Genetic tools for studying cochlear inhibition.

Efferent feedback to the mammalian cochlea includes cholinergic medial olivocochlear neurons (MOCs) that release ACh to hyperpolarize and shunt the voltage change that drives electromotility of outer hair cells (OHCs). Via brainstem connectivity, MOCs are activated by sound in a frequency- and intensity-dependent manner, thereby reducing the amplification of cochlear vibration provided by OHC electromotility. Among other roles, this efferent feedback protects the cochlea from acoustic trauma. Lesion studies, as well as a variety of genetic mouse models, support the hypothesis of efferent protection from acoustic trauma. Genetic knockout and gain-of-function knockin of the unique α9α10-containing nicotinic acetylcholine receptor (nAChR) in hair cells show that acoustic protection correlates with the efficacy of cholinergic inhibition of OHCs. This protective effect was replicated by viral transduction of the gain-of-function α9L9'T nAChR into α9-knockout mice. Continued progress with "efferent gene therapy" will require a reliable method for visualizing nAChR expression in cochlear hair cells. To that end, mice expressing HA-tagged α9 or α10 nAChRs were generated using CRISPR technology. This progress will facilitate continued study of the hair cell nAChR as a therapeutic target to prevent hearing loss and potentially to ameliorate associated pathologies such as hyperacusis.

BAI1 localizes AMPA receptors at the cochlear afferent post-synaptic density and is essential for hearing.

Type I spiral ganglion neurons (SGNs) convey sound information to the central auditory pathway by forming synapses with inner hair cells (IHCs) in the mammalian cochlea. The molecular mechanisms regulating the formation of the post-synaptic density (PSD) in the SGN afferent terminals are still unclear. Here, we demonstrate that brain-specific angiogenesis inhibitor 1 (BAI1) is required for the clustering of AMPA receptors GluR2-4 (glutamate receptors 2-4) at the PSD. Adult Bai1-deficient mice have functional IHCs but fail to transmit information to the SGNs, leading to highly raised hearing thresholds. Despite the almost complete absence of AMPA receptor subunits, the SGN fibers innervating the IHCs do not degenerate. Furthermore, we show that AMPA receptors are still expressed in the cochlea of Bai1-deficient mice, highlighting a role for BAI1 in trafficking or anchoring GluR2-4 to the PSDs. These findings identify molecular and functional mechanisms required for sound encoding at cochlear ribbon synapses.

Ptch1 is essential for cochlear marginal cell differentiation and stria vascularis formation.

A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.

Fabrication and Characterization of PDMS Waveguides for Flexible Optrodes.

With the growth of optogenetic research, the demand for optical probes tailored to specific applications is ever rising. Specifically, for applications like the coiled cochlea of the inner ear, where planar, stiff and non-conformable probes can hardly be used, transitioning from commonly used stiff glass fibers to flexible probes is required, especially for long-term use. Following this demand, Polydimethylsiloxane (PDMS) with its lower Young's modulus compared to glass fibers could serve as material of choice. Hence, we investigated the long-term usability of PDMS as a waveguide material with respect to variations in transmission and refractive index over time. Different manufacturing methods for PDMS-based flexible waveguides were established and compared with the aim to minimize optical losses and thus maximize optical output power. Finally, the waveguides with lowest optical losses (-4.8 dB/cm ± 1.3 dB/cm at 472 nm) were successfully inserted into the optogenetically modified cochlea of a Mongolian gerbil (meriones unguiculatus), where optical stimuli delivered by the waveguides evoked robust neuronal responses in the auditory pathway. This article is protected by copyright. All rights reserved.

Microanatomy of the human tunnel of Corti structures and cochlear partition-tonotopic variations and transcellular signaling.

Auditory sensitivity and frequency resolution depend on the optimal transfer of sound-induced vibrations from the basilar membrane (BM) to the inner hair cells (IHCs), the principal auditory receptors. There remains a paucity of information on how this is accomplished along the frequency range in the human cochlea. Most of the current knowledge is derived either from animal experiments or human tissue processed after death, offering limited structural preservation and optical resolution. In our study, we analyzed the cytoarchitecture of the human cochlear partition at different frequency locations using high-resolution microscopy of uniquely preserved normal human tissue. The results may have clinical implications and increase our understanding of how frequency-dependent acoustic vibrations are carried to human IHCs. A 1-micron-thick plastic-embedded section (mid-modiolar) from a normal human cochlea uniquely preserved at lateral skull base surgery was analyzed using light and transmission electron microscopy (LM, TEM). Frequency locations were estimated using synchrotron radiation phase-contrast imaging (SR-PCI). Archival human tissue prepared for scanning electron microscopy (SEM) and super-resolution structured illumination microscopy (SR-SIM) were also used and compared in this study. Microscopy demonstrated great variations in the dimension and architecture of the human cochlear partition along the frequency range. Pillar cell geometry was closely regulated and depended on the reticular lamina slope and tympanic lip angle. A type II collagen-expressing lamina extended medially from the tympanic lip under the inner sulcus, here named "accessory basilar membrane." It was linked to the tympanic lip and inner pillar foot, and it may contribute to the overall compliance of the cochlear partition. Based on the findings, we speculate on the remarkable microanatomic inflections and geometric relationships which relay different sound-induced vibrations to the IHCs, including their relevance for the evolution of human speech reception and electric stimulation with auditory implants. The inner pillar transcellular microtubule/actin system's role of directly converting vibration energy to the IHC cuticular plate and ciliary bundle is highlighted.

