The UmboMic: A PVDF Cantilever Microphone.

Objective: We present the "UmboMic," a prototype piezoelectric cantilever microphone designed for future use with totally-implantable cochlear implants. Methods: The UmboMic sensor is made from polyvinylidene difluoride (PVDF) because of its low Young's modulus and biocompatibility. The sensor is designed to fit in the middle ear and measure the motion of the underside of the eardrum at the umbo. To maximize its performance, we developed a low noise charge amplifier in tandem with the UmboMic sensor. This paper presents the performance of the UmboMic sensor and amplifier in fresh cadaveric human temporal bones. Results: When tested in human temporal bones, the UmboMic apparatus achieves an equivalent input noise of 32.3 dB SPL over the frequency range 100 Hz to 7 kHz, good linearity, and a flat frequency response to within 10 dB from about 100 Hz to 6 kHz. Conclusion: These results demonstrate the feasibility of a PVDF-based microphone when paired with a low-noise amplifier. The reported UmboMic apparatus is comparable in performance to a conventional hearing aid microphone. Significance: The proof-of-concept UmboMic apparatus is a promising step towards creating a totally-implantable cochlear implant. A completely internal system would enhance the quality of life of cochlear implant users.

Reconstruction of transverse-longitudinal vibrations in the organ of Corti complex via optical coherence tomography.

Optical coherence tomography (OCT) is a common modality for measuring vibrations within the organ of Corti complex (OCC) in vivo. OCT's uniaxial nature leads to limitations that complicate the interpretation of data from cochlear mechanics experiments. The relationship between the optical axis (axis of motion measurement) and anatomically relevant axes in the cochlea varies across experiments, and generally is not known. This leads to characteristically different motion measurements taken from the same structure at different orientations. We present a method that can reconstruct two-dimensional (2-D) motion of intra-OCC structures in the cochlea's longitudinal-transverse plane. The method requires only a single, unmodified OCT system, and does not require any prior knowledge of precise structural locations or measurement angles. It uses the cochlea's traveling wave to register points between measurements taken at multiple viewing angles. We use this method to reconstruct 2-D motion at the outer hair cell/Deiters cell junction in the gerbil base, and show that reconstructed transverse motion resembles directly measured transverse motion, thus validating the method. The technique clarifies the interpretation of OCT measurements, enhancing their utility in probing the micromechanics of the cochlea.

Using volumetric optical coherence tomography to achieve spatially resolved organ of Corti vibration measurements.

Optical coherence tomography (OCT) has become a powerful tool for measuring vibrations within the organ of Corti complex (OCC) in cochlear mechanics experiments. However, the one-dimensional nature of OCT measurements, combined with experimental and anatomical constraints, make these data ambiguous: Both the relative positions of measured structures and their orientation relative to the direction of measured vibrations are not known a priori. We present a method by which these measurement features can be determined via the use of a volumetric OCT scan to determine the relationship between the imaging/measurement axes and the canonical anatomical axes. We provide evidence that the method is functional by replicating previously measured radial vibration patterns of the basilar membrane (BM). We used the method to compare outer hair cell and BM vibration phase in the same anatomical cross section (but different optical cross sections), and found that outer hair cell region vibrations lead those of the BM across the entire measured frequency range. In contrast, a phase lead is only present at low frequencies in measurements taken within a single optical cross section. Relative phase is critical to the workings of the cochlea, and these results emphasize the importance of anatomically oriented measurement and analysis.

Model of cochlear microphonic explores the tuning and magnitude of hair cell transduction current.

The mammalian cochlea relies on the active forcing of sensory outer hair cells (OHCs) to amplify traveling wave responses along the basilar membrane. These forces are the result of electromotility, wherein current through the OHCs leads to conformational changes in the cells that provide stresses on surrounding structures. OHC transducer current can be detected via the voltage in the scala tympani (the cochlear microphonic, CM), and the CM can be used as an indicator of healthy cochlear operation. The CM represents a summation of OHC currents (the inner hair cell contribution is known to be small) and to use CM to probe the properties of OHC transduction requires a model that simulates that summation. We developed a finite element model for that purpose. The pattern of current generators (the model input) was initially based on basilar membrane displacement, with the current size based on in vitro data. The model was able to reproduce the amplitude of experimental CM results reasonably well when the input tuning was enhanced slightly (peak increased by ∼6 dB), which can be regarded as additional hair bundle tuning, and with a current/input value of 200-260 pA/nm, which is ∼4 times greater than the largest in vitro measures.

