Effect Of Early Auditory Deprivation On The Development Of Auditory Pathway And Transplantation Of Neural Stem Cells To The Cochlea Nucleus

Objective:Auditory deprivation effect is a significant reduce of acoustic message, which lead to the decrease of hearing ability. People have already noticed that the input signal of olfactory, vision and audition had an important role in the development of its neural pathway. Experts had thought that 2-3year-old was the perfect time for the artificial cochlea implantation, and FDA also suggested 12month-old as the lower limits of implantation. Now, people incline to take the implantation at an early age to reduce the effect of early auditory deprivation and get a better rehabilitation. More and more literatures shew that it was beneficial to our hearing reconstruction and language learning to take the implantation at an early age. Bilateral chronic SOM of chilidren and congenital microtia are the common cause of conductive hearing loss of children. We have found some mature ideas for the treatment of these, but when to interfer is still uncertain. Studies revealed that the reduce of acoustic message might cause the disorder of auditory center. But these studies were performed under the circumstances of destroying the cochlea. In our study, we prepared the auditory deprivation animal models by plugging their ears, which made it possible to study the changes of both auditory pathway and the modality and function of cochlea.In recent years, scientists have been going in for a new therapy with stem cells transplantation for those awkward diseases, and have made some progress. In the same time, otologists also turn to stem cells transplantation in order to find a new therapy for sensorineural hearing loss. However, the transplanted stem cells must carries their own tag of identity, thus the scientists could observe their biological characters through their specific identity. There are a lot of labeling methods for stem cells, all with its advantages and disadvantages. In our experiment, we compared three of those methods to detect the different results of NSEs transplantation in adult rats, and provide support for the different results of NSEs transplantation into the AD animal models.Methods:1. The experiment group underwent an auditory deprivation with a plugging in the external acoustic meatus form 12 days postnatal to 42 days, raised in a acoustic chamber. Then animals were tested for ABR threshold and DPOAE, and perfused by formalin before preparing the frozen sections of cochlea and brain stem.2. The location and expression of F-actin in the Corti organ. We observed hair cells and Cytoskeleton of both groups with a laser scanning confocal microscope after immunohistochemistry, immuno-fluorescence and Phalloidin stain of F-actin. Western blot was used to the semiquantitative analysis of F-actin.3. TUNEL was used to test the cell apoptosis. Transmission electron microscope was used to observe the neuron of spiral ganglion. Then we took the immunohistochemical staining of spiral ganglion with NSE antibody, and count the number of its neuron.4. FJC was used to test the neuron degeneration of cochlea nucleus, while Nissl stain was used to observe the cochlea modality and count the number of octopus-like cells.5. In vitro culture, differentiation and identification of neural stem cells derived from the olfactory bulb.We derived the neural stem cells from the olfactory bulb of SD rats and C57BL/6-gfp mice embryos, observed the culture, differentiation progress and viability of stem cells with a fluorescence inverted microscope, and then identified the stem cells with immunocytochemistry and immunofluorescence.6. Labeling of the olfactory bulb neural stem cells derived from SD rats embryos.We transfected the olfactory bulb neural stem cells derived from SD rats embryos with liposome2000-mediated PIRES-EGFP-C2 plasmid, and passaged them for 1 month after screening by G-418.Transfection efficiency of the stem cells was evaluated with flow cytometer. Before collecting the cells, we added Hoechst 33342 to the culture medium to label the transplanted cells, then 37℃incubation for 1 hour.7. Stereotaxic injection of labelled olfactory bulb neural stem cells to the cochlea.The C57BL/6-gfp mice NSCs, SD rats NSCs transfected with green fluorescent protein and stained with Hoechst 33342 were collected and injected to the rats cochlear nucleus. We anesthetized SD rats, fixed them on the stereotaxic apparatus, and located their left cochlea nucleus according to the brain atlas solid positioner. NSCs suspension was drew out into a microinjector with a glass microelectrode in the tip, and injected slowly into the cochlea nucleus. Post-operation animals were bred in normal environment and fixed by perfusion 2 and 10 days after operation. 40μm brain sections were made and observed with immunofluorescence staining.Results:1. DPOAE results shew that rate of control group in the frequency of 0.5,1,2,4,8kHz compared with AD group, there was significant difference between two groups in the frequency of 0.5,1,4kHz. There was significant difference between two groups on the ABR threshold: the control group was 18.6538±1.58785 dB SPL and the AD group was 28.4000±1.33775 dB SPL. The latency ABRⅠ-Ⅴwave of control group compared with those of AD group, there was no significant difference between two groups. The ABRⅠ-Ⅴwave amplitude of control group compared with those of AD group, there was significant difference between two groups inⅡ,Ⅲ,Ⅴwave amplitude. And there was significant difference between two groups inⅠ-Ⅴwave interval. TheⅠ-Ⅴwave interval of control group was 3.978±0.435, while that of AD group was 3.629±0.416.2. F-actin can express in alll kinds of cells of Corti organ, mostly in stereocilium, inner pillar cells and outer pillar cells, little in the outer hair cells. F-actin expressed in Corti organ of AD was less than that of control group, which was more significant in the concentration position. Western blot results shew F-actin express less in the basilar membrane of AD rats cochlea. And