Patrick D. Raphael

Noninvasive in vivo imaging reveals differences between tectorial membrane and basilar membrane traveling waves in the mouse cochlea.

Significance The membranes within the cochlea vibrate in response to sound. However, measuring these vibrations to study the sense of hearing has been a technological challenge because invasive techniques have been required. Herein, we describe a new technique capable of depth-resolved displacement measurements in 3D space with picometer sensitivity within the unopened mouse cochlea. We used this technique to make, to our knowledge, the first measurements of the tectorial membrane, the structure that overlies the sensory hair cell stereociliary bundles, within a healthy cochlea. We found that the tectorial membrane sustains traveling wave propagation differently than the more commonly measured basilar membrane. This finding provides a clearer understanding of the mechanical stimulus at the level of the inner hair cell responsible for non-linear sound encoding. Sound is encoded within the auditory portion of the inner ear, the cochlea, after propagating down its length as a traveling wave. For over half a century, vibratory measurements to study cochlear traveling waves have been made using invasive approaches such as laser Doppler vibrometry. Although these studies have provided critical information regarding the nonlinear processes within the living cochlea that increase the amplitude of vibration and sharpen frequency tuning, the data have typically been limited to point measurements of basilar membrane vibration. In addition, opening the cochlea may alter its function and affect the findings. Here we describe volumetric optical coherence tomography vibrometry, a technique that overcomes these limitations by providing depth-resolved displacement measurements at 200 kHz inside a 3D volume of tissue with picometer sensitivity. We studied the mouse cochlea by imaging noninvasively through the surrounding bone to measure sound-induced vibrations of the sensory structures in vivo, and report, to our knowledge, the first measures of tectorial membrane vibration within the unopened cochlea. We found that the tectorial membrane sustains traveling wave propagation. Compared with basilar membrane traveling waves, tectorial membrane traveling waves have larger dynamic ranges, sharper frequency tuning, and apically shifted positions of peak vibration. These findings explain discrepancies between previously published basilar membrane vibration and auditory nerve single unit data. Because the tectorial membrane directly overlies the inner hair cell stereociliary bundles, these data provide the most accurate characterization of the stimulus shaping the afferent auditory response available to date.

Mechanisms of hearing loss after blast injury to the ear.

Given the frequent use of improvised explosive devices (IEDs) around the world, the study of traumatic blast injuries is of increasing interest. The ear is the most common organ affected by blast injury because it is the body’s most sensitive pressure transducer. We fabricated a blast chamber to re-create blast profiles similar to that of IEDs and used it to develop a reproducible mouse model to study blast-induced hearing loss. The tympanic membrane was perforated in all mice after blast exposure and found to heal spontaneously. Micro-computed tomography demonstrated no evidence for middle ear or otic capsule injuries; however, the healed tympanic membrane was thickened. Auditory brainstem response and distortion product otoacoustic emission threshold shifts were found to be correlated with blast intensity. As well, these threshold shifts were larger than those found in control mice that underwent surgical perforation of their tympanic membranes, indicating cochlear trauma. Histological studies one week and three months after the blast demonstrated no disruption or damage to the intra-cochlear membranes. However, there was loss of outer hair cells (OHCs) within the basal turn of the cochlea and decreased spiral ganglion neurons (SGNs) and afferent nerve synapses. Using our mouse model that recapitulates human IED exposure, our results identify that the mechanisms underlying blast-induced hearing loss does not include gross membranous rupture as is commonly believed. Instead, there is both OHC and SGN loss that produce auditory dysfunction.

Quantitative imaging of cochlear soft tissues in wild-type and hearing-impaired transgenic mice by spectral domain optical coherence tomography

Human hearing loss often occurs as a result of damage or malformations to the functional soft tissues within the cochlea, but these changes are not appreciable with current medical imaging modalities. We sought to determine whether optical coherence tomography (OCT) could assess the soft tissue structures relevant to hearing using mouse models. We imaged excised cochleae with an altered tectorial membrane and during normal development. The soft tissue structures and expected anatomical variations were visible using OCT, and quantitative measurements confirmed the ability to detect critical changes relevant to hearing.

In vivo vibrometry inside the apex of the mouse cochlea using spectral domain optical coherence tomography

Sound transduction within the auditory portion of the inner ear, the cochlea, is a complex nonlinear process. The study of cochlear mechanics in large rodents has provided important insights into cochlear function. However, technological and experimental limitations have restricted studies in mice due to their smaller cochlea. These challenges are important to overcome because of the wide variety of transgenic mouse strains with hearing loss mutations that are available for study. To accomplish this goal, we used spectral domain optical coherence tomography to visualize and measure sound-induced vibrations of intracochlear tissues. We present, to our knowledge, the first vibration measurements from the apex of an unopened mouse cochlea.

