This autonomic healing explains why the hearing function remained unchanged. Moreover, the ABR test results, which revealed no threshold difference between operated and contralateral ears, indicated that the injury caused by RWM puncture had healed by 2 weeks post-surgery. We used a micromanipulator to hold and advance the glass needle so that the RWM perforation was extremely small, as determined by the needle tip diameter, which was approximately. We determined that this hole was well sealed after the needle was removed because any observed perilymph leakage was not significant. RWM treatment using 90 mg/mL collagenase caused over-digestion and significant hearing loss. A comparison of the trans-RWM and RWM-puncture methods in terms of hearing function maintenance, transfection efficiency, and surgical difficulty revealed that both procedures preserved hearing function equally well, although the trans-RWM method did not disturb the perilymph with the administration of a large volume of exogenous agent. Our findings suggest that transfection via RWM puncture is more effective than that via the trans-RWM method, based on equal amounts of viral solution. However, more viral solution reached the perilymph in the RWM puncture group than in the trans-RWM diffusion group. The difficulty of the surgery was comparable in both approaches. The collagenase treatment and application of the virus increased the surgical time in the trans-RWM procedure. However, the vertical insertion of the needle tip across the RWM, which is hidden approximately 1 mm below the RW niche, required great caution and skill. Our results indicated that cochlear transfection in neonatal mice is characterized by an even longitudinal transfection in the hair cells and low transfection efficiency in the SGNs. The mechanisms underlying the even transfection are not clear. In guinea pigs, transfection rates are reportedly higher in the basal turn, which is closer to the site of virus application. One explanation for this is that the vector was not fully diffused to the apex of the cochlea. However, evidence for quick longitude transportation of material in the perilymph has been reported, so the diffusion distance does not explain the transfection gradient. AAV entry into the cell is mediated by special receptors; differences in receptor distribution may establish a gradient of cellular tropism to AAVs along the cochlea. The third possible reason is a difference in basilar membrane permeability to AAVs across the cochlea. However, no evidence for AAV tropism or basilar membrane permeability along the BAY-60-7550 cochlea has been reported in either guinea pigs or mice. The reasons for the low SGN transfection rate in neonatal mice observed in the present study are also not clear. 2 pathways have been proposed for AAV transportation to the organ of Corti: diffusion across the basilar membrane, which lacks tight junctions; and transport along nerve fibers via the habenula perforata after AAV transfection of the SGNs. The 2nd approach was proposed to explain the higher transduction rates in IHCs than in OHCs following AAV transfection, suggesting that IHCs receive richer innervations from thick type I fibers. However, no clear evidence exists to support this postulation. The low SGN transfection rate observed in the present study and in others essentially refutes the possibility of the latter pathway. This was likely the result of the connection between the cochlear fluid and cerebrospinal fluid via the cochlear aqueduct, and the fact that the 1 mL of viral solution injected in the present study was greater than the perilymph volume, which is reportedly 0.62 mL in adult mice. We injected a large volume of viral solution to maximize gene transfection.