Impact of Systemic versus Intratympanic Dexamethasone Administration on the Perilymph Proteome.

Glucocorticoids are the first-line treatment for sensorineural hearing loss, but little is known about the mechanism of their protective effect or the impact of route of administration. The recent development of hollow microneedles enables safe and reliable sampling of perilymph for proteomic analysis. Using these microneedles, we investigate the effect of intratympanic (IT) versus intraperitoneal (IP) dexamethasone administration on guinea pig perilymph proteome. Guinea pigs were treated with IT dexamethasone (n = 6), IP dexamethasone (n = 8), or untreated for control (n = 8) 6 h prior to aspiration. The round window membrane (RWM) was accessed via a postauricular approach, and hollow microneedles were used to perforate the RWM and aspirate 1 µL of perilymph. Perilymph samples were analyzed by liquid chromatography-mass spectrometry-based label-free quantitative proteomics. Mass spectrometry raw data files have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under data set # MSV000086887. In the 22 samples of perilymph analyzed, 632 proteins were detected, including the inner ear protein cochlin, a perilymph marker. Of these, 14 proteins were modulated by IP, and three proteins were modulated by IT dexamethasone. In both IP and IT dexamethasone groups, VGF nerve growth factor inducible was significantly upregulated compared to control. The remaining adjusted proteins modulate neurons, inflammation, or protein synthesis. Proteome analysis facilitated by the use of hollow microneedles shows that route of dexamethasone administration impacts changes seen in perilymph proteome. Compared to IT administration, the IP route was associated with greater changes in protein expression, including proteins involved in neuroprotection, inflammatory pathway, and protein synthesis. Our findings show that microneedles can mediate safe and effective intracochlear sampling and hold promise for inner ear diagnostics.

An Implantable Umbo Microphone For Fully-Implantable Assistive Hearing Devices.

This paper presents an implantable microphone for sensing the displacement of the umbo, the end of the malleus where it attaches to the center tip of the cone-shaped tympanic membrane. The sensor comprises a piezoelectric polyvinylidene fluoride (PVDF) film with copper-nickel electrodes suspended across a brass cylinder. The cylinder is oriented so that the umbo pushes on the film center, causing a static and acoustically-driven dynamic film displacement. An amplifier filters the resulting piezoelectric charge to produce an output signal. The sensor enables the full implantation of assistive hearing devices, which can restore hearing without inhibiting the user's lifestyle, while enabling better sound localization in noisy environments.

Manipulation of the Endocochlear Potential Reveals Two Distinct Types of Cochlear Nonlinearity.

The mammalian hearing organ, the cochlea, contains an active amplifier to boost the vibrational response to low level sounds. Hallmarks of this active process are sharp location-dependent frequency tuning and compressive nonlinearity over a wide stimulus range. The amplifier relies on outer hair cell (OHC)-generated forces driven in part by the endocochlear potential, the ∼+80 mV potential maintained in scala media, generated by the stria vascularis. We transiently eliminated the endocochlear potential in vivo by an intravenous injection of furosemide and measured the vibrations of different layers in the cochlea's organ of Corti using optical coherence tomography. Distortion product otoacoustic emissions were also monitored. After furosemide injection, the vibrations of the basilar membrane lost the best frequency (BF) peak and showed broad tuning similar to a passive cochlea. The intra-organ of Corti vibrations measured in the region of the OHCs lost the BF peak and showed low-pass responses but retained nonlinearity. This strongly suggests that OHC electromotility was operating and being driven by nonlinear OHC current. Thus, although electromotility is presumably necessary to produce a healthy BF peak, the mere presence of electromotility is not sufficient. The BF peak recovered nearly fully within 2 h, along with the recovery of odd-order distortion product otoacoustic emissions. The recovery pattern suggests that physical shifts in operating condition are a critical step in the recovery process.

Experimental and Theoretical Explorations of Traveling Waves and Tuning in the Bushcricket Ear.

