Effective, reproducible, and safe delivery of therapeutics into the inner ear is required for the prevention and treatment of hearing loss. A commonly used delivery method is via the posterior semicircular canal (PSCC); however, its specific targeting within the cochlea remains unclear, impacting precision and reproducibility. To assess safety and target specificity, we conducted in vivo recordings of the pharmacological manipulations delivered through the PSCC. Measurements of auditory brainstem response (ABR), vibrometry, and vestibular behavioral and sensory-evoked potential (VsEP) revealed preserved hearing and vestibular functions after artificial perilymph injections. Injection of curare, a mechanoelectrical transducer (MET) channel blocker that affects hearing when in the endolymph, had no effect on ABR or VsEP thresholds. Conversely, injection of CNQX, an AMPA receptor blocker, or lidocaine, a Na ⁺ channel blocker, which affects hearing when in the perilymph, significantly increased both thresholds, indicating that PSCC injections selectively target the perilymphatic space. In vivo tracking of gold nanoparticles confirmed their exclusive distribution in the perilymph during PSCC injection, supporting the pharmacological finding. Together, PSCC injection is a safe method for inner ear delivery, specifically targeting the perilymphatic space. Our findings will allow for precise delivery of therapeutics within the inner ear for therapeutic and research purposes.

The mammalian cochlea contains three rows of outer hair cells (OHCs) that amplify the basilar membrane traveling wave with high gain and exquisite tuning. The pattern of OHC loss caused by typical methods of producing hearing loss in animal models (noise, ototoxic exposure, or aging) is variable and not consistent along the length of the cochlea. Thus, it is difficult to use these approaches to understand how forces from multiple OHCs summate to create normal cochlear amplification. Here, we selectively removed the third row of OHCs and Deiters' cells in adult mice and measured cochlear amplification. In the mature cochlear epithelia, expression of the Wnt target gene Lgr5 is restricted to the third row of Deiters' cells, the supporting cells directly underneath the OHCs. Diphtheria toxin administration to Lgr5^DTR-EGFP/+ mice selectively ablated the third row of Deiters' cells and the third row of OHCs. Basilar membrane vibration in vivo demonstrated disproportionately lower reduction in cochlear amplification by about 13.5 dB. On a linear scale, this means that the 33% reduction in OHC number led to a 79% reduction in gain. Thus, these experimental data describe the impact of reducing the force of cochlear amplification by a specific amount. Furthermore, these data argue that because OHC forces progressively and sequentially amplify the traveling wave as it travels to its peak, the loss of even a relatively small number of OHCs, when evenly distributed longitudinally, will cause a substantial reduction in cochlear amplification.

Novel therapeutic delivery systems and delivery methods to the inner ear are necessary to treat hearing loss and inner ear disorders. However, numerous barriers exist to therapeutic delivery into the bone-encased and immune-privileged environment of the inner ear and cochlea, which makes treating inner ear disorders challenging. Nanoparticles (NPs) are a type of therapeutic delivery system that can be engineered for multiple purposes, and posterior semicircular canal (PSCC) infusion is a method to directly deposit them into the cochlea. We sought to assess PSCC infusion of gold NPs into the cochlea, including the NPs’ distribution and effect on cochlear mechanics. We performed optical coherence tomography (OCT) imaging to monitor PSCC infusion of gold NPs into the cochlear chambers. OCT imaging demonstrated that the infusion specifically targeted the perilymphatic spaces within the cochlea. We assessed cochlear mechanics by using OCT vibrometry to measure sound-evoked movements of the basilar membrane. We found no changes in cochlear mechanics between measurements at baseline, after the PSCC canalostomy, immediately after the infusion, and 1 h after the infusion of gold NPs (p > 0.05, paired t-test). These findings validate the PSCC infusion approach for perfusing the cochlear perilymphatic space with a nanoparticle delivery system. Thus, PSCC infusion of nanoparticles is a feasible therapeutic delivery technique for treating inner ear disorders while preserving residual cochlear function.

SignificanceOur understanding of mechanotransduction in mammalian inner ears remains incomplete, in part due to imaging limitations: current systems cannot simultaneously provide high-resolution images needed for subcellular analysis and the deep focus required for structural mechanics. Optical coherence tomography (OCT) enables structural and vibrational imaging through the bone of the intact cochlea in models such as mice, supporting studies of cochlear mechanics in animals with functional hearing. However, capturing both cellular (200 μm) details requires rapid switching between optical configurations with numerical apertures ranging from 0.13 to 0.8. A spectral-domain OCT system combined with two-photon fluorescence microscopy (TPM) and interchangeable objectives could overcome this challenge, enabling high-precision vibration and fluorescence imaging across multiple scales in a single experiment.AimWe aim to develop an integrated OCT and two-photon microscope optimized for imaging the morphology and function of the cochlea.ApproachWe integrated a custom SD-OCT/TPM system into an upright microscope with a high-precision stage for animal positioning. The system uses two tunable liquid lenses to form a beam expander, enabling dynamic adjustment of the beam diameter at the back aperture of each objective. This optimized light throughput and maintained a high signal-to-noise ratio (SNR) across all objectives. In addition, we automated optical adjustments to facilitate seamless imaging with a wide range of objectives.ResultsFor each objective, we measured the SNR difference between a beam expanded to match the largest back aperture and a beam adjusted to match the back aperture of the objective. Except for the 4× objective, the measured SNR improvements closely matched theoretical predictions. Using four selected objectives spanning the required numerical aperture (NA) range, we successfully imaged excised murine cochlea samples, obtaining relevant structural information across scales. In living murine models, we used TPM to locate fluorescent outer hair cells and make vibrometry measurements through the round window membrane. We found that hair cells, the basilar membrane, and the reticular lamina moved in phase in response to a 70 kHz stimulus at 90 dB SPL, consistent with expected cochlear mechanics.ConclusionsAutomation and optimization of the optical system enabled seamless multiscale imaging of the murine cochlea, providing high-quality morphological, functional, and two-photon fluorescence images. The dynamic adjustment of the beam diameter within the microscope was essential for maintaining high SNR across a wide range of numerical apertures.