Generation of a zebrafish neurofibromatosis model via inducible knockout of nf2.

Neurofibromatosis Type 2 (NF-2) is a dominantly inherited genetic disorder that results from mutations in the tumor suppressor gene, neurofibromin 2 (NF2) gene. Here, we report the generation of a conditional zebrafish model of neurofibromatosis established by an inducible genetic knockout of nf2a/b, the zebrafish homolog of human NF2. Analysis of nf2a and nf2b expression reveals ubiquitous expression of nf2b in the early embryo, with overlapping expression in the neural crest and its derivatives and in the cranial mesenchyme. In contrast, nf2a displays lower expression levels. Induction of nf2a/b knockout at early stages increases the proliferation of larval Schwann cells and meningeal fibroblasts. Subsequently, in adult zebrafish, nf2a/b knockout triggers the development of a spectrum of tumors, including vestibular schwannomas, spinal schwannomas, meningiomas, and retinal hamartomas, mirroring the tumor manifestations observed in patients with NF-2. Collectively, these findings highlight the generation of a novel zebrafish model that mimics the complexities of the human NF-2 disorder. Consequently, this model holds significant potential for facilitating therapeutic screening and elucidating key driver genes implicated in NF-2 onset.

COVID-19 in the Clinic: Trial of an Aerosol Containment Mask for Endoscopic Clinic Procedures.

OBJECTIVE: Create an aerosol containment mask (ACM) for common otolaryngologic endoscopic procedures which also provides nanoparticle-level protection to patients. STUDY DESIGN: Prospective feasibility study. SETTING: In-person testing with a novel ACM. METHODS: The mask was designed in Solidworks and 3-dimensional printed. Measurements were made on 100 consecutive clinic patients who underwent medically necessarily endoscopy, 50 rigid nasal and 50 flexible, by 9 surgeons. RESULTS: Of the 50 patients who underwent rigid nasal endoscopy with the ACM, 0 of 25 patients with the suction off and 0 of 25 patients with the suction on had evidence of leakage of 0.3 µm particles. Of the 50 patients who underwent flexible endoscopy with the ACM, 0 of 25 patients with the suction off and 0 of 25 patients with the suction on had evidence of leakage of 0.3 µm particles. In terms of comfort, 73% of patients found the ACM somewhat or very comfortable without suction, compared to 86% with the suction on. Surgeons were able to visualize all necessary anatomic areas in 98% of procedures. In 97% of procedures, the masks were able to be placed easily. CONCLUSION: ACM can accommodate rigid nasal and flexible endoscopes and may prevent leakage of patient-generated aerosols, thus avoiding contamination of the room and protecting health care workers from airborne contagions. LEVEL OF EVIDENCE: The level of evidence is 2.

Fabrication of waveguide directional couplers using 2-photon lithography.

Advances in 2-photon lithography have enabled in-lab production of sub-micron resolution and millimeter scale 3D optical components. The potential complex geometries are well suited to rapid prototyping and production of waveguide structures, interconnects, and waveguide directional couplers, furthering future development and miniaturization of waveguide-based imaging technologies. System alignment is inherent to the 2-photon process, obviating the need for manual assembly and allowing precise micron scale waveguide geometries not possible in traditional fused fiber coupler fabrication. Here we present the use of 2-photon lithography for direct printing of multi-mode waveguide couplers with air cladding and single mode waveguide couplers with uncured liquid photoresin cladding. Experimental results show reproducible coupling which can be modified by selected design parameters.

Porcine vocal fold elasticity evaluation using Brillouin spectroscopy.

Significance: The vocal folds are critically important structures within the larynx which serve the essential functions of supporting the airway, preventing aspiration, and phonation. The vocal fold mucosa has a unique multilayered architecture whose layers have discrete viscoelastic properties facilitating sound production. Perturbations in these properties lead to voice loss. Currently, vocal fold pliability is inferred clinically using laryngeal videostroboscopy and no tools are available for in vivo objective assessment. Aim: The main objective of the present study is to evaluate viability of Brillouin microspectroscopy for differentiating vocal folds' mechanical properties against surrounding tissues. Approach: We used Brillouin microspectroscopy as an emerging optical imaging modality capable of providing information about local viscoelastic properties of tissues in noninvasive and remote manner. Results: Brillouin measurements of the porcine larynx vocal folds were performed. Elasticity-driven Brillouin spectral shifts were recorded and analyzed. Elastic properties, as assessed by Brillouin spectroscopy, strongly correlate with those acquired using classical elasticity measurements. Conclusions: These results demonstrate the feasibility of Brillouin spectroscopy for vocal fold imaging. With more extensive research, this technique may provide noninvasive objective assessment of vocal fold mucosal pliability toward objective diagnoses and more targeted treatments.

