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Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Capital Medical University

Vesicular glutamate transporter 3 (VGLUT3) plays an important role in hearing, and VGLUT3 knockout mice are deaf. However, the mechanisms whereby VGLUT3 exerts its effects in the cochlea are not well established. Elucidating the developmental and aging dynamics of VGLUT3 localization and expression in the cochlea would aid a functional understanding of auditory glutamatergic transmission. In this study, we characterized the expression of VGLUT3 in rat auditory epithelium and spiral ganglion cells (SGCs) during postnatal development and with age using immunohistochemistry and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We observed VGLUT3 expression in cochlea inner hair cells (IHCs) and SGCs as early as postnatally, and its expression increased gradually with maturity. However, the expression of VGLUT3 in the IHCs decreased in the aging rats compared to postnatal day 60 (P60), and VGLUT3 was still expressed in the remaining SGCs in the aging rats. VGLUT3-immunopositive punctate structures were only found in the cytoplasm of SGCs, not IHCs, at P7, or later. Variation in VGLUT3 mRNA expression evaluated by quantitative real time RT-PCR was consistent with the immunohistochemical data except for no statistical difference between P60 and aging rats in the whole cochlea level. Our data suggest that VGLUT3 likely has developmental and physiological roles in the rat cochlea during postnatal development as well as later in life. VGLUT3 may have additional unknown roles in SGCs besides glutamate transport.

Temporospatial expression and cellular localization of VGLUT3 in the rat cochlea.

Tumor necrosis factor (TNF)-α is a cytokine involved in acute inflammatory phase reactions, and is the primary upstream mediator in the cochlear inflammatory response. Treatment of the organ of Corti with TNF-α can induce hair cell damage. However, the resulting morphological changes have not been systematically examined. In the present study, cochlear organotypic cultures from neonatal mice were treated with various concentrations and durations of TNF-α to induce inflammatory responses. Confocal microscopy was used to evaluate the condition of hair cells and supporting cells following immunohistochemical staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy. TNF-α treatment resulted in a fusion and loss of stereocilia bundles in hair cells, swelling of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. Disruption of tight junctions between hair cells and supporting cells was also observed at high concentrations. Hair cell loss was preceded by apoptosis of Deiters' and pillar cells. Taken together, these findings detail the morphological changes in the organ of Corti after TNF-α treatment, and provide an in vitro model of inflammatory-induced ototoxicity. 

Tumor Necrosis Factor-α-Induced Ototoxicity in Mouse Cochlear Organotypic Culture.

Cochlear hair cell loss results in the secondary loss of spiral ganglion cells (SGCs). The death of these SGCs is due to apoptosis. The E2F1-cyclin dependent kinase 1 (CDK1) pathway is believed to represent an important mechanism of neuronal cell death. However, the role of this pathway in spiral ganglion neuronal apoptosis has not yet been reported. In this study, we deafened guinea pigs with a subcutaneous injection of kanamycin followed by an intravenous infusion of furosemide and then assayed the expression levels of cleaved caspase-3, E2F1, CDK1 and cleaved caspase-9 during the induced SGC apoptosis. Our results revealed that co-administration of kanamycin and furosemide rapidly induced hair cell loss in the guinea pigs and then resulted in a progressive loss of SGCs. Expression levels of E2F1 and CDK1 were obviously up-regulated at 1 and 3 days after deafening. Cleaved caspase-9 also increased robustly 1 or 2 weeks after the deafening procedure. The up-regulation of E2F1, CDK1 and cleaved caspase-9 was significantly attenuated by the systemic injection of CR8 (1mg/kg/day, intraperitoneally) starting at 5min after deafening. These findings indicate that the activation of the E2F1-CDK1 pathway and cell cycle re-entry contributes to the apoptosis of SGCs and that the selective inhibition of this signaling cascade may represent an attractive therapeutic strategy. CR8 has the potential to protect SGCs from apoptosis. 

E2F1-CDK1 pathway activation in kanamycin-induced spiral ganglion cell apoptosis and the protective effect of CR8.

Impairments of the inner ear result in sensorineural hearing loss and vestibular dysfunction in humans. A large proportion of these disorders are congenital, and involve both auditory and vestibular systems. Therefore, genetic interventions to correct deficits must be administered during early developmental stages. In this study, we evaluated inner ear gene transfer in neonatal mice by canalostomy using an adeno-associated virus serotype 8 (AAV8) vector. AAV8 with the green fluorescence protein (GFP) gene was inoculated into the inner ear of the neonatal mice through the posterior semicircular canal (canalostomy). At 30 days following surgery, animals were subjected to swim tests and auditory brainstem response measurements. Then, the animals were euthanized and temporal bones were harvested for whole-mount preparation. GFP expression and morphological changes in the inner ear were assessed by immunohistochemistry. After surgery, no signs of vestibular dysfunction were found, and there were no significant differences in the auditory brainstem response threshold between AAV8-inoculated ears and nonsurgery ears. In the surgery ears, extensive GFP expression and no morphological lesions were detected in the cochlear and vestibular end organs. Robust GFP expression was found in inner hair cells, marginal cells, vestibular hair cells, and vestibular supporting cells. In conclusion, AAV8 inoculation through canalostomy into the inner ears of neonatal mice led to extensive overexpression of exogenous genes in the inner ear without affecting hearing or vestibular function. It serves as a promising approach for gene therapy of congenital cochleovestibular diseases.

Cochleovestibular gene transfer in neonatal mice by canalostomy.

Noise-induced hearing loss (NIHL) severely impacts the quality of life of affected individuals. Oxidative stress resulting from noise exposure is a significant cause of NIHL. Although histone deacetylase (HDAC) inhibitors were shown to protect against NIHL, the underlying mechanism remains unclear, and it is not known how they act on noise-induced oxidative stress. In the current study, we investigated the expression levels of acetyl-histone H3 (Lys9) (H3-AcK9), histone deacetylase 1 (HDAC1), and 3-nitrotyrosine (3-NT), an oxidative stress marker, in a guinea pig model of NIHL using immunohistology and Western blotting. We then assessed the effects of systemic administration of the HDAC inhibitor, sodium butyrate (SB), on noise-induced permanent threshold shifts (PTS), hair cell (HC) loss, and changes in the above mentioned markers. The results showed that SB attenuated noise-induced PTS and outer hair cell loss. SB treatment promoted H3-AcK9 expression and repressed HDAC1 expression in the nuclei of HCs and Hensen's cells after noise exposure. Furthermore, SB attenuated the noise-induced increase of 3-NT expression in HCs and Hensen's cells. These findings suggest that SB protects against NIHL by reversing the noise-induced histone acetylation imbalance and inhibiting oxidative stress in cochlear HCs and Hensen's cells. SB treatment may represent a potential strategy to prevent and treat NIHL.

Article	Year
Temporospatial expression and cellular localization of VGLUT3 in the rat cochlea. Brain research\| PubMed ID: 24064385	2013
Tumor Necrosis Factor-α-Induced Ototoxicity in Mouse Cochlear Organotypic Culture. PloS one\| PubMed ID: 26000970	2015
E2F1-CDK1 pathway activation in kanamycin-induced spiral ganglion cell apoptosis and the protective effect of CR8. Neuroscience letters\| PubMed ID: 26905670	2016
Cochleovestibular gene transfer in neonatal mice by canalostomy. Neuroreport\| PubMed ID: 28614181	2017
The histone deacetylase inhibitor sodium butyrate protects against noise-induced hearing loss in Guinea pigs. Neuroscience letters\| PubMed ID: 28928030	2017

Jing-Ying Guo

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