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.

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.

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.

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.

Osmotic stabilization prevents cochlear synaptopathy after blast trauma.

Traumatic noise causes hearing loss by damaging sensory hair cells and their auditory synapses. There are no treatments. Here, we investigated mice exposed to a blast wave approximating a roadside bomb. In vivo cochlear imaging revealed an increase in the volume of endolymph, the fluid within scala media, termed endolymphatic hydrops. Endolymphatic hydrops, hair cell loss, and cochlear synaptopathy were initiated by trauma to the mechanosensitive hair cell stereocilia and were K+-dependent. Increasing the osmolality of the adjacent perilymph treated endolymphatic hydrops and prevented synaptopathy, but did not prevent hair cell loss. Conversely, inducing endolymphatic hydrops in control mice by lowering perilymph osmolality caused cochlear synaptopathy that was glutamate-dependent, but did not cause hair cell loss. Thus, endolymphatic hydrops is a surrogate marker for synaptic bouton swelling after hair cells release excitotoxic levels of glutamate. Because osmotic stabilization prevents neural damage, it is a potential treatment to reduce hearing loss after noise exposure.

Amplification and Suppression of Traveling Waves along the Mouse Organ of Corti: Evidence for Spatial Variation in the Longitudinal Coupling of Outer Hair Cell-Generated Forces.

Mammalian hearing sensitivity and frequency selectivity depend on a mechanical amplification process mediated by outer hair cells (OHCs). OHCs are situated within the organ of Corti atop the basilar membrane (BM), which supports sound-evoked traveling waves. It is well established that OHCs generate force to selectively amplify BM traveling waves where they peak, and that amplification accumulates from one location to the next over this narrow cochlear region. However, recent measurements demonstrate that traveling waves along the apical surface of the organ of Corti, the reticular lamina (RL), are amplified over a much broader region. Whether OHC forces accumulate along the length of the RL traveling wave to provide a form of "global" cochlear amplification is unclear. Here we examined the spatial accumulation of RL amplification. In mice of either sex, we used tones to suppress amplification from different cochlear regions and examined the effect on RL vibrations near and far from the traveling-wave peak. We found that although OHC forces amplify the entire RL traveling wave, amplification only accumulates near the peak, over the same region where BM motion is amplified. This contradicts the notion that RL motion is involved in a global amplification mechanism and reveals that the mechanical properties of the BM and organ of Corti tune how OHC forces accumulate spatially. Restricting the spatial buildup of amplification enhances frequency selectivity by sharpening the peaks of cochlear traveling waves and constrains the number of OHCs responsible for mechanical sensitivity at each location.SIGNIFICANCE STATEMENT Outer hair cells generate force to amplify traveling waves within the mammalian cochlea. This force generation is critical to the ability to detect and discriminate sounds. Nevertheless, how these forces couple to the motions of the surrounding structures and integrate along the cochlear length remains poorly understood. Here we demonstrate that outer hair cell-generated forces amplify traveling-wave motion on the organ of Corti throughout the wave's extent, but that these forces only accumulate longitudinally over a region near the wave's peak. The longitudinal coupling of outer hair cell-generated forces is therefore spatially tuned, likely by the mechanical properties of the basilar membrane and organ of Corti. Our findings provide new insight into the mechanical processes that underlie sensitive hearing.

Enhanced coupling of light into a turbid medium through microscopic interface engineering.

There are many optical detection and sensing methods used today that provide powerful ways to diagnose, characterize, and study materials. For example, the measurement of spontaneous Raman scattering allows for remote detection and identification of chemicals. Many other optical techniques provide unique solutions to learn about biological, chemical, and even structural systems. However, when these systems exist in a highly scattering or turbid medium, the optical scattering effects reduce the effectiveness of these methods. In this article, we demonstrate a method to engineer the geometry of the optical interface of a turbid medium, thereby drastically enhancing the coupling efficiency of light into the material. This enhanced optical coupling means that light incident on the material will penetrate deeper into (and through) the medium. It also means that light thus injected into the material will have an enhanced interaction time with particles contained within the material. These results show that, by using the multiple scattering of light in a turbid medium, enhanced light-matter interaction can be achieved; this has a direct impact on spectroscopic methods such as Raman scattering and fluorescence detection in highly scattering regimes. Furthermore, the enhanced penetration depth achieved by this method will directly impact optical techniques that have previously been limited by the inability to deposit sufficient amounts of optical energy below or through highly scattering layers.

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.

Endoscopic optical coherence tomography enables morphological and subnanometer vibratory imaging of the porcine cochlea through the round window.