Multi-window approach enables two-fold improvement in OCT axial resolution with strong side-lobe suppression and improved phase sensitivity.

A common processing approach for optical coherence tomography (OCT) uses a window function (e.g., Hann or rectangular window) for spectral shaping prior to calculating the Fourier transform. Here we build on a multi-window approach [Opt. Express8, 5267 (2017)10.1364/BOE.8.005267] that enables improved resolution while still suppressing side-lobe intensity. The shape of the window function defines the trade-off between main-lobe width (resolution) and side-lobe intensity. We have extended the approach to include the interferometric phase for phase-sensitive applications like vibrometry and Doppler OCT. Using the Hann window as a reference, we show that 11 Taylor windows are sufficient to achieve 50% improvement in axial resolution, -31 dB side-lobe intensity, and 20% improvement in phase sensitivity with low computational cost.

Electronic frequency shifting enables long, variable working distance optical coherence tomography.

Increased imaging range is of growing interest in many applications of optical coherence tomography to reduce constraints on sample location, size, and topography. The design of optical coherence tomography systems with sufficient imaging range (e.g., 10s of centimeters) is a significant challenge due to the direct link between imaging range and acquisition bandwidth. We have developed a novel and flexible method to extend the imaging range in optical coherence tomography using electronic frequency shifting, enabling imaging in dynamic environments. In our approach, a laser with a quasi-linear sweep is used to limit the interferometric bandwidth, enabling decoupling of imaging range and acquisition bandwidth, while a tunable lens allows dynamic refocusing in the sample arm. Electronic frequency shifting then removes the need for high frequency digitization. This strategy is demonstrated to achieve high contrast morphological imaging over a > 21 cm working distance range, while maintaining high resolution and phase sensitivity. The system design is flexible to the application while requiring only a simple phase correction in post-processing. By implementing this approach in an auto-focusing paradigm, the proposed method demonstrates strong potential for the translation of optical coherence tomography into emerging applications requiring variable and centimeter-scale imaging ranges.

The impact of targeted ablation of one row of outer hair cells and Deiters' cells on cochlear amplification.

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 Lgr5DTR-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.NEW & NOTEWORTHY Normal cochlear physiology involves force production from three rows of outer hair cells to amplify and tune the traveling wave. Here, we used a genetic approach to target and ablate the third row of outer hair cells in the mouse cochlea and found it reduced cochlear amplification by 79%. This means that the loss of even a relatively small number of OHCs, when evenly distributed, causes a substantial reduction in cochlear amplification.

Interplay between traveling wave propagation and amplification at the apex of the mouse cochlea.

Sounds entering the mammalian ear produce waves that travel from the base to the apex of the cochlea. An electromechanical active process amplifies traveling wave motions and enables sound processing over a broad range of frequencies and intensities. The cochlear amplifier requires combining the global traveling wave with the local cellular processes that change along the length of the cochlea given the gradual changes in hair cell and supporting cell anatomy and physiology. Thus, we measured basilar membrane (BM) traveling waves in vivo along the apical turn of the mouse cochlea using volumetric optical coherence tomography and vibrometry. We found that there was a gradual reduction in key features of the active process toward the apex. For example, the gain decreased from 23 to 19 dB and tuning sharpness decreased from 2.5 to 1.4. Furthermore, we measured the frequency and intensity dependence of traveling wave properties. The phase velocity was larger than the group velocity, and both quantities gradually decrease from the base to the apex denoting a strong dispersion characteristic near the helicotrema. Moreover, we found that the spatial wavelength along the BM was highly level dependent in vivo, such that increasing the sound intensity from 30 to 90 dB sound pressure level increased the wavelength from 504 to 874 µm, a factor of 1.73. We hypothesize that this wavelength variation with sound intensity gives rise to an increase of the fluid-loaded mass on the BM and tunes its local resonance frequency. Together, these data demonstrate a strong interplay between the traveling wave propagation and amplification along the length of the cochlea.

Vector of motion measurements in the living cochlea using a 3D OCT vibrometry system.