Two-Dimensional Cochlear Micromechanics Measured In Vivo Demonstrate Radial Tuning within the Mouse Organ of Corti

The exquisite sensitivity and frequency discrimination of mammalian hearing underlie the ability to understand complex speech in noise. This requires force generation by cochlear outer hair cells (OHCs) to amplify the basilar membrane traveling wave; however, it is unclear how amplification is achieved with sharp frequency tuning. Here we investigated the origin of tuning by measuring sound-induced 2-D vibrations within the mouse organ of Corti in vivo. Our goal was to determine the transfer function relating the radial shear between the structures that deflect the OHC bundle, the tectorial membrane and reticular lamina, to the transverse motion of the basilar membrane. We found that, after normalizing their responses to the vibration of the basilar membrane, the radial vibrations of the tectorial membrane and reticular lamina were tuned. The radial tuning peaked at a higher frequency than transverse basilar membrane tuning in the passive, postmortem condition. The radial tuning was similar in dead mice, indicating that this reflected passive, not active, mechanics. These findings were exaggerated in TectaC1509G/C1509G mice, where the tectorial membrane is detached from OHC stereocilia, arguing that the tuning of radial vibrations within the hair cell epithelium is distinct from tectorial membrane tuning. Together, these results reveal a passive, frequency-dependent contribution to cochlear filtering that is independent of basilar membrane filtering. These data argue that passive mechanics within the organ of Corti sharpen frequency selectivity by defining which OHCs enhance the vibration of the basilar membrane, thereby tuning the gain of cochlear amplification. SIGNIFICANCE STATEMENT Outer hair cells amplify the traveling wave within the mammalian cochlea. The resultant gain and frequency sharpening are necessary for speech discrimination, particularly in the presence of background noise. Here we measured the 2-D motion of the organ of Corti in mice and found that the structures that stimulate the outer hair cell stereocilia, the tectorial membrane and reticular lamina, were sharply tuned in the radial direction. Radial tuning was similar in dead mice and in mice lacking a tectorial membrane. This suggests that radial tuning comes from passive mechanics within the hair cell epithelium, and that these mechanics, at least in part, may tune the gain of cochlear amplification.

Prestin Regulation and Function in Residual Outer Hair Cells after Noise-Induced Hearing Loss

The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. TectaC1509G transgenic mice have hearing loss, and surprisingly have increased OHC prestin levels. We hypothesized, therefore, that prestin up-regulation may represent a generalized response to compensate for a state of hearing loss. In the present study, we sought to determine the effects of noise-induced hearing loss on prestin expression. After noise exposure, we performed cytocochleograms and observed OHC loss only in the basal region of the cochlea. Next, we patch clamped OHCs from the apical turn (9–12 kHz region), where no OHCs were lost, in noise-exposed and age-matched control mice. The non-linear capacitance was significantly higher in noise-exposed mice, consistent with higher functional prestin levels. We then measured prestin protein and mRNA levels in whole-cochlea specimens. Both Western blot and qPCR studies demonstrated increased prestin expression after noise exposure. Finally, we examined the effect of the prestin increase in vivo following noise damage. Immediately after noise exposure, ABR and DPOAE thresholds were elevated by 30–40 dB. While most of the temporary threshold shifts recovered within 3 days, there were additional improvements over the next month. However, DPOAE magnitudes, basilar membrane vibration, and CAP tuning curve measurements from the 9–12 kHz cochlear region demonstrated no differences between noise-exposed mice and control mice. Taken together, these data indicate that prestin is up-regulated by 32–58% in residual OHCs after noise exposure and that the prestin is functional. These findings are consistent with the notion that prestin increases in an attempt to partially compensate for reduced force production because of missing OHCs. However, in regions where there is no OHC loss, the cochlea is able to compensate for the excess prestin in order to maintain stable auditory thresholds and frequency discrimination.

Hair cell force generation does not amplify or tune vibrations within the chicken basilar papilla.

Frequency tuning within the auditory papilla of most non-mammalian species is electrical, deriving from ion-channel resonance within their sensory hair cells. In contrast, tuning within the mammalian cochlea is mechanical, stemming from active mechanisms within outer hair cells that amplify the basilar membrane travelling wave. Interestingly, hair cells in the avian basilar papilla demonstrate both electrical resonance and force-generation, making it unclear which mechanism creates sharp frequency tuning. Here, we measured sound-induced vibrations within the apical half of the chicken basilar papilla in vivo and found broadly-tuned travelling waves that were not amplified. However, distortion products were found in live but not dead chickens. These findings support the idea that avian hair cells do produce force, but that their effects on vibration are small and do not sharpen tuning. Therefore, frequency tuning within the apical avian basilar papilla is not mechanical, and likely derives from hair cell electrical resonance.

High-speed spectral calibration by complex FIR filter in phase-sensitive optical coherence tomography.