A highly phase stable hand-held (HH) endoscopic system has been developed for optical coherence tomography and vibrometry. Designed to transit the ear canal to the middle ear space and peer through the round window (RW), it is capable of imaging the vibratory function of the cochlear soft tissues with subnanometer scale sensitivity. A side-looking, 9 cm long rigid endoscope with a distal diameter of 1.2 mm, was able to fit within the RW niche and provide imaging access. The phase stability was achieved in part by fully integrating a Michelson interferometer into the HH device. Ex vivo imaging of a domestic pig demonstrated the system's ability for functional vibratory imaging of the cochlea via the RW.

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

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.

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

UNLABELLED: 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 Tecta(C1509G/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.

Hybrid nonlinear photoacoustic and reflectance confocal microscopy for label-free subcellular imaging with a single light source.

Nonlinear photoacoustic microscopy is capable of achieving subcellular optically resolved absorption contrast in three dimensions but cannot provide structural context for the acquired images. We have developed a dual-modality imaging system that combines the optical absorption contrast of a nonlinear photoacoustic microscope with the optical scattering contrast of a reflectance confocal microscope. By integrating the confocal detection optics into the optical setup of the nonlinear photoacoustic microscope, the two systems were co-registered and may be acquired at the same time and with the same light source. Simultaneous images of fixed erythrocytes and fibroblasts were measured to demonstrate the complementary information that is provided by the two modalities.

Lensless, ultra-wideband fiber optic rotary joint for biomedical applications.

The demands of optical fiber-based biomedical applications can, in many cases, outstrip the capabilities of lens-based commercially available fiber optic rotary joints. In some circumstances, it is necessary to use very broad spectral bandwidths (near UV to short-wave IR) and specialized optical fibers, such as double-clad fiber, and have the capacity to accommodate high rotational velocities. The broad spectrum, stretching down into the UV, presents two problems: (1) adequate chromatic correction in the lenses across the entire bandwidth and (2) strong UV absorption by the fluids used to lubricate the rotary joint. To accommodate these types of applications, we have developed an ultra-wideband lensless fiber optic rotary joint based on the principle that when two optical fibers are coaligned and placed in contact (or very close), the optical losses at the junction are very low. The advances demonstrated here enable excellent performance (<0.2 dB insertion loss), even down into the UV and spanning a wavelength range of at least 355-1360 nm with single-mode, multimode, and double-clad fibers. We also demonstrate excellent performance, ∼0.38 dB insertion loss, at rotational velocities up to 8800 rpm (146 Hz). To the best of our knowledge, this is the first demonstration of this type of rotary joint capable of such a wide bandwidth and high rotational velocities.

In vivo molecular contrast OCT imaging of methylene blue.

An 830-nm spectral-domain optical coherence tomography (OCT) system with an integrated 663-nm diode pump laser has been developed to enable molecular contrast OCT imaging of methylene blue (MB), a common vital dye used clinically. The introduction of the 663-nm diode laser, which acts as the pump in this implementation of pump-probe OCT (PPOCT), represents a minor modification to an otherwise typical OCT system. A newly developed background subtraction technique completely removes all background from intensity noise at the pump modulation frequency, simplifying the interpretation of PPOCT images. These developments have enabled the first in vivo imaging of MB with PPOCT. Volumetric images of a zebrafish, stained by submersion in a 0.01% (w/v) solution of MB for 6 h, show accumulation of MB in the mesonephros, the primordial filtration organ.

Vibration of the organ of Corti within the cochlear apex in mice.

The tonotopic map of the mammalian cochlea is commonly thought to be determined by the passive mechanical properties of the basilar membrane. The other tissues and cells that make up the organ of Corti also have passive mechanical properties; however, their roles are less well understood. In addition, active forces produced by outer hair cells (OHCs) enhance the vibration of the basilar membrane, termed cochlear amplification. Here, we studied how these biomechanical components interact using optical coherence tomography, which permits vibratory measurements within tissue. We measured not only classical basilar membrane tuning curves, but also vibratory responses from the rest of the organ of Corti within the mouse cochlear apex in vivo. As expected, basilar membrane tuning was sharp in live mice and broad in dead mice. Interestingly, the vibratory response of the region lateral to the OHCs, the "lateral compartment," demonstrated frequency-dependent phase differences relative to the basilar membrane. This was sharply tuned in both live and dead mice. We then measured basilar membrane and lateral compartment vibration in transgenic mice with targeted alterations in cochlear mechanics. Prestin(499/499), Prestin(-/-), and Tecta(C1509G/C1509G) mice demonstrated no cochlear amplification but maintained the lateral compartment phase difference. In contrast, Sfswap(Tg/Tg) mice maintained cochlear amplification but did not demonstrate the lateral compartment phase difference. These data indicate that the organ of Corti has complex micromechanical vibratory characteristics, with passive, yet sharply tuned, vibratory characteristics associated with the supporting cells. These characteristics may tune OHC force generation to produce the sharp frequency selectivity of mammalian hearing.

