neurons By Electrostimulation Of Artificial Cochlea In Vitro

Objective 1?To establish and optimize the artificial cochlear-graphene-electrical stimulation device and provide an ideal platform for electrical stimulation of spiral ganglion neurons in vitro.2?To identify the artificial cochlear-graphene-electrical stimulation device from the perspective of physics and biology.3?To stimulate the spiral ganglion neurons in vitro at regular hours and in a fixed quantity using the artificial cochlear-graphene-electrical stimulation device and observe whether the growth is more mature or not.Methods 1?We isolated the hippocampus,olfactory bulb and cortical tissues from SPF FVB fetal mice and the original neural stem cells were extracted by mechanical separation and trypsin digestion.The neural stem cells were cultured by means of serum-free culture,mechanical blowing and enzymatic digestion.We also isolated the cochlear shaft tissues from SPF FVB P1 mice and used mechanical separation and trypsin digestion to extract the original spiral neurons which were cultured in serum-free culture medium.2?We placed the Australia artificial cochlear Nucleus CI24RE(ST)on the circuit board and connected the reference electrode and the graphene on the cell dish bottom by electric wires.Meanwhile,we connected the stimulating electrode and the platinum electrode on the cell dish by electric wires.In this way the circuit loop were formed by conductive cell culture system.The cultured neural stem cells and spiral ganglion neurons were in an electric field environment.We chose American singer Michael Jackson's song "Heal the World" as acoustic stimulus material,and used collier company of cochlear debug software Custom Sound 4.0 to make artificial cochlear acoustoelectric conversion.Therefore we could stimulate the neural stem cells and spiral ganglion neurons in the culture dish.Results 1? We observe the current waveform from the oscilloscope and we prove that the artificial cochlear-graphene-electrical stimulation device designed by us is in good condition from the perspective of physics.We detect the electric potential range of the device is between-560 mv and +560m V.And the electric potential range on the surface of graphene is between-60 mv and +60m V.2? We choose Fluo-4,AM as fluorescent probes to detect the concentration changes of calcium ion in neural stem cells.There are strong fluorescence when the Fluo-4 free ligands combine with the internal calcium ions of the neural stem cells,and we play the song to make artificial cochlear acoustoelectric conversion when the fluorescence quench gradually.Then we can observe the fluorescence enhance markedly because the neural stem cells receive the electrical stimulation and make the internal calcium ions flow.We prove that the artificial cochlear-graphene-electrical stimulation device designed by us is in good condition from the perspective of biology.3? The spiral ganglion neurons isolated from SPF FVB P1 mice cultured in the artificial cochlear-graphene-electrical stimulation device are given a two-day electrical stimulation(10 hours/day).And we observe the area of growth cones and the length of the filopodia increase comparing with the spiral ganglion neurons on the surface of graphene without electrical stimulation.The expression quantity of myosin-X?fscn2 and integrin?1 was higher than that in the control group through real-time fluorescence quantitative PCR detection and there is no significant difference of diap3 expression.The spiral ganglion neurons isolated from SPF FVB P1 mice cultured in the artificial cochlear-graphene-electrical stimulation device are given a seven-day electrical stimulation(2 hours/day)and there is no significant difference of the length of the neurite comparing with the group without electrical stimulation.And the length of neurite in the experimental group is increased statistically significant which is given a five-day electrical stimulation(10 hours/day).There is statistically significant of the length of the neurite in the experimental group ES1(10 hours per day)and ES2(stop 1 hour after 2 hours stimulation,a total of 10 hours per day)both given a seven-day electrical stimulation comparing with the control group and it is more significant in ES2 group.Conclusions 1? We establish and optimize the artificial cochlear-graphene-electrical stimulation device successfully and it can be used in the experimental study of electrical stimulation of cell culture in vitro.2? The cells cultured on the surface of graphene are given electrical stimulation through acoustoelectric conversion by artificial cochlea and the feasibility of the device is proven from the perspective of physics and biology.3? It can promote the growth and development of spiral ganglion neurons in vitro through the electrostimulation of cochlea combined with the high conductivity of graphene.