Study Of Highly Proliferative Cells From The Cochlea Of The Newborn Rats And Neomycin's Effect On The Nestin Expression In The Cochlea

Part I The Study of the Highly Proliferative Cells from the Cochlea of Newborn RatsObjectives: 1.To study if there are highly proliferative cells in the cochlea of the new born rat; what kinds of cells were differentiated from the highly proliferative cells. At the same time, effect of fibronectin, polyornithine and polylysine on the attachment and growth of the highly proliferative cell spheres was explored. 2.To study EGF and bFGF's effect on the highly proliferative cells. 3.To observe ultrastructure of the highly proliferative cell spheres and the differentiated cells. Our aims are to supply some experimental evidences for application of the highly proliferative cells from the cochlea in treatment of sensorineural deafness by cell transplantation.Methods: 1. The cochlear basilar membranes from P1～3 SD rats were dissected. The tissues were dissociated by digestion and trituration and made into single cell suspension, which were cultured in DMEM/F12 media containing B27 and N2 supplements. After cell spheres were formed in the cultures, they were plated onto coverglasses coated by fibronectin, polyornithine and polylysine, and differentiated in the adherent culture conditions, the attachment and growth of cell spheres were examined by microscopic observation,the number of cell spheres attached on coverglasses were counted after 16 hours, the data were analysed statistically. The proliferative condition of cells was tested by infusing the BrdU into the culture medium. The varieties of the cell spheres and differentiated cells were identified by immunocytochemistry. 2. In the presence of fibroblast growth factor b(b-FGF) or/and epidermal growth factor(EGF), the dissociated single cells from single basilar membrane were cultured for 7 days, effect of EGF and bFGF on the proliferative cells were observed by counting the number of cell spheres. 3. The cell spheres and the differentiated cells were observed by scanning electron microscopy and transmission electron microscopy respectively.Results: 1. In the above culture conditions, cells continuously proliferated and formed suspended spheres, 90% cells of spheres were labeled by BrdU, and cells of spheres were also nestin positive. In the differentiated cells, we find: ①the marker of hair cells (including: myosin7A, espin, and Phalloidin) were expressed positively in some cells, and some differentiated cells were doubly labeled for both myosin 7A andBrdU, or both espin and BrdU; ②the marker of neuron(including: NF-M or tubulin-β-III or nestin) were also positive in some cells, some differentiated cells were doubly labeled for both NF-M and BrdU;③the marker of supporting cells or astrocytes(GFAP) was positive in some cells. 2. The number of cell spheres attached on coverglasses were 44.12±8.04 in fibronectin group, 11±2.10 in polyornithine group and 7.33±1.51 in polylysine group per coverglass respectively. A distinctly significant difference was observed between fibronectin group and polyornithine group, and fibronectin group and polylysine group, however, a difference between polyornithine group and polylysine group had no statistically significance. 3. The average 45.25±22.95 cell spheres were observed in cell culture medium of a single cochlear basilar membrane. The cell spheres increased obviously in the presence of fibroblast growth factor b(bFGF) or/and epidermal growth factor (EGF). Significant differences were seen between control group and every experimental group including EGF group, bFGF group, and EGF+bFGF group, significant differences were also seen between EGF group and EGF+bFGF group, bFGF group and EGF+bFGF group respectively. 4. Scanning electron microscopy showed that cells of the spheres were round and have the same size, many short and thin microvilli were observed on the surface of cells. Many round-shape cells adhered with each other. Transmission electron microscope showed cells of spheres adhered with each other, tight junction, desmosomes and gap junctions between two adjacent cells were seen. The large nucleus with little plasm were observed in the cells, the microvilli were observed on the surface of cells. The cells were rich of organellae such as endoplasmic reticulum, at same time, mitochondrion, microfilament, microtube in the cells were seen in the cytoplasm. The differentiated cells also exhibited many short and thin microvilli under scanning electron microscope.Conclusions: 1. The highly Proliferative cells were present in the cochlea of newborn SD rats, which are able to differentiate into hair cells with bundle-like structure, neuron and astrocytes or supporting cells. 2. Fibronectin was helpful for the attachment and growth of the highly Proliferative cells. 3. A single cochlear basilar membrane may form average 45.25±22.95 cell spheres. EGF and/or bFGF possess the promoting effects for proliferation on the highly Proliferative cells. 4. The study of ultrastructure displays that the highly Proliferative cells have characters of earlier immature cells.Part II Neomycin's Effect on Nestin Expressionin the Cochlea of RatsObjectives: To investigate the changes of nestin expression in the cochlear sensory epithelia and spiral ganglion neurons in the normal developing rats and the rats damaged by neomycin. Our aims are to understand neomycin's effect on the nestin expression in the cochlea.Methods: 1. Immunohistochemistry was made on the normal cochlea of different days' rats (P1, P14, P30, P60), the location and change of the nestin expression in the cochlear sensory epithelia and spiral ganglion neurons were observed.2. Twelve P7 SD rats were divided into the experimental group and control randomly. The experimental rats were injected with neomycin hypodermically 200mg/kg/d for 12 days, while the control group was treated with equal amount of physiological saline. The rats were sacrificed on the 1st and 14th day after the drug administration respectively. Immunohistochemistry was made on the cochlea. The location and change of the nestin and GFAP expression in the cochlear sensory epithelia and spiral ganglion neurons were observed.Results: 1. The nestin expression positive cells were found in the cochlea sensory epithelia of P1 SD rats. The cells at the apical turn of cochlea demonstrated the strongest immunoreactivity for nestin, then immunoreactivity for nestin decreased at the middle turn, the cells at the basal turn of cochlea showed weaker reactivity for nestin. Not only the supporting cells but also the inner hair cells and outer hair cells expressed nestin epitopes. Immunoreactivity for nestin significantly decreased in the cochlear sensory epithelia of P30 rats. Cells lost immunoreactivity for nestin in P60 rats. 2. Expression of nestin epitopes was observed in the every turn's spiral ganglion neurons of P1 and P14, the neurons of the apexturn demonstrated the strongest immunoreactivity for nestin, immunoreactivity for nestin in neurons became weaker in P30 rats, but expression of nestin epitopes in neurons was still detectable in P60 rats. 3. In the experimental rats, expression of nestin epitopes in the cochlea sensory epithelia was not detected on the 1th day after injection as compared with control rats, and nestin positive cells were still not found on the 14th day after the drugadminstration. 4. In the experimental rats, expression of nestin epitopes in the spiral ganglion neurons decreased on the 14th day after the drug adminstration as compared with control rats, but it still was detectable. 5. Immunostaining for GFAP was observed and identical in the cochlear sensory epithelia of both the experimental and control rats on the 14th day after the drug adminstration. Expression of GFAP was not observed in the spiral ganglion neurons of both groups.Conclusions: 1. Expression of nestin epitopes is detectable in the cochlear sensory epithelia and spiral ganglion neurons of the postnatal rats; and immunoreactivitiy for nestin decreases as the inner ear matures; 2. In neomycin-damaged rats, immunoreactivitiy for nestin decreased in the cochlear sensory epithelia and spiral ganglion neurons after treatment, whereas immunoreactivitiy for GFAP in the cochlear sensory epithelia was not affected.