Simplified method for ultra high-resolution photoacoustic microscopy via transient absorption.

Photoacoustic microscopy (PAM) is a hybrid imaging modality that combines optical illumination with ultrasonic detection to achieve absorption contrast imaging of endogenous and exogenous chromophores. Optical resolution PAM achieves high lateral-resolution by tightly focusing the excitation light; however the axial resolution is still dependent upon the bandwidth of the ultrasonic transducer. As a result, PAM images have highly asymmetric voxels with submicron lateral resolution and axial resolution typically limited to tens of microns. We have previously reported on a resonant multiphoton approach to PAM called transient absorption ultrasonic microscopy (TAUM), which enables high axial resolution by frequency encoding the photoacoustic signal at the overlap of a pump and a probe beam. This approach enables photoacoustic imaging with subcellular resolution on par with other multiphoton microscopy techniques. Here, we report on an innovation that enables TAUM imaging with a much less sophisticated optical system than previously reported. If we allow the time delay between the pump and probe to collapse to zero, the pump and probe optical paths can be combined. An amplitude modulator in the single beam path is sufficient to encode the TAUM signal at the second harmonic of the modulation frequency. The resulting system is essentially a standard optical resolution PAM system that incorporates an amplitude modulator and utilizes a Fourier post processing algorithm to improve the axial resolution by approximately an order of magnitude. A prototype system based on this approach has been assembled and tested on fixed bovine erythrocytes.

Molecular specificity in photoacoustic microscopy by time-resolved transient absorption.

We have recently harnessed transient absorption, a resonant two-photon process, for ultrahigh resolution photoacoustic microscopy, achieving nearly an order of magnitude improvement in axial resolution. The axial resolution is optically constrained due to the two-photon process unlike traditional photoacoustic microscopy where the axial resolution is inversely proportional to the frequency bandwidth of the detector. As a resonant process, the arrival time of the two photons need not be instantaneous. Systematically recording the signal as a function of the delay between two pulses will result in the measurement of an exponential decay whose time constant is related to the molecular dynamics. This time constant, analogous to the fluorescence lifetime, but encompassing nonradiative decay as well, can be used to differentiate between molecular systems with overlapping absorption spectra. This is frequently the situation for closely related yet distinct molecules such as redox pairs. In order to enable the measure of the exponential decay, we have reconfigured our transient absorption ultrasonic microscopy (TAUM) system to incorporate two laser sources with precisely controlled pulse trains. The system was tested by measuring Rhodamine 6G, an efficient laser dye where the molecular dynamics are dominated by the fluorescence pathway. As expected, the measured exponential time constant or ground state recovery time, 3.3±0.7 ns, was similar to the well-known fluorescence lifetime, 4.11±0.05 ns. Oxy- and deoxy-hemoglobin are the quintessential pair whose relative concentration is related to the local blood oxygen saturation. We have measured the ground state recovery times of these two species in fully oxygenated and deoxygenated bovine whole blood to be 3.7±0.8 ns and 7.9±1.0 ns, respectively. Hence, even very closely related pairs of molecules may be differentiated with this technique.

Phase-sensitive optical coherence tomography using an Vernier-tuned distributed Bragg reflector swept laser in the mouse middle ear.

Phase-sensitive optical coherence tomography (PhOCT) offers exquisite sensitivity to mechanical vibration in biological tissues. There is growing interest in using PhOCT for imaging the nanometer scale vibrations of the ear in animal models of hearing disorders. Swept-source-based systems offer fast acquisition speeds, suppression of common mode noise via balanced detection, and good signal roll-off. However, achieving high phase stability is difficult due to nonlinear laser sweeps and trigger jitter in a typical swept laser source. Here, we report on the initial application of a Vernier-tuned distributed Bragg reflector (VT-DBR) swept laser as the source for a fiber-based PhOCT system. The VT-DBR swept laser is electronically tuned and precisely controls sweeps without mechanical movement, resulting in highly linear sweeps with high wavelength stability and repeatability. We experimentally measured a phase sensitivity of 0.4 pm standard deviation, within a factor of less than 2 of the computed shot-noise limit. We further demonstrated the system by making ex vivo measurements of the vibrations of the mouse middle ear structures.

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.

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.