Swept-laser sources offer a number of advantages for Phase-sensitive Optical Coherence Tomography (PhOCT). However, inter- and intra-sweep variability leads to calibration errors that adversely affect phase sensitivity. While there are several approaches to overcoming this problem, our preferred method is to simply calibrate every sweep of the laser. This approach offers high accuracy and phase stability at the expense of a substantial processing burden. In this approach, the Hilbert phase of the interferogram from a reference interferometer provides the instantaneous wavenumber of the laser, but is computationally expensive. Fortunately, the Hilbert transform may be approximated by a Finite Impulse-Response (FIR) filter. Here we explore the use of several FIR filter based Hilbert transforms for calibration, explicitly considering the impact of filter choice on phase sensitivity and OCT image quality. Our results indicate that the complex FIR filter approach is the most robust and accurate among those considered. It provides similar image quality and slightly better phase sensitivity than the traditional FFT-IFFT based Hilbert transform while consuming fewer resources in an FPGA implementation. We also explored utilizing the Hilbert magnitude of the reference interferogram to calculate an ideal window function for spectral amplitude calibration. The ideal window function is designed to carefully control sidelobes on the axial point spread function. We found that after a simple chromatic correction, calculating the window function using the complex FIR filter and the reference interferometer gave similar results to window functions calculated using a mirror sample and the FFT-IFFT Hilbert transform. Hence, the complex FIR filter can enable accurate and high-speed calibration of the magnitude and phase of spectral interferograms.

Methodology for assessment of structural vibrations by spectral domain optical coherence tomography

Clinical diagnosis of cochlear dysfunction typically remains incomplete due to a lack of proper diagnostic methods. Medical imaging modalities can only detect gross changes in the cochlea, and non-invasive in vivo cochlear measurements are scarce. As a result, extensive efforts have been made to adapt optical coherence tomography (OCT) techniques to analyze and study the cochlea. Herein, we detail the methods for measuring vibration using OCT. We used spectral domain OCT with ~950 nm as the center wavelength and a bandwidth of ~80 nm. The custom spectrometer used was based on a high speed line scan camera which is capable of line rates up to 28 kHz. The signal-to- noise ratio of the system was ~90 dB. The data collection and processing software was written in LabVIEW and MATLAB. We tested whether streaming directly from the camera, writing the data to multiple hard drives in the RAID- 0 configuration, and processing using the GPU shortened experiment times. We then analyzed the A-line phase noise over several hundred milliseconds and growth curves from a piezoelectric element. We believe this is the first step towards a diagnostic device which generates vibration information of cochlear structures.

Basilar membrane vibration after targeted removal of the third row of OHCs and Deiters cells

The mammalian cochlea has three rows of outer hair cells (OHCs) that amplify the basilar membrane (BM) traveling wave with high gain and exquisite sharpness. However, it is unclear why three rows of OHCs are needed to achieve this. We used a novel transgenic mouse with the diphtheria toxin receptor in Lgr5-positive cells (Lgr5DTR-EGFP/+ mouse) that allowed us to ablate the third row of OHCs and Deiters cells (D) in adulthood via DT injection, after normal cochlear function had developed. We then used volumetric optical coherence tomography (VOCTV) to investigate the impacts of this manipulation of cochlear amplification in the apical turn. As expected, Lgr5DTR-EGFP/+ control mice had sharply-tuned vibratory responses. However, Lgr5DTR-EGFP/+ mice had broad tuning with a 20 dB increase in vibratory thresholds. The Q10dB was ∼1 in Lgr5DTR-EGFP/+ mice, whereas it was ∼3 in control mice. The characteristic frequency was lower in Lgr5DTR-EGFP/+ mice compared to controls (7.5 vs. 9.0 kHz). The gain of cochlear amplification was substantially lower in Lgr5DTR-EGFP/+ mice compared to controls (22 vs. 50). In the post-mortem period, the vibratory responses in Lgr5DTR-EGFP/+ mice were identical to controls. Together, these results demonstrate the substantial importance of the third row of OHCs and Deiters cells to normal cochlear amplification.The mammalian cochlea has three rows of outer hair cells (OHCs) that amplify the basilar membrane (BM) traveling wave with high gain and exquisite sharpness. However, it is unclear why three rows of OHCs are needed to achieve this. We used a novel transgenic mouse with the diphtheria toxin receptor in Lgr5-positive cells (Lgr5DTR-EGFP/+ mouse) that allowed us to ablate the third row of OHCs and Deiters cells (D) in adulthood via DT injection, after normal cochlear function had developed. We then used volumetric optical coherence tomography (VOCTV) to investigate the impacts of this manipulation of cochlear amplification in the apical turn. As expected, Lgr5DTR-EGFP/+ control mice had sharply-tuned vibratory responses. However, Lgr5DTR-EGFP/+ mice had broad tuning with a 20 dB increase in vibratory thresholds. The Q10dB was ∼1 in Lgr5DTR-EGFP/+ mice, whereas it was ∼3 in control mice. The characteristic frequency was lower in Lgr5DTR-EGFP/+ mice compared to controls (7.5 vs. 9.0 kHz). The gain of cochlear ...