The Culture, Natural Differentiation And Transplantation Of The Otocyst Cells Of The Mouse And The Plasticity In The Central Auditory System After Cochlear Ablation

Loss of hair cells and spiral ganglion cells which involve sensorineural hearing impairment, is irreversible, and is usually followed with changes in auditory center. Just only for the characteristic of the mentioned above, stem-cell-implantation is the most hopeful methods commended in therapy of sensorineural hearing impairment. Up to now, some researches have showed that stem cells possess potentials to differentiate into hair-cell-like cells or neurons after cochlear implantation, however, people little knows about the corresponding auditory center. So, to study cells implantation in hearing loss causing the effect of auditory center, we attempt to cultivate cells from otocyst and further explore the effect of auditory deprivation in morphology along with the feasibility in implanting otocyst cells and otocyst by cochlear operation.PartⅠ: Culture and Induced Differentiation of Otocyst Cells from MouseObjective: To study the feasibility of isolation and culture of stem cells from the otocyst of mouse embryos, and characterize the differentiation of Cells from otocyt. Methods: 22 pregnant adult mice were enrolled with study. At 9.5-10.5 days post coitum, otocysts were dissected out successfully from embryos, and then were dissociated in Hank's Buffered Salt Solution (HBSS). After digestion, cells from otocyst were cultured in serum free medium with BFGF and EGF in vitro. After formed otosphere, cells were purified in the form of otosphere and then passaged with neurosphere assay. Immunocytochemistry (ICC) and RT-PCR were taken. To explore the ability of differentiation, cells from otosphere were cultured with serum medium and examed with ICC and RT-PCR after 15days in serum medium. Antibodies against nestin, NSE, myosinVIIa and synaptophysin were used being the marker of stem cell and inner ear cells and neurons. Results: Otocyst was formed by invagination of the embryonic ectodermal tissue near the hindbrain. 22 Otocysts were obtained successfully from embryonic mouse. Otospheres were fromed after 3 days cultured in serum free medium in vitro. Results of ICC and RT-PCR suggest nestin was expressed in cells from otoshperes. After 15 days culture on adherent in the medium containing serum, cells were differentiated into cells with positive marker expression of myosinVIIa and NSE and synaptophysin. Some transcription factor including BMP7, Nestin, SOX2, PAX6, myosinVIIa and jagged-1 were presented with RT-PCR. Conclution: Cells with self-renewed capacity and potential multi-differentiation exist in the otocyst of mouse. Cells of otocyst show the general charactization of common stem cells. They can especially differentiate into hair cells and neuron in vitro.PartⅡ: Transplantation of Otocyst and Cells from Otocyt into the Cochlear of MouseObjective: To study the characters of migration and survival of Cells from otocyt s in cochlear and explore the ability of differentiation for inner ear cell and neurons in vivo. Methods: After labeled with Hoechst 33342, cells from otocyst were transplanted into the cochlear of healthy adult mouse. Meanwhile, tissues of otocyst were transplanted into the cochlear of normal adult mouse. At one week post-grafting, after perfusion in heart with 4% paraformaldehyde in 0.01 M phosphate-buffered saline, The temporal bones were collected and immersed in the 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) at pH 7.4 for 4 h at 4℃. Cryostat sections 3μm thick were made, and the mid-modiolus sections were used for histological analysis. Antibody aginst myosinVIIa and synaptophysin were used to test the differentiation of Cells from otocyt s invivo. Results: At one week post-grafting, Cells from otocyt were localized on the surface of basilar membrane of cochlear, and migrate into scala tympani and scala vestibuli. For the tissues of otocyst, survival and proliferation were also found in scala tympani. Conclusion: Cells from otocyt can survive on the surface of base membrane of cochlear and migrate into scala tympani and scala vestibuli,. The tissues of otocyst were also found in scala tympani, and can proliferate. These cells have the possibility of treatment of deafness.PartⅢ: Morphometric Study of the Cochlear Nucleus of Mouse Model of Cochlear AblationObjective: To study the morphological features of the auditory centre induced by the mouse model of auditory deprived. Methods: 20 BABL/c mice were used in the present study. Mice were checked for normal hearing by auditory brainstem response (ABR) prior to the study, were divided into experimental group and control group at random. Afer the lateral wall of the dorsal and mastoid bulla mouse was removed, the cochlea was destoried in experimental group. The two groups mice were also checked by auditory brainstem response after operation. The animals were allowed to survive 4 months. The brain stems were continuously sectioned. Histological sections of the cochlear nucleus were evaluated with serial sections and several strains for neurons, and fibers. The volume of cochlear nucleus and number of neurons in cochlear nucleus were evaluated four months later. Results: We establish a stable auditory deprived mouse model with cochlea ablation. A decrease in volume of the CN and a loss of neurons in the CN were observed. Conclusion: The cochlea ablation was an excellent method for establishing the auditory deprived animal model. The degeneration of cochlea nucleus can be induced by auditory deprived even for a long time in adult mouse.