Optical coherence tomography (OCT) has become an important tool for measuring the vibratory response of the living cochlea. It stands alone in its capacity to measure the intricate motion of the hearing organ through the surrounding otic capsule bone. Nevertheless, as an extension of phase-sensitive OCT, it is only capable of measuring motion along the optical axis. Hence, measurements are 1-D. To overcome this limitation and provide a measure of the 3-D vector of motion in the cochlea, we developed an OCT system with three sample arms in a single interferometer. Taking advantage of the long coherence length of our swept laser, we depth (frequency) encode the three channels. An algorithm to depth decode and coregister the three channels is followed by a coordinate transformation that takes the vibrational data from the experimental coordinate system to Cartesian or spherical polar coordinates. The system was validated using a piezo as a known vibrating element that could be positioned at various angles. The angular measurement on the piezo was shown to have an RMSE of ≤ 0.30° (5.2 mrad) with a standard deviation of the amplitude of ≤ 120 pm. Finally, we demonstrate the system for in vivo imaging by measuring the vector of motion over a volume image in the apex of the mouse cochlea.

Unloading outer hair cell bundles in vivo does not yield evidence of spontaneous oscillations in the mouse cochlea.

Along with outer hair cell (OHC) somatic electromotility as the actuator of cochlear amplification, active hair bundle motility may be a complementary mechanism in the mammalian auditory system. Here, we searched the mouse cochlea for the presence of spontaneous bundle oscillations that have been observed in non-mammalian ears. In those systems, removal of the overlying membrane is necessary for spontaneous bundle oscillations to manifest. Thus, we used a genetic mouse model with a C1509G (cysteine-to-glycine) point mutation in the Tecta gene where the tectorial (TM) is lifted away from the OHC bundles, allowing us to explore whether unloaded bundles spontaneously oscillate. We used VOCTV in vivo to detect OHC length changes due to electromotility as a proxy for the spontaneous opening and closing of the mechanoelectrical transduction (MET) channels associated with bundle oscillation. In wild type mice with the TM attached to OHC bundles, we did find peaks in vibratory magnitude spectra. Such peaks were not observed in the mutants where the TM is detached from the OHC bundles. Statistical analysis of the time signals indicates that these peaks do not signify active oscillations. Rather, they are filtered responses of the sensitive wild type cochlea to weak background noise. We therefore conclude that, to the limits of our system (∼30 pm), there is no spontaneous mechanical activity that manifests as oscillations in OHC electromotility within the mouse cochlea, arguing that unloaded OHC bundles do not oscillate in vivo. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

COVID-19 in the Clinic: Human Testing of an Aerosol Containment Mask for Endoscopic Clinic Procedures.

OBJECTIVE: To create an aerosol containment mask (ACM) for common otolaryngologic endoscopic procedures that also provides nanoparticle-level protection to patients. STUDY DESIGN: Prospective feasibility study . SETTING: In-person testing with a novel ACM. METHODS: The mask was designed in Solidworks and 3D printed. Measurements were made on 10 healthy volunteers who wore the ACM while reading the Rainbow Passage repeatedly and performing a forced cough or sneeze at 5-second intervals over 1 minute with an endoscope in place. RESULTS: There was a large variation in the number of aerosol particles generated among the volunteers. Only the sneeze task showed a significant increase compared with normal breathing in the 0.3-µm particle size when compared with a 1-tailed t test (P = .013). Both the 0.5-µm and 2.5-µm particle sizes showed significant increases for all tasks, while the 2 largest particle sizes, 5 and 10 µm, showed no significant increase (both P < .01). With the suction off, 3 of 30 events (2 sneeze events and 1 cough event) had increases in particle counts, both inside and outside the mask. With the suction on, 2 of 30 events had an increase in particle counts outside the mask without a corresponding increase in particle counts inside the mask. Therefore, these fluctuations in particle counts were determined to be due to random fluctuation in room particle levels. CONCLUSION: ACM will accommodate rigid and flexible endoscopes plus instruments and may prevent the leakage of patient-generated aerosols, thus avoiding contamination of the room and protecting health care workers from airborne contagions. LEVEL OF EVIDENCE: 2.

COVID-19 in the Clinic: Aerosol Containment Mask for Endoscopic Otolaryngologic Clinic Procedures.

OBJECTIVE: To create an aerosol containment mask (ACM) that contains aerosols during common otolaryngologic endoscopic procedures while protecting patients from environmental aerosols. STUDY DESIGN: Bench testing. SETTING: Mannequin testing. METHODS: The mask was designed in SolidWorks and 3-dimensional printed. Mannequins were fitted with a nebulizer to generate aerosols. Commercial particle counters were used to measure mask performance. RESULTS: The ACM has 2 ports on either side for instruments and endoscopes, a port for a filter, and a port that can evacuate aerosols contained within the mask via a standard suction pump. The mask contained aerosols on a mannequin with and without facial hair when the suction was set to 18.5 L/min. Other types of masks demonstrated substantial aerosol leakage under similar conditions. In a subsequent experiment, the ACM contained aerosols generated by a nebulizer up to the saturation of the particle detector without measurable leakage with or without suction. CONCLUSION: The ACM will accommodate rigid and flexible endoscopes plus instruments and prevent leakage of patient-generated aerosols, thus avoiding contamination of the room and protecting health care workers from airborne contagions. LEVEL OF EVIDENCE: 2.