Not an immune cell, but they may act like one-cells with immune properties outside the immune system.

The cells presented in this work are not classified as cells that make up the immune system. They, however, present functions and molecules, which are characteristic of immune cells. These characteristic functions are, for example, sensing threat, performing phagocytosis, presentation of foreign antigens, cytokine release or enhancing immune memory. The enlisted immune response mechanisms are carried out by the possession of molecules such as Toll-like receptors, receptors for the Fc fragment of IgG, major histocompatibility complex class II molecules, costimulatory CD80/CD86 proteins and molecules needed for NLRP3 (NOD-like family pyrin domain containing 3) inflammasome activation. Thanks to these properties, the described nonimmune cells play an important role in the local immune response and support of the entire body in the fight against pathogens. They constitute the first line of defense of tissues and organs against pathogens and molecules recognized as harmful. The cells described in this article are particularly important in immunologically privileged places (e.g. the Bowman's capsule in the kidney), where "typical" immune cells normally do not have access. In this paper, we present immune-like functions and molecule suites of resident kidney cells (podocytes and mesangial cells), cochlear resident cells, fibrocytes and fibroblasts, as well as some stem cells (mesenchymal stem cells and umbilical cord Wharton's jelly-derived cells).

Characteristics of spatial protein expression in the mouse cochlear sensory epithelia: Implications for age-related hearing loss.

Hair cells in the cochlear sensory epithelia serve as mechanosensory receptors, converting sound into neuronal signals. The basal sensory epithelia are responsible for transducing high-frequency sounds, while the apex handles low-frequency sounds. Age-related hearing loss predominantly affects hearing at high frequencies and is indicative of damage to the basal sensory epithelia. However, the precise mechanism underlying this site-selective injury remains unclear. In this study, we employed a microscale proteomics approach to examine and compare protein expression in different regions of the cochlear sensory epithelia (upper half and lower half) in 1.5-month-old (normal hearing) and 6-month-old (severe high-frequency hearing loss without hair cell loss) C57BL/6J mice. A total of 2,386 proteins were detected, and no significant differences in protein expression were detected in the upper half of the cochlear sensory epithelia between the two age groups. The expression of 20 proteins in the lower half of the cochlear sensory epithelia significantly differed between the two age groups (e.g., MATN1, MATN4, and AQP1). Moreover, there were 311 and 226 differentially expressed proteins between the upper and lower halves of the cochlear sensory epithelia in 1.5-month-old and 6-month-old mice, respectively. The expression levels of selected proteins were validated by Western blotting. These findings suggest that the spatial differences in protein expression within the cochlear sensory epithelia may play a role in determining the susceptibility of cells at different sites of the cochlea to age-related damage.

High-frequency hearing vulnerability associated with the different supporting potential of Hensen's cells: SMART-Seq2 RNA sequencing.

Hearing loss is the third most prevalent physical condition affecting communication, well-being, and healthcare costs. Sensorineural hearing loss often occurs first in the high-frequency region (basal turn), then towards the low-frequency region (apical turn). However, the mechanism is still unclear. Supporting cells play a critical role in the maintenance of normal cochlear function. The function and supporting capacity of these cells may be different from different frequency regions. Hensen's cells are one of the unique supporting cell types characterized by lipid droplets (LDs) in the cytoplasm. Here, we investigated the morphological and gene expression differences of Hensen's cells along the cochlear axis. We observed a gradient change in the morphological characteristics of Hensen's cells along the cochlear tonotopic axis, with larger and more abundant LDs observed in apical Hensen's cells. Smart-seq2 RNA-seq revealed differentially expressed genes (DEGs) between apical and basal Hensen's cells that clustered in several pathways, including unsaturated fatty acid biosynthesis, cholesterol metabolism, and fatty acid catabolism, which are associated with different energy storage capacities and metabolic potential. These findings suggest potential differences in lipid metabolism and oxidative energy supply between apical and basal Hensen's cells, which is consistent with the morphological differences of Hensen's cells. We also found differential expression patterns of candidate genes associated with hereditary hearing loss (HHL), noise-induced hearing loss (NIHL), and age-related hearing loss (ARHL). These findings indicate functional heterogeneity of SCs along the cochlear axis, contribute to our understanding of cochlear physiology and provide molecular basis evidence for future studies of hearing loss.