The ability to detect airborne sound is essential for many animals. Examples from the inner ear of mammals and bushcrickets demonstrate that similar detection strategies evolved in taxonomically distant species. Both mammalian and bushcricket ears possess a narrow strip of sensory tissue that exhibits an anatomical gradient and traveling wave motion responses used for frequency discrimination. We measured pressure and motion in the bushcricket ear to investigate physical properties, stiffness, and mass, which govern the mechanical responses to sound. As in the mammalian cochlea, sound-induced fluid pressure and motion responses were tonotopically organized along the longitudinal axis of the crista acustica, the bushcricket's hearing organ. The fluid pressure at the crista and crista motion were used to calculate the acoustic impedance of the organ-bounded fluid mass (Zmass). We used a theoretical wave analysis of wavelength data from a previous study to predict the crista acustica stiffness. The wave analysis also predicts Zmass, and that result agreed reasonably well with the directly measured Zmass, lending support to the theoretical wave analysis. The magnitude of the crista stiffness was similar to basilar membrane stiffness in mammals, and as in mammals, the stiffness decreased from the high-frequency to the low-frequency region. At a given location, the stiffness increased with increasing frequency, corresponding to increasing curvature of the traveling wave (decreasing wavelength), indicating that longitudinal coupling plays a substantial role in determining crista stiffness. This is in contrast to the mammalian ear, in which stiffness is independent of frequency and longitudinal coupling is relatively small.

Adaptation of Cochlear Amplification to Low Endocochlear Potential.

Endocochlear potential (EP) is essential for cochlear amplification by providing the voltage source needed to drive outer hair cell (OHC) transducer current, which leads to OHC electromechanical force. An early study using furosemide to reversibly reduce EP showed that distortion product otoacoustic emissions (DPOAEs) recovered before EP. This indicated that cochlear amplification may be able to adjust to a new, lower EP. To investigate the mechanism of this adjustment, the extracellular OHC voltage, which we term local cochlear microphonic (LCM), was measured simultaneously with DPOAE and EP while using intraperitoneal (IP) and intravenous injection of furosemide to reversibly reduce EP. With IP injection, the DPOAEs recovered fully, whereas the EP was reduced, but LCM showed a similar time course as EP. The DPOAEs failed to accurately report the variation of cochlear amplification. With intravenous injection, for which both reduction and recovery of EP are known to occur relatively quickly compared to IP, the cochlear amplification observed in LCM could attain nearly full or even full recovery with reduced EP. This showed the cochlea has an ability to adjust to diminished operating condition. Furthermore, the cochlear amplifier and EP recovered with different time courses: cochlear amplification just started to recover after the EP was nearly fully recovered and stabilized. Using a Boltzmann model and the second harmonic of the LCM to estimate the mechanoelectric transducer channel operating point, we found that the recovery of cochlear amplification occurred with recentering of the operating point.

Signal competition in optical coherence tomography and its relevance for cochlear vibrometry.

The usual technique for measuring vibration within the cochlear partition is heterodyne interferometry. Recently, spectral domain phase microscopy (SDPM) was introduced and offers improvements over standard heterodyne interferometry. In particular, it has a penetration depth of several mm due to working in the infrared range, has narrow and steep optical sectioning due to using a wideband light source, and is able to measure from several cochlear layers simultaneously. However, SDPM is susceptible to systematic error due to "phase leakage," in which the signal from one layer competes with the signal from other layers. Here, phase leakage is explored in vibration measurements in the cochlea and a model structure. The similarity between phase leakage and signal competition in heterodyne interferometry is demonstrated both experimentally and theoretically. Due to phase leakage, erroneous vibration amplitudes can be reported in regions of low reflectivity that are near structures of high reflectivity. When vibration amplitudes are greater than ∼0.1 of the light source wavelength, phase leakage can cause reported vibration waveforms to be distorted. To aid in the screening of phase leakage in experimental results, the error is plotted and discussed as a function of the important parameters of signal strength and vibration amplitude.

Two-Tone Suppression of Simultaneous Electrical and Mechanical Responses in the Cochlea.