Endolymphatic Hydrops is a Marker of Synaptopathy Following Traumatic Noise Exposure.

After acoustic trauma, there can be loss of synaptic connections between inner hair cells and auditory neurons in the cochlea, which may lead to hearing abnormalities including speech-in-noise difficulties, tinnitus, and hyperacusis. We have previously studied mice with blast-induced cochlear synaptopathy and found that they also developed a build-up of endolymph, termed endolymphatic hydrops. In this study, we used optical coherence tomography to measure endolymph volume in live CBA/CaJ mice exposed to various noise intensities. We quantified the number of synaptic ribbons and postsynaptic densities under the inner hair cells 1 week after noise exposure to determine if they correlated with acute changes in endolymph volume measured in the hours after the noise exposure. After 2 h of noise at an intensity of 95 dB SPL or below, both endolymph volume and synaptic counts remained normal. After exposure to 2 h of 100 dB SPL noise, mice developed endolymphatic hydrops and had reduced synaptic counts in the basal and middle regions of the cochlea. Furthermore, round-window application of hypertonic saline reduced the degree of endolymphatic hydrops that developed after 100 dB SPL noise exposure and partially prevented the reduction in synaptic counts in the cochlear base. Taken together, these results indicate that endolymphatic hydrops correlates with noise-induced cochlear synaptopathy, suggesting that these two pathologic findings have a common mechanistic basis.

Cochlear outer hair cell electromotility enhances organ of Corti motion on a cycle-by-cycle basis at high frequencies in vivo.

Mammalian hearing depends on an amplification process involving prestin, a voltage-sensitive motor protein that enables cochlear outer hair cells (OHCs) to change length and generate force. However, it has been questioned whether this prestin-based somatic electromotility can operate fast enough in vivo to amplify cochlear vibrations at the high frequencies that mammals hear. In this study, we measured sound-evoked vibrations from within the living mouse cochlea and found that the top and bottom of the OHCs move in opposite directions at frequencies exceeding 20 kHz, consistent with fast somatic length changes. These motions are physiologically vulnerable, depend on prestin, and dominate the cochlea's vibratory response to high-frequency sound. This dominance was observed despite mechanisms that clearly low-pass filter the in vivo electromotile response. Low-pass filtering therefore does not critically limit the OHC's ability to move the organ of Corti on a cycle-by-cycle basis. Our data argue that electromotility serves as the primary high-frequency amplifying mechanism within the mammalian cochlea.

In vivo functional imaging of the human middle ear with a hand-held optical coherence tomography device.

We describe an optical coherence tomography and vibrometry system designed for portable hand-held usage in the otology clinic on awake patients. The system provides clinically relevant point-of-care morphological imaging with 14-44 µm resolution and functional vibratory measures with sub-nanometer sensitivity. We evaluated various new approaches for extracting functional information including a multi-tone stimulus, a continuous chirp stimulus, and alternating air and bone stimulus. We also explored the vibratory response over an area of the tympanic membrane (TM) and generated TM thickness maps. Our results suggest that the system can provide real-time in vivo imaging and vibrometry of the ear and could prove useful for investigating otologic pathology in the clinic setting.

Development and Characterization of PLGA-Based Multistage Delivery System for Enhanced Payload Delivery to Targeted Vascular Endothelium.

Vascular-targeted drug delivery remains an attractive platform for therapeutic and diagnostic interventions in human diseases. This work focuses on the development of a poly-lactic-co-glycolic-acid (PLGA)-based multistage delivery system (MDS). MDS consists of two stages: a micron-sized PLGA outer shell and encapsulated drug-loaded PLGA nanoparticles. Nanoparticles with average diameters of 76, 119, and 193 nm are successfully encapsulated into 3-6 µm MDS. Sustained in vitro release of nanoparticles from MDS is observed for up to 7 days. Both MDS and nanoparticles arebiocompatible with human endothelial cells. Sialyl-Lewis-A (sLeA ) is successfully immobilized on the MDS and nanoparticle surfaces to enable specific targeting of inflamed endothelium. Functionalized MDS demonstrates a 2.7-fold improvement in endothelial binding compared to PLGA nanoparticles from human blood laminar flow. Overall, the presented results demonstrate successful development and characterization of MDS and suggest that MDS can serve as an effective drug carrier, which can enhance the margination of nanoparticles to the targeted vascular wall.