Exploring Trauma Patterns and Contributing Factors With Slim Straight Electrode Array After Cochlear Implantation.

OBJECTIVE: To assess trauma patterns associated with the insertion of lateral wall electrode arrays. The study focused on 3 categories-scala tympani (ST), intermediate, and scala vestibuli (SV)-to identify traumatic patterns and contributing factors. STUDY DESIGN: Retrospective study. SETTING: Data from 106 cochlear implant recipients at a tertiary otologic center. METHODS: Demographic and surgical data were collected from recipients who underwent cochlear implantation manually and with RobOtol®. Measurements included cochlear dimensions, angular depth of insertion, and position of the first electrode. Three-dimensional reconstructions were used to analyze the electrode array location relative to the basilar membrane, categorized into ST, intermediate, and SV electrodes. Nontraumatic insertion was defined as all electrodes in the ST, while traumatic insertions had 1 or more electrodes in intermediate or SV locations. RESULTS: Out of 106 cases, 44% had nontraumatic and 56% had traumatic insertions. Demographic and surgical characteristics showed no association with traumatic insertions. A deeper position of the first electrode, relative to the round window, was associated with traumatic insertions (P = .03). Three trauma patterns were observed: distal (facing the apical electrodes), proximal (facing the middle electrodes around 180°), and distal/proximal. CONCLUSION: This study considers the intermediate position which could be associated with basilar membrane lesions. Risk zones for intracochlear trauma with lateral wall arrays were identified distally and proximally. Traumatic insertions were independently linked to deeper array placement. Future studies should explore whether gentler insertion, without insisting on further electrode array insertion depth, could reduce the trauma during cochlear implantation.

Recent advances in Otology: Current landscape and future direction.

Hearing is an essential sensation, and its deterioration leads to a significant decrease in the quality of life. Thus, great efforts have been made by otologists to preserve and recover hearing. Our knowledge regarding the field of otology has progressed with advances in technology, and otologists have sought to develop novel approaches in the field of otologic surgery to achieve higher hearing recovery or preservation rates. This requires knowledge regarding the anatomy of the temporal bone and the physiology of hearing. Basic research in the field of otology has progressed with advances in molecular biology and genetics. This review summarizes the current views and recent advances in the field of otology and otologic surgery, especially from the viewpoint of young Japanese clinician-scientists, and presents the perspectives and future directions for several topics in the field of otology. This review will aid next-generation researchers in understanding the recent advances and future challenges in the field of otology.

Deciphering the genetic interactions between Pou4f3, Gfi1, and Rbm24 in maintaining mouse cochlear hair cell survival.

Mammals harbor a limited number of sound-receptor hair cells (HCs) that cannot be regenerated after damage. Thus, investigating the underlying molecular mechanisms that maintain HC survival is crucial for preventing hearing impairment. Intriguingly, Pou4f3-/- or Gfi1-/- HCs form initially but then rapidly degenerate, whereas Rbm24-/- HCs degenerate considerably later. However, the transcriptional cascades involving Pou4f3, Gfi1, and Rbm24 remain undescribed. Here, we demonstrate that Rbm24 expression is completely repressed in Pou4f3-/- HCs but unaltered in Gfi1-/- HCs, and further that the expression of both POU4F3 and GFI1 is intact in Rbm24-/- HCs. Moreover, by using in vivo mouse transgenic reporter assays, we identify three Rbm24 enhancers to which POU4F3 binds. Lastly, through in vivo genetic testing of whether Rbm24 restoration alleviates the degeneration of Pou4f3-/- HCs, we show that ectopic Rbm24 alone cannot prevent Pou4f3-/- HCs from degenerating. Collectively, our findings provide new molecular and genetic insights into how HC survival is regulated.

Endoplasmic reticulum stress-induced necroptosis promotes cochlear inflammation: Implications for age-related hearing loss.

Age-related hearing loss (ARHL) is the most common sensory disorder associated with human aging. Chronic inflammation is supposed to be an important contributor to ARHL. Yet, the underlying mechanisms of developing cochlear inflammation are still not well understood. In this study, we found that the inflammation, endoplasmic reticulum (ER) stress and necroptosis signalings are activated in the cochlea of aged C57BL/6 mice. ER stress activator tunicamycin (TM) induced necroptosis in cochlear HEI-OC1 cells and cochlear explants, while necroptosis inhibitors protected cochlear cells from ER stress-induced cell death. The antioxidants inhibited necroptosis and protected HEI-OC1 cells from TM insults. Necroptotic HEI-OC1 cells promoted the activation of the co-cultured macrophages via Myd88 signaling. Moreover, necroptosis inhibitor protected from TM-induced hearing loss, and inhibited inflammation in C57BL/6 mice. These findings suggest that ER stress-induced necroptosis promotes cochlear inflammation and hearing loss. Targeting necroptosis serves as a potential approach for the treatment of cochlear inflammation and ARHL.

