2. Aim Examine the possibility of restoration of spiral ganglion neurons by transplantation of fetal neural stem cells (NSCs) into the modiolus of cochlea.

3. Materials-donor Fetal mouse neural stem cells (NSCs) have the potential for differentiation into neurons expressing green fluorescence were used as donor cells. NSC spheres were obtained from the neuroepithelium of the dorsal telencephalon of embryos at embryonic day 11.5 of C57BL/6JTg14 (Act-EGFP) Obs-Y01 transgenic mice using the neurosphere culture medium. Secondary neurospheres, which exhibited expression of nestin, but no expression of TuJ1 or GFAP, were collected, dissociated and suspended for transplantation at a density of 1×10 5 cells/ml in the neurosphere culture medium.

4. Materials-recipient C57BL/6 mice at 6 weeks of age affected by cisplatin, in which severe degeneration of SGNs is induced. In this model, about 60% of SGNs disappear at 14 days after cisplatin treatment.

5. Surgery procedure A cisplatin solution (2.5 mg/ml) was injected from the left posterior semicircular canal. The left cochlea of recipient animals was exposed 14 days after deafening. 2 μl NSC was injected into the cochlea through the round window toward the direction of the cochlear modiolus by Hamilton syringe and infusion pump. 14 days after transplantation, the left cochlea was re-exposed and 4% paraformaldehyde was gently perfused into the perilymph from the round window. The animals were then sacrificed. The temporal bones were collected and immersed in the same fixative for 4 h at 4 ℃. Cryostat sections 10 μm thick were made, and the mid- modiolus sections were used for histological analysis.

6. Staining The cell fates of grafted NSCs were determined by immunohistochemistry for Tuj 1 (marker for neurons) or GFAP (marker of glial cells). Counterstaining with DAPI. The numbers of transplant-derived cells in the modiolus in one section and the ratios of positivity for each marker in transplant-derived cells were examined. Both EGFP- and DAPI-positive cells were defined as transplant- derived cells. The average of two sections for each animal was defined as the number for that animal. The ratio of positivity for each marker in transplant-derived cells in the modiolus was then calculated.

7. Result Robust survival of the injected cells was found in cochleae. NSC-derived cells expressing GFP located in the modiolus of cochleae or scala tympani, settled in the apical portion of the modiolus. The mean and standard deviation of numbers of NSC derived cells in the modiolus was 190.5 and 60.8.

8. NSC-derived cells in the modiolus exhibited expression of TuJ1. TuJ1-positive grafted cells were predominantly located in the osseous spiral lamina or spiral ganglion Expression of TuJ1 was observed in about 10% of NSC-derived cells.

9. NSC-derived cells in the modiolus exhibited expression of GFAP. GFAP-positive grafted cells were found in the modiolus and spiral ganglion.

10. TuJ1-positive grafted cells were positioned on the periphery of grafted cells (Fig2A), GFAP-positive grafted cells were mainly located in the center of grafted cells (Fig. 2B).

11. Discussion NSCs injected in the basal portion of the modiolus settled in the apical end of the modiolus and osseous spiral lamina, indicating the high potential of NSCs for migration. NSC-derived cells differentiated into neurons in the modiolus, indicating that NSC transplantation into the modiolus may be utilized for restoration of SGNs.

12. Discussion Promotion of the activity of neural differentiation or increase of numbers of surviving NSC-derived cells was crucial for functional recovery of SGNs. -Neurotrophins The microenvironments where NSCs settled influence the fate of grafted NSCs, Cytokines associated with inflammation influence the survival and differentiation of NSCs.

13. Conclusion Injection of NSCs into the modiolus of injured cochleae results in robust survival of grafted NSCs in the modiolus. Grafted NSCs differentiate into neurons in the modiolus, although their number is limited. NSC is a possible candidate of cell therapy for restoration of SGNs.

14. My plan Examine the possibility of restoration of spiral ganglion neurons and function reservation by transplantation of human neuronal precursor cells (NPCs) into the internal auditory canal (IAC) thru cochleostomy in the Guinea Pig. Examine the morphology/function change of the brain/contralateral cochlea after Ouabain deafening. (The animals are hard to anesthetize after Ouabain application. )

15. Materials Donor cells: Human NPCs Recipient: Hartley Guinea Pigs 2~3 weeks old

16. Procedure 1.18 animals, 6 deafened with cell implantation,( 3 for 1w, 3 for 4w), 6 undeafened with cell implantation,(3 for 1w, 3 for 4w), 3 deafened without cell implantation for 4w, 3 undeafened without cell implantation for 4w. 2.BL-clk ABR for both side of ear. 3.10M Ouabain 5/10μl deafen left ear thru round window membrane for 8 animals. 4.Post-deafening (2week) clk ABR for both ear. 5.5 μl NPCs injection thru cochleostomy by Hamilton syringe and pump. 6.Post-implant (1,2,4 week) clk ABR for both ear. 7.Sacrifice 1(4) week(s) and harvest both side of cochlea with BS. 8. Staining.

17. Tips and Hurdles Accurate puncture into the IAC with little cochleostomy and try to avoid the leakage of perilymph and CSF. Accurate volume for the NPCs is hard as the needle was inside the cochlea and could not be show by microscope. Anesthetized by Xylazine & Ketamine is not strong enough to do the the surgery as it would last for 2 hours or more, could we use gas? How to care the immune action to the NPCs? Should antibody and immune suppressor be applied? Is it available for click ABR to appraise the hearing fuction after cell implantation?