Experimental Study On Co-culture Of Neural Stem Cells And Corti's Organs Of Newborn Rats

In nowdays, cochlear implant is the main solution for patients suffered with severe sensorineural deafness. It is well known that the integrality of spiral ganglion neuron is the precondition of cochlear implant and affects the prognosis. Patients with severe sensorineural deafness often have a certain extent damnification or degeneration of spiral ganglion neurons no matter what pathogeny is. Therefor, it is a challenge to recover the integrality of spiral ganglion neuron for otologist. Neural stem cells have characters of multipotential differentiation and excellent tissue compatibility. They exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including humans. Neural stem cells can also be made to differentiate into a large variety of terminal cell types in vitro such as: neurons, astrocytes and oligodendrocytes. Embryonic neural stem cells which transplanted into mammalian nervous system, especially the centrol nervous system, such as brain can survive, develop and repair some functional deficits by replacement the damaged neurons and construction of new neural circuits.Many studies show that the mechanisms which control the stem cells to keep proliferation and polarization is very complicated. A lot of factors may work on it. It is also a hotspot that how the stem cells can be induced to differentiate to what we hoped. In the light of the characters of neural stem cells , we put forward a assume that whether we can induce neural stem cells differentiate into spiral ganglion neurons through simulating environment of inner ear in vitro. In this study, we hope to find a new way for managing severe sensorineural deafness through inducing neural stem cells to differentiate into spiral ganglion neurons by the coculture of neural stem cells and Corti's organ of newborn rat.Part I Study on the Cultivation and Differentiation of Neural Stem Cells Derived from Embryonic Rats in VitroObjective: To establish procedures of neural stem cells cultivation and discuss their characters of proliferation and differentiation in vitro.Methods: Neural stem cells were separated from the hippocampus in SD rat embryos(E 14) mechanically and were cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor. Single cell cloning was undertaken in order to purify the cells. The culture conditions of neural stem cells were explored and the passaging methods were investigated. Then, all neural spheres were attached to the bottom and were induced to differentiate. The indirect immunofluorescence cytochemistry were used to identify the neural stem cells and the differentiated cells with the marker of nestin for neural stem cell, neurofllament(NF) for neurons , glial fibrillary acidicprotein(GFAP) for astrocytes and Galactocerebroside(GalC) for oligodendrocytes.Results: Plentyof neural stem cells were obtained from the hippocampus by means of the mechanical dissociation and the serum-free medium. The obvious neural spheres can be observed in about 10 days and can be maintained in vitro for more than 8 weeks. The neural stem cells induced to differentiate in vitro can produce various types of neural cells. The indirect immunofluorescence cytochemistry showed Nestin positive for the neural stem cells and NF, GFAP and GalC positive for the differentiated cells.Conclusions: Plenty of neural stem cells can be obtained from the embryonic rats .These cells showed multipotential differentiation and can be induced to differentiate into various types of neural cells such as neurons and astrocytes.Part II Study on the Separation and Cultivation of Corti's Organs Derivedfrom Newborn SD RatsObjective: To observe Corti's organs derived from newborn SD rats living in the medium of neural stem cells and make preparation for the later coculture of neural stem cells and Corti's organs.Methods: Firstly, we isolated the Corti's organs of newborn SD rats (p 1) by means of microsurgery manipulation; then put them into the medium of neural stem cells (DMEM/F₁₂+B₂₇) using the approach of adherent culture; finally, we observed how the Corti's organs lived in this medium through covert discrepancy microscope and electrical microscope.Results: The Corti's organs of newborn SD rats can survive more than three weeks in the medium of neural stem cells and maintained excellent growing state. Cultivation for one week, hair cells and their cilias developed well like in vivo. As time went on, a certain extent absence of hair cells and their cilias can be observed, but the Corti's organs maintained good growing trend.Conclusions: The Corti's organs of newborn SD rats can survive in the medium of neural stem cells and maintained excellent growing trend. Hair cells and their cilias in the Corti's organs developed well like in vivo.Part III Study on Co-culture of Neural Stem Cells and Corti's Organs ofNewborn RatsObjective: To study the co-culture of neural stem cells and Corti's organ and the differentiation of neural stem cells.Methods: We co-cultivated neural stem cells and the Corti's organs of newborn SD rats (p 1) in the neuralbasical medium, then plused transwayfilter membrane to observe the co-cultivation. Mysin VIIA (marker for hair cells) and Dil (marker for neurons) were used for indirect immunofluorescence cytochemistry. , we observed how the neural stem cells differentiated in this environment through covert discrepancy microscope , electrical microscope and confocal microscope. Results: When co-cultivation for 3 to 5 days, the part of neural sphere close to Corti's organs began to differentiate and showed the chemotaxis towards the Corti's organs. On the tenth day, the fibres differentiated from neural stem cells immerged into the Corti's organ. On the fourteenth day, we observed 3 flash stripes in the area of out hair cells and 1 flash stripes in the area of inner hair cells; under light microscope we found fibres surrounded hair cells and formed clear strias. Under electrical microscope we found the fibres differentiated from neural stem cells grew towards Corti's organs through holes on the transwayfilter membrane. Immunofluorescence cytochemistry and confocal microscope showed connection had came into being between neural stem cells and hair cells.Conclusions: Neural stem cells and Corti's organs of newborn rats can be co-cultivated under neuralbasical medium; furthermore, under the induction of Corti's organs the fibres differentiated from neural stem cells showed chemotaxis. As time went on, fibres immerged into the Corti's organ and formed connections with hair cells. It merits further study to determine whether the differentiaed neurons are spiral ganglion cells .