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Inner ear gene therapy using adeno-associated viral vectors (AAVs) in neonatal mice can alleviate hearing loss in mouse models of deafness. However, efficient and safe transgene delivery to the adult mouse cochlea is critical for the effectiveness of AAV-mediated therapy. Here, we examined three gene delivery approaches including posterior semicircular canal (PSCC) canalostomy, round window membrane (RWM) injection, and tubing-RWM+PSCC (t-RP) in adult mice. Transduction rates and survival rates of cochlear hair cells were analyzed, hearing function was recorded, AAV distribution in the sagittal brain sections was evaluated, and cochlear histopathologic images were appraised. We found that an injection volume of 1 μL AAV through the PSCC is safe and highly efficient and does not impair hearing function in adult mice, but local injection allows AAV vectors to spread slightly into the brain. We then tested five AAV serotypes (PHP.eB, IE, Anc80L65, AAV2, and PHP.s) in parallel and observed the most robust eGFP expression in inner hair cells, outer hair cells, and spiral ganglion neurons throughout the cochlea after AAV-Anc80L65 injection. Thus, PSCC-injected Anc80L65 provides a foundation for gene therapy in the adult cochlea and will facilitate the development of inner ear gene therapy.

Delivery of substances into the inner ear via local routes is increasingly being used in clinical treatment. Studies have focused on methods to increase permeability through the round window membrane (RWM) and enhance drug diffusion into the inner ear. However, the clinical applications of those methods have been unclear and few studies have investigated the efficacy of methods in an inner ear injury model. Here, we employed the medium chain fatty acid caprate, a biologically safe, clinically applicable substance, to modulate tight junctions of the RWM. Intratympanic treatment of sodium caprate (SC) induced transient, but wider, gaps in intercellular spaces of the RWM epithelial layer and enhanced the perilymph and cochlear concentrations/uptake of dexamethasone. Importantly, dexamethasone co-administered with SC led to significantly more rapid recovery from noise-induced hearing loss at 4 and 8 kHz, compared with the dexamethasone-only group. Taken together, our data indicate that junctional modulation of the RWM by SC enhances dexamethasone uptake into the inner ear, thereby hastening the recovery of hearing sensitivity after noise trauma.

Clinically, gentamicin has been used extensively to treat the debilitating symptoms of Mèniére's disease and is well known for its vestibulotoxic properties. Until recently, it was widely accepted that the round window membrane (RWM) was the primary entry route into the inner ear following intratympanic drug administration. In the current study, gentamicin was delivered to either the RWM or the stapes footplate of guinea pigs (GPs) to assess the associated hearing loss and histopathology associated with each procedure. Vestibulotoxicity of the utricular macula, saccular macula, and crista ampullaris in the posterior semicircular canal were assessed quantitatively with density counts of hair cells, supporting cells, and stereocilia in histological sections. Cochleotoxicity was assessed quantitatively by changes in threshold of auditory brainstem responses (ABR), along with hair cell and spiral ganglion cell counts in the basal and second turns of the cochlea. Animals receiving gentamicin applied to the stapes footplate exhibited markedly higher levels of hearing loss between 8 and 32 kHz, a greater reduction of outer hair cells in the basal turn of the cochlea and fewer normal type I cells in the utricle in the vestibule than those receiving gentamicin on the RWM or saline controls. This suggests that gentamicin more readily enters the ear when applied to the stapes footplate compared with RWM application. These data provide a potential explanation for why gentamicin preferentially ablates vestibular function while preserving hearing following transtympanic administration in humans., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Unlabelled: NADPH oxidases are enzymes that transport electrons across the plasma membrane and generate superoxide radical from molecular oxygen. The current study investigated the expression and distribution of NOX/DUOX members of the NADPH oxidase family (NOX1-5 and DUOX1-2) in the rat cochlea and their regulation in response to noise. Wistar rats (8-10 weeks) were exposed for 24 h to band noise (8-12 kHz) at moderate (100 dB) or traumatic (110 dB) sound pressure levels (SPL). Animals exposed to ambient noise (45-55 dB SPL) served as controls. Immunohistochemistry demonstrated predominant expression of all NOX/DUOX isoforms in the sensory and supporting cells of the organ of Corti, with very limited immunoexpression in the lateral wall tissues and spiral ganglion neurons. Noise exposure induced up-regulation of NOX1 and DUOX2 in the cochlea, whereas NOX3 was down-regulated. A significant reduction in the intensity of NOX3 immunolabeling was observed in the inner sulcus region of the cochlea after exposure to noise. Post-exposure inhibition of NADPH oxidases by Diphenyleneiodonium (DPI), a broadly selective NADPH oxidase inhibitor, mitigated noise-induced hearing loss., Conclusion: Noise-induced up-regulation of NOX1 and DUOX2 could be linked to cochlear injury. In contrast, down-regulation of NOX3 may represent an endogenous protective mechanism to reduce oxidative stress in the noise-exposed cochlea. Inhibition of NADPH oxidases is potentially a novel pathway for therapeutic management of noise-induced hearing loss., (Copyright © 2013 Elsevier B.V. All rights reserved.)