Simon S. Gao

Deficient forward transduction and enhanced reverse transduction in the alpha tectorin C1509G human hearing loss mutation

SUMMARY Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliary bundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and the C1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice and found that the TM was shortened in heterozygous TectaC1509G/+ mice, reaching only the first row of OHCs. Thus, deficient forward transduction renders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both TectaC1509G/+ and TectaC1509G/C1509G mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that an increase in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstrates a human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.

Optimization of the split-spectrum amplitude-decorrelation angiography algorithm on a spectral optical coherence tomography system

The split-spectrum amplitude-decorrelation angiography algorithm was optimized on a spectral optical coherence tomography system using a flow phantom. The number of times the spectrum was split and the bandwidth of each split were adjusted to maximize the flow phantom decorrelation signal-to-noise ratio. The improvement in flow detection was then demonstrated with en face retinal angiograms. The optimized algorithm increased the detectable retinal microvascular flow and decreased the variability of the quantified vessel density in OCT retinal angiograms of healthy human subjects.

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.

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.

Imaging high-frequency periodic motion in the mouse ear with coherently interleaved optical coherence tomography.

Vibratory measurements of the structures of the ear are key to understanding much of the pathology in mouse models of hearing loss. Unfortunately the high-speed sampling required to interrogate the high end of the mouse hearing spectrum is beyond the reach of most optical coherence tomography (OCT) systems. To address this issue, we have developed an algorithm that enables phase-sensitive OCT measurements over the full range of the mouse hearing spectrum (4-90 kHz). The algorithm phase-locks the line-trigger to the acoustic stimulation and then uses interleaved sampling to reconstruct the signal with higher temporal sampling. The algorithm was evaluated by measuring the vibratory response of mouse tympanic membrane to a pure tone stimulus.

Calcium imaging of inner ear hair cells within the cochlear epithelium of mice using two-photon microscopy

Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing, loss are commonly available. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes study of their hair cells difficult. We develop a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae are harvested, left intact within their otic capsule bone, and fixed in a recording chamber. The bone overlying the cochlear epithelium is opened and Reissners membrane is incised. A fluorescent calcium indicator is applied to the preparation. A custom-built upright two-photon microscope was used to image the preparation using 3-D scanning. We are able to image about one third of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we find that outer hair cells demonstrate increased fluorescence compared with surrounding supporting cells. This methodology is then used to visualize hair cell calcium changes during mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are expected to be reduced.

The candidate splicing factor Sfswap regulates growth and patterning of inner ear sensory organs.

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.

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.