there was significant difference between two groups.3. In the Cordi organ of control group rats, we can see TUNEL-positive cells in the stria vascularis, but no in the basilar membrane. In the AD group rats, we can see TUNEL-positive cells along the spiral limbus surface and in supporting cells of the basilar membrane, but not in the IHCs,OHCs.4. There were two kinds of spiral ganglion neurons: typeⅠand typeⅡ. Transmission electron microscope revealed smaller cell body, bigger cell interval and degeneration of AD spiral ganglion neurons. membrane like tissues proliferated along the plasma. Star cells didn't develop well, and not form myelin sheath. Cell organs degenerated and decreased. The NSEs immunohistochemical staining shew that NSEs primarily located in cytoplasm of normal neuron. Cells bodies painted remarkably and necleus was faint staining. Arrangement of cells bodies was some kind of intensive. We couldn't tell the two kinds of neuron according to their modality. The neural fiber was faint staining. The NSEs immunohistochemical staining of AD group shew that the neurons were fainter staining with the fuzzy modality and disorder arrangement. There was significant difference between two groups on the results of neurons counting: AD group were 10.643±1.104, while that of the control group were 7.883±0.987.5.We could find green FJC-positive progress in the cochlea nucleus in the immunohistochemical staining of brain stem sections. Some of the progress was thin and branching. But we didn't find the FJC-positive neural bodies.6. The neuron counting of cochlea nucleus of AD group was significant smaller than that of the control group. And the neuron density was also smaller than that of the control group, but not significant. The counting of octopus like cells and the cells size of AD group was significant smaller than that of the control group. The cells density was also smaller, but not significant.7. Olfactory bulb neural stem cells of C57BL/6-gfp mice and SD rats embryos formed cell spheres in culture and were nestin positive. They could differentiate into MSE positive neurons and GFAP, Galc positive glial cells.8. Olfactory bulb neural stem cells of C57BL/6-gfp mice presented light green fluorescence in the burst of ultraviolel; EGFP transfected rats NSCs presented light green fluorescence with irregular or ring shape, and were GFP positive. Some smaller cell spheres shew darker fluorescence. Rats NSCs labelled with Hoechst 33342 presented light green fluorescence.9. Two days after injection, pin holes and cells gathering were observed in the injection cite of three group animals. 10days after injection, no positive cells were found in the injection cite of C57BL/6-gfp mice NSCs. Only some positive cells with slender green processes were found in the injection cite of EGFP transfected rats NSCs. NSE and GFAP positive cells were observed in the green fluorescence positive cells with the immunofluorescence stain, no Galc positive ones. A lot of blue fluorescence shew in the injection cites of Hoechst 33342 labelled SD NSCs and the surface of brainstem, and the latter extended to distant places along the meninges. Cells of blood vessels also shew a blue fluorescence positive. In the pin hole, blue fluorescence positive cells gathered and tended to extend.Conclusion:1. F-actin can express in all kinds of cells of Corti organ, mostly in inner pillar cells and outer pillar cells. They were light red fluorescence. Deiters cells were middle light red fluorescence, while the other supporting cells were faint. The OHCs were faint straining in the cytoplasm. The staining of OHCs cilium, and IHCs were the same as that of pillar cells.2. Early auditory deprivation of air-conduction could lead to the F-actin loss of Corti organ. The loss of F-actin in the cuticular plates and pillar cells could cause the disorder of cytoskeleton, the compliance of basal membrance and its converting function of mechanical energy and electric energy, which resulted in the abnormal ABR and DPOAE.F-actin expressed in Corti organ of AD was less than that of control group, which was more significant in the concentration position. Western blot results shew F-actin express less in the basilar membrane of AD rats cochlea. And there was significant difference between two groups.3. Auditory deprivation of air-conduction which lead to the reduce of the auditory signal didn't result in the neuron degeneration and apoptosis. But we found the degeneration of neural fibers in the cochlea nucleus. In the AD group, NSE stained faint in the spiral ganglion. The number of neurons decreased and the size smaller. Both neurons and star cells degenerated. 4. Auditory deprivation of air-conduction resulted in the loss of neuron and its density. In the AD group, the PVCN octopus like cells was smaller, less number and density. All of these indicated that the auditory deprivation of air-conduction in the crucial time might cause the great effect on the development of auditory neural pathway and its function.5. Olfactory bulb neural stem cells of C57BL/6-gfp mice and SD rats embryos formed cell spheres in culture and were nestin positive. They could differentiate into MSE positive neurons and GFAP, Galc positive glial cells. These indicated that those cells were from nervous systerm and could self-renewal and generated other cells through asymmetric cell divisions.6. It was easy and convenient to derive the NSCs from C57BL/6-gfp mice. The C57BL/6-gfp mice NSCs could present stable green flurescence. But the animal races were limited. And we estimated that the race difference was the main reason of NSCs death.7. It was simple and safe in the process of transfectting the NSCs with EGFP, which was less immunogenicity or toxicity to NSCs and higher transfection efficiency. It could wonderfully display the modality of tagged cells and could be taken as a perfect way to tag the cells.8. It was also simple, low toxicity and high efficiency to label NSCs with Hoechst 33342. But Hoechst 33342 stain may stain the cells in the injection cite and lead to false positive.