In Vivo Cochlear imaging provides a tool to study endolymphatic hydrops.

Exposure to noise trauma, such as that from improvised explosive devices, can lead to sensorineural hearing loss and a reduced quality of life. In order to elucidate the mechanisms underlying noise-induced hearing loss, we have adapted optical coherence tomography (OCT) for real-time cochlear visualization in live mice after blast exposure. We demonstrated that endolymphatic hydrops develops following blast injury, and that this phenomenon may be associated with glutamate excitotoxicity and cochlear synaptopathy. Additionally, osmotic stabilization of endolymphatic hydrops partially rescues cochlear synapses after blast trauma. OCT is thus a valuable research tool for investigating the mechanisms underlying acoustic trauma and dynamic changes in endolymph volume. It may also help with the diagnosis and treatment of human hearing loss and/or vertigo in the near future.

Methylene blue-filled biodegradable polymer particles as a contrast agent for optical coherence tomography.

Optical coherence tomography (OCT) images largely lack molecular information or molecular contrast. We address that issue here, reporting on the development of biodegradable micro and nano-spheres loaded with methylene blue (MB) as molecular contrast agents for OCT. MB is a constituent of FDA approved therapies and widely used as a dye in off-label clinical applications. The sequestration of MB within the polymer reduced toxicity and improved signal strength by drastically reducing the production of singlet oxygen and leuco-MB. The former leads to tissue damage and the latter to reduced image contrast. The spheres are also strongly scattering which improves molecular contrast signal localization and enhances signal strength. We demonstrate that these contrast agents may be imaged using both pump-probe OCT and photothermal OCT, using a 830 nm frequency domain OCT system and a 1.3 µm swept source OCT system. We also show that these contrast agents may be functionalized and targeted to specific receptors, e.g. the VCAM receptor known to be overexpressed in inflammation.

Direct frequency domain fluorescence lifetime imaging using field programmable gate arrays for real time processing.

Frequency domain (FD) fluorescence lifetime imaging (FLIM) involves the excitation of the sample of interest with a modulated light source and digitization of the fluorescence emission for further analysis. Traditional FD-FLIM systems use heterodyne or homodyne detection, where the excitation light source and detector are modulated at specific frequency(s). More recently, FD-FLIM systems that use reflection of the light source as a trigger or phase reference for lifetime calculations have been developed. These detection schemes, however, require extra components that increase the cost and complexity of the FD-FLIM system. Here, we report a novel FD-FLIM detection scheme whereby the light source modulation and emission digitization are implemented using Field Programmable Gate Arrays (FPGAs), and fixed gain avalanche photodiodes are used for fluorescence detection. The reported FD-FLIM system was designed for probing nanosecond lifetime fluorophores (2-10 ns) at three emission bands simultaneously. The system utilizes a 375 nm diode laser for excitation at multiple simultaneous modulation frequencies (between 1 MHz and 83 MHz, bandwidth limited intentionally by using a lowpass filter) and three fixed gain avalanche photodiodes for simultaneous detection of three emission bands: 405/20 nm, 440/40 nm, and 525/50 nm (center/FWHM). Real-time computation of the modulation and phase lifetimes is simply performed by direct application of the discrete Fourier transform (max. of 10 frequencies) to the digitized fluorescence emission signals. The accuracy and sensitivity of this novel FD-FLIM detection scheme was demonstrated by imaging standard fluorophores and ex vivo unfixed human coronary artery tissue samples.

Noise and sensitivity in optical coherence tomography based vibrometry.

There is growing interest in using the exquisite phase sensitivity of optical coherence tomography (OCT) to measure the vibratory response in organ systems such as the middle and inner ear. Using frequency domain analysis, it is possible to achieve picometer sensitivity to vibration over a wide frequency band. Here we explore the limits of the frequency domain vibratory sensitivity due to additive noise and consider the implication of phase noise statistics on the estimation of vibratory amplitude and phase. Noise statistics are derived in both the Rayleigh (s/n = 0) and Normal distribution (s/n > 3) limits. These theoretical findings are explored using simulation and verified with experiments using a swept-laser system and a piezo electric element. A metric for sensitivity is proposed based on the 98% confidence interval for the Rayleigh distribution.