Cochlear frequency tuning is based on a mildly tuned traveling-wave response that is enhanced in amplitude and sharpness by outer hair cell (OHC)-based forces. The nonlinear and active character of this enhancement is the fundamental manifestation of cochlear amplification. Recently, mechanical (pressure) and electrical (extracellular OHC-generated voltage) responses were simultaneously measured close to the sensory tissue's basilar membrane. Both pressure and voltage were tuned and showed traveling-wave phase accumulation, evidence that they were locally generated responses. Approximately at the frequency where nonlinearity commenced, the phase of extracellular voltage shifted up, to lead pressure by >1/4 cycle. Based on established and fundamental relationships among voltage, force, pressure, displacement, and power, the observed phase shift was identified as the activation of cochlear amplification. In this study, the operation of the cochlear amplifier was further explored, via changes in pressure and voltage responses upon delivery of a second, suppressor tone. Two different suppression paradigms were used, one with a low-frequency suppressor and a swept-frequency probe, the other with two swept-frequency tones, either of which can be considered as probe or suppressor. In the presence of a high-level low-frequency suppressor, extracellular voltage responses at probe-tone frequencies were greatly reduced, and the pressure responses were reduced nearly to their linear, passive level. On the other hand, the amplifier-activating phase shift between pressure and voltage responses was still present in probe-tone responses. These findings are consistent with low-frequency suppression being caused by the saturation of OHC electrical responses and not by a change in the power-enabling phasing of the underlying mechanics. In the two-tone swept-frequency suppression paradigm, mild suppression was apparent in the pressure responses, while deep notches could develop in the voltage responses. A simple analysis, based on a two-wave differencing scheme, was used to explore the observations.

Intracochlear Scala Media Pressure Measurement: Implications for Models of Cochlear Mechanics.

Models of the active cochlea build upon the underlying passive mechanics. Passive cochlear mechanics is based on physical and geometrical properties of the cochlea and the fluid-tissue interaction between the cochlear partition and the surrounding fluid. Although the fluid-tissue interaction between the basilar membrane and the fluid in scala tympani (ST) has been explored in both active and passive cochleae, there was no experimental data on the fluid-tissue interaction on the scala media (SM) side of the partition. To this aim, we measured sound-evoked intracochlear pressure in SM close to the partition using micropressure sensors. All the SM pressure data are from passive cochleae, likely because the SM cochleostomy led to loss of endocochlear potential. Thus, these experiments are studies of passive cochlear mechanics. SM pressure close to the tissue showed a pattern of peaks and notches, which could be explained as an interaction between fast and slow (i.e., traveling wave) pressure modes. In several animals SM and ST pressure were measured in the same cochlea. Similar to previous studies, ST-pressure was dominated by a slow, traveling wave mode at stimulus frequencies in the vicinity of the best frequency of the measurement location, and by a fast mode above best frequency. Antisymmetric pressure between SM and ST supported the classic single-partition cochlear models, or a dual-partition model with tight coupling between partitions. From the SM and ST pressure we calculated slow and fast modes, and from active ST pressure we extrapolated the passive findings to the active case. The passive slow mode estimated from SM and ST data was low-pass in nature, as predicted by cochlear models.

A study of sound transmission in an abstract middle ear using physical and finite element models.

The classical picture of middle ear (ME) transmission has the tympanic membrane (TM) as a piston and the ME cavity as a vacuum. In reality, the TM moves in a complex multiphasic pattern and substantial pressure is radiated into the ME cavity by the motion of the TM. This study explores ME transmission with a simple model, using a tube terminated with a plastic membrane. Membrane motion was measured with a laser interferometer and pressure on both sides of the membrane with micro-sensors that could be positioned close to the membrane without disturbance. A finite element model of the system explored the experimental results. Both experimental and theoretical results show resonances that are in some cases primarily acoustical or mechanical and sometimes produced by coupled acousto-mechanics. The largest membrane motions were a result of the membrane's mechanical resonances. At these resonant frequencies, sound transmission through the system was larger with the membrane in place than it was when the membrane was absent.

External and middle ear sound pressure distribution and acoustic coupling to the tympanic membrane.

Sound energy is conveyed to the inner ear by the diaphanous, cone-shaped tympanic membrane (TM). The TM moves in a complex manner and transmits sound signals to the inner ear with high fidelity, pressure gain, and a short delay. Miniaturized sensors allowing high spatial resolution in small spaces and sensitivity to high frequencies were used to explore how pressure drives the TM. Salient findings are: (1) A substantial pressure drop exists across the TM, and varies in frequency from ∼10 to 30 dB. It thus appears reasonable to approximate the drive to the TM as being defined solely by the pressure in the ear canal (EC) close to the TM. (2) Within the middle ear cavity (MEC), spatial variations in sound pressure could vary by more than 20 dB, and the MEC pressure at certain locations/frequencies was as large as in the EC. (3) Spatial variations in pressure along the TM surface on the EC-side were typically less than 5 dB up to 50 kHz. Larger surface variations were observed on the MEC-side.

Regional differences in cochlear nonlinearity across the basal organ of Corti of gerbil: Regional differences in cochlear nonlinearity.

