Tatsuya Yamasoba

Ebselen attenuates cochlear damage caused by acoustic trauma

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound, mimics glutathione peroxidase and reacts with peroxynitrite. It is reported to protect against gentamicin- and cisplatin-induced ototoxicity. We investigated whether it protects the cochlea from acoustic trauma. Male pigmented guinea pigs (250-300 g) with normal auditory brainstem response (ABR) thresholds were exposed for 5 h to 125 dB sound pressure level octave band noise centered at 4 kHz. One hour before and 18 h after exposure, they received orally 0.25 ml chloroform solution containing 0, 10, or 30 mg/kg ebselen (n=6, 5 and 5, respectively). The protective effect of ebselen was evaluated by ABR measurement and quantitative hair cell assessment. Treatment significantly (P<0.01) reduced the extent of permanent threshold shifts and outer hair cell loss. Interestingly, the protective effect of a 30 mg/kg dose was less than that of a 10 mg/kg dose. There were no adverse systemic or auditory function effects in three unexposed control subjects given 30 mg/kg ebselen. These findings indicate that ebselen attenuates noise-induced cochlear damage. The concentration that provides optimal protection against such damage has now to be determined.

Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac.

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.

A Glucocorticoid Reduces Adverse Effects of Adenovirus Vectors in the Cochlea

Gene transfer using a recombinant adenovirus is a powerful tool for research and clinical applications, but its cytotoxicity and immune response limit its use, especially when repeated application of the vector is necessary. This study investigated the effects of dexamethasone (DEX)-induced immunosuppression on the outcome of adenovirus gene transfer in guinea pig inner ears. Animals received DEX for 29 days. Their inner ear was inoculated with 5 µl of adenovirus vector twice, on days 5 and 26. Auditory brainstem response was measured on days 1, 8 and 29. The animals were sacrificed on day 29, and reporter gene expression was evaluated. In control animals that received no DEX, postinoculation threshold shifts and lesions in the organ of Corti were observed and reporter gene expression was absent. In contrast, DEX-treated ears were largely protected, and transduction of inner ear cells was readily apparent. These data demonstrate that immunosuppressive treatment can reduce the negative consequences of repeated adenovirus-mediated gene therapy.

Changes in cell proliferation in rat and guinea pig cochlea after aminoglycoside-induced damage

Cell proliferation in the cochleae of guinea pigs and rats was investigated after systemic application of kanamycin sulfate (KM) and ethacrynic acid (EA). Bromodeoxyuridine (BrdU) was injected daily for 10 days, after which the number of BrdU-positive cells was counted in paraffin sections of the cochlea. Only a few BrdU-positive cells were present in the spiral ligament and among the acoustic nerve fibers in the non-deafened control animals. Animals treated with KM and EA had profound hearing loss and significant increases in the number of BrdU-positive cells in the spiral ligament and among the acoustic nerve fibers. No BrdU-positive cells were found in the auditory sensory epithelium of any animal. These findings suggest that in the mature mammalian cochlea cell proliferation increases in nonsensory regions after ototoxic damage but may not occur in the auditory sensory epithelium.

Transgene expression in mature guinea pig cochlear cells in vitro

Inoculation of adenovirus vectors in vivo has induced transgene expression in a variety of cochlear cells, but not hair cells or supporting cells in most previous studies. Specific hair cell inoculation by viral vectors has not been demonstrated in the mature guinea pig cochlea in vitro. We injected an Adex1CAlacZ into the mature guinea pig cochlear explants, which were incubated for 24-72 h. We found many lacZ-positive cells in a variety of tissues including the spiral ganglion and stria vascularis. Transgene expression was also found in the outer hair cells and supporting cells, such as the Deiters cells and pillar cells. These findings indicate that adenovirus vectors can be transfected into mature guinea pig hair cells and supporting cells in culture.

Transfection of young guinea pig vestibular cells in vitro with an adenovirus vector

This study shows distributions of lacZ-positive cells in the vestibular labyrinthine explants of young guinea pigs with mature ears. When adenovirus lacZ vectors were administered to the vestibular labyrinth following removal of the otoconial membrane, lacZ-positive cells were observed in the mesothelial cells in the perilymphatic space, in the sensory and supporting cells in the utricle and ampulla, and in the transitional and dark cells in the ampulla. When the otoconial membrane was preserved, lacZ-positive cells were not distributed in the utricular sensory epithelium. These findings suggest that adenovirus vectors can transform a variety of vestibular epithelial cells, but that it is difficult for adenovirus vectors to pass through the otoconial membrane.

Morphological and physiological effects of long duration infusion of strychnine into the organ of Corti

Acute strychnine administration has long been used as a method to eliminate the effects of efferent activity. It has been shown that long after termination of chronic strychnine infusion into the cochlea, the ear becomes more susceptible to acoustic trauma suggesting that chronic strychnine infusion results in long lasting or permanent disruption of efferent function. Much research has been directed towards the functional significance of the olivocochlear system. However, there is little information concerning the effect of long duration inactivation of the medial olivocochlear system in an awake behaving animal. This study was designed to determine the structural and functional consequences of inactivation of the efferents by chronic infusion of strychnine into the cochlear perilymph of guinea pigs for two weeks via an osmotic pump. Physiological evaluations showed that the strychnine infusion eliminated the efferent induced reduction of the cochlear whole-nerve action potential three weeks after cessation of strychnine infusion. Contralateral efferent function remained unaltered. Histological evaluation at the light and electron microscopic levels revealed disoriented efferent synapses under the outer hair cells.