Auditory sensation is based in nanoscale vibration of the sensory tissue of the cochlea, the organ of Corti complex (OCC). Motion within the OCC is now observable due to optical coherence tomography. In a previous study (Cooper et al., 2018), the region that includes the electro-motile outer hair cells (OHC) and Deiters cells (DC) was observed to move with larger amplitude than the basilar membrane (BM) and surrounding regions and was termed the "hotspot." In addition to this quantitative distinction, the hotspot moved qualitatively differently than the BM, in that its motion scaled nonlinearly with stimulus level at all frequencies, evincing sub-BF activity. Sub-BF activity enhances non-BF motion; thus the frequency tuning of the OHC/DC region was reduced relative to the BM. In this work we further explore the motion of the gerbil basal OCC and find that regions that lack significant sub-BF activity include the BM, the medial and lateral OCC, and the reticular lamina (RL) region. The observation that the RL region does not move actively sub-BF (already observed in Cho and Puria 2022), suggests that hair cell stereocilia are not exposed to sub-BF activity in the cochlear base. The observation that the lateral and RL regions move approximately linearly sub-BF indicates that linear forces dominate non-linear OHC-based forces on these components at sub-BF frequencies. A complex difference analysis was performed to reveal the internal motion of the OHC/DC region and showed that amplitude structure and phase shifts in the directly measured OHC/DC motion emerge due to the internal OHC/DC motion destructively interfering with BM motion.

Physiologic Effects of Microneedle-Mediated Intracochlear Dexamethasone Injection in the Guinea Pig.

OBJECTIVES: Oral or intratympanic corticosteroids are commonly used to treat sudden sensorineural hearing loss (SSHL), tinnitus, and Meniere disease. Direct intracochlear delivery has been proposed to overcome the variability in bioavailability and efficacy of systemic or middle ear delivery. In this study, we aim to characterize the physiologic consequences of microneedle-mediated direct intracochlear injection of dexamethasone through the round window membrane (RWM). METHODS: In Hartley guinea pigs (n = 5), a post-auricular incision followed by bullostomy was made to access the round window membrane. Using 100 µm diameter hollow microneedles, 1.0 µl of 10 mg/ml dexamethasone was injected through the RWM over 1 min. Compound action potential (CAP) and distortion product otoacoustic action emissions (DPOAE) were measured before perforation, at 1 h, and at 5 h following injection. CAP hearing thresholds were measured from 0.5 to 40 kHz, and DPOAE f2 frequencies ranged from 1.0 and 32 kHz. Repeated measures ANOVA followed by pairwise t-tests were used for statistical analysis. RESULTS: ANOVA identified significant CAP threshold shifts at four frequencies (4, 16, 36, and 40 kHz) and differences in DPOAE at 1 frequency (6 kHz). Paired t-tests revealed differences between the pre-perforation and 1 h time point. By 5 h post injection, both CAP hearing thresholds and DPOAE recover and are not significantly different from baseline thresholds. CONCLUSION: Direct intracochlear delivery of dexamethasone via microneedles results in temporary shifts in hearing thresholds that resolve by 5 hours, thus supporting microneedle technology for the treatment of inner ear disorders. LEVEL OF EVIDENCE: NA Laryngoscope, 134:388-392, 2024.

Hampshire Sheep as a Large-Animal Model for Cochlear Implantation.

BACKGROUND: Sheep have been proposed as a large-animal model for studying cochlear implantation. However, prior sheep studies report that the facial nerve (FN) obscures the round window membrane (RWM), requiring FN sacrifice or a retrofacial opening to access the middle-ear cavity posterior to the FN for cochlear implantation. We investigated surgical access to the RWM in Hampshire sheep compared to Suffolk-Dorset sheep and the feasibility of Hampshire sheep for cochlear implantation via a facial recess approach. METHODS: Sixteen temporal bones from cadaveric sheep heads (ten Hampshire and six Suffolk-Dorset) were dissected to gain surgical access to the RWM via an extended facial recess approach. RWM visibility was graded using St. Thomas' Hospital (STH) classification. Cochlear implant (CI) electrode array insertion was performed in two Hampshire specimens. Micro-CT scans were obtained for each temporal bone, with confirmation of appropriate electrode array placement and segmentation of the inner ear structures. RESULTS: Visibility of the RWM on average was 83% in Hampshire specimens and 59% in Suffolk-Dorset specimens (p = 0.0262). Hampshire RWM visibility was Type I (100% visibility) for three specimens and Type IIa (> 50% visibility) for seven specimens. Suffolk-Dorset RWM visibility was Type IIa for four specimens and Type IIb (< 50% visibility) for two specimens. FN appeared to course more anterolaterally in Suffolk-Dorset specimens. Micro-CT confirmed appropriate CI electrode array placement in the scala tympani without apparent basilar membrane rupture. CONCLUSIONS: Hampshire sheep appear to be a suitable large-animal model for CI electrode insertion via an extended facial recess approach without sacrificing the FN. In this small sample, Hampshire specimens had improved RWM visibility compared to Suffolk-Dorset. Thus, Hampshire sheep may be superior to other breeds for ease of cochlear implantation, with FN and facial recess anatomy more similar to humans.

An Implantable Piezofilm Middle Ear Microphone: Performance in Human Cadaveric Temporal Bones.

PURPOSE: One of the major reasons that totally implantable cochlear microphones are not readily available is the lack of good implantable microphones. An implantable microphone has the potential to provide a range of benefits over external microphones for cochlear implant users including the filtering ability of the outer ear, cosmetics, and usability in all situations. This paper presents results from experiments in human cadaveric ears of a piezofilm microphone concept under development as a possible component of a future implantable microphone system for use with cochlear implants. This microphone is referred to here as a drum microphone (DrumMic) that senses the robust and predictable motion of the umbo, the tip of the malleus. METHODS: The performance was measured by five DrumMics inserted in four different human cadaveric temporal bones. Sensitivity, linearity, bandwidth, and equivalent input noise were measured during these experiments using a sound stimulus and measurement setup. RESULTS: The sensitivity of the DrumMics was found to be tightly clustered across different microphones and ears despite differences in umbo and middle ear anatomy. The DrumMics were shown to behave linearly across a large dynamic range (46 dB SPL to 100 dB SPL) across a wide bandwidth (100 Hz to 8 kHz). The equivalent input noise (over a bandwidth of 0.1-10 kHz) of the DrumMic and amplifier referenced to the ear canal was measured to be about 54 dB SPL in the temporal bone experiment and estimated to be 46 dB SPL after accounting for the pressure gain of the outer ear. CONCLUSION: The results demonstrate that the DrumMic behaves robustly across ears and fabrication. The equivalent input noise performance (related to the lowest level of sound measurable) was shown to approach that of commercial hearing aid microphones. To advance this demonstration of the DrumMic concept to a future prototype implantable in humans, work on encapsulation, biocompatibility, and connectorization will be required.

Microneedle-Mediated Delivery of siRNA via Liposomal-Based Transfection for Inner Ear Gene Therapy.

HYPOTHESIS: Microneedle-mediated intracochlear injection of siRNA-Lipofectamine through the round window membrane (RWM) can be used to transfect cells within the cochlea. BACKGROUND: Our laboratory has developed 100-µm diameter hollow microneedles for intracochlear injection through the guinea pig RWM. In this study, we test the feasibility of microneedle-mediated injection of siRNA and Lipofectamine, a commonly used reagent with known cellular toxicity, through the RWM for cochlear transfection. METHODS: Fluorescently labeled scramble siRNA was diluted into Lipofectamine RNAiMax and OptiMEM. One microliter of 5 µM siRNA was injected through the RWM of Hartley guinea pigs at a rate of 1 µl/min (n = 22). In a control group, 1.0 µl of Lipofectamine, with no siRNA, was diluted into OptiMEM and injected in a similar fashion (n = 5). Hearing tests were performed before and either at 24 hours, 48 hours, or 5 days after injection. Afterward, animals were euthanized, and cochleae were harvested for imaging. Control cochleae were processed in parallel to untreated guinea pigs. RESULTS: Fluorescence, indicating successful transfection, was observed within the basal and middle turns of the cochlea with limited distribution in the apex at 24 and 48 hours. Signal was most intense in the organ of Corti, spiral ligament, and spiral ganglion. Little to no fluorescence was observed at 5 days post-injection. No significant changes in auditory brainstem response (ABR) were noted post-perforation at 5 days, suggesting that siRNA-Lipofectamine at low doses does not cause cochlear toxicity. CONCLUSIONS: Small volumes of siRNA and Lipofectamine can be effectively delivered to cochlear structures using microneedles, paving the way for atraumatic cochlear gene therapy.