OCT-based intra-cochlear imaging and 3D reconstruction: ex vivo validation of a robotic platform.

PURPOSE: The small size of the cochlea, and its location deeply embedded in thick temporal bone, poses a challenge for intra-cochlear guidance and diagnostics. Current radiological imaging techniques are not able to visualize the cochlear microstructures in detail. Rotational optical coherence tomography (OCT) fibers show great potential for intra-cochlear guidance. The generated images could be used to map, and study, the tiny cochlear microstructures relevant for hearing. METHODS: This work describes the design of a rotational OCT probe with an outer diameter of 0.9 mm. It further discusses a robotic system, which features a remote center of motion mechanism, dedicated to the probe's positioning, fine manipulation and stable insertion into the cochlear micro-spaces. Furthermore, the necessary calibration steps for 3D reconstruction are described, followed by a detailed quantitative analysis, comparing the 3D reconstructions using a synthetic, 2:1 scaled scala tympani model with a reconstruction from micro-CT, serving as the ground truth. Finally, the potential of the system is demonstrated by scanning a single ex vivo cadaveric human cochlea. RESULTS: The study investigates five insertions in the same 2:1 scaled tympani model, along with their corresponding 3D reconstruction. The comparison with micro-CT results in an average root-mean-square error of 74.2 µm, a signed distance error of 38.1 µm and a standard deviation of 63.6 µm. The average F-score of the reconstructions, using a distance threshold of 100 and 74.2 µm, resulted in 83.0% and 71.8%, respectively. Insertion in the cadaveric human cochlea showed the challenges for straight insertion, i.e., navigating the hook region. CONCLUSION: Overall, the system shows great potential for intra-cochlear guidance and diagnostics, due to the system's capability for precise and stable insertion into the basal turn in the scala tympani. The system, combined with the calibration procedure, results in detailed and precise 3D reconstructions.

Loss of Esr1 Does Not Affect Hearing and Balance.

Although estrogen affects the structure and function of the nervous system and brain and has a number of effects on cognition, its roles in the auditory and vestibular systems remain unclear. The actions of estrogen are mediated predominately through two classical nuclear estrogen receptors, estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2). In the current study, we investigated the roles of ESR1 in normal auditory function and balance performance using 3-month-old wild-type (WT) and Esr1 knockout (KO) mice on a CBA/CaJ background, a normal-hearing strain. As expected, body weight of Esr1 KO females was lower than that of Esr1 KO males. Body weight of Esr1 KO females was higher than that of WT females, while there was no difference in body weight between WT and Esr1 KO males. Similarly, head diameter was higher in Esr1 KO vs. WT females. Contrary to our expectations, there were no differences in auditory brainstem response (ABR) thresholds, ABR waves I-V amplitudes and ABR waves I-V latencies at 8, 16, 32, and 48 kHz, distortion product otoacoustic emission (DPOAE) thresholds and amplitudes at 8, 16, and 32 kHz, and rotarod balance performance (latency to fall) between WT and Esr1 KO mice. Furthermore, there were no sex differences in ABRs, DPOAEs, and rotarod balance performance in Esr1 KO mice. Taken together, our findings show that Esr1 deficiency does not affect auditory function or balance performance in normal hearing mice, and suggest that loss of Esr1 is likely compensated by ESR2 or other estrogen receptors to maintain the structure and function of the auditory and vestibular systems under normal physiological conditions.

The Impact of the Fluid-Solid Coupling Behavior of Macro and Microstructures in the Spiral Cochlea on Hearing.

The cilia of the outer hair cells (OHCs) are the key microstructures involved in cochlear acoustic function, and their interactions with lymph in the cochlea involve complex, highly nonlinear, coupled motion and energy conversions, including macroscopic fluid-solid coupling. Recent optical measurements have shown that the frequency selectivity of the cochlea at high sound levels is entirely mechanical and is determined by the interactions of the hair bundles with the surrounding fluid. In this paper, an analytical mathematical model of the spiral cochlea containing macro- and micromeasurements was developed to investigate how the phonosensitive function of OHCs' motions is influenced by the macrostructural and microstructural fluid-solid coupling in the spiral cochlea. The results showed that the macrostructural and microstructural fluid-solid coupling exerted the radial forces of OHCs through the flow field, deflecting the cilia and generating frequency-selective properties of the microstructures. This finding showed that microstructural frequency selectivity arises from the radial motions of stereocilia hair bundles and enhances the hearing of sound signals at specific frequencies. It also implied that the macrostructural and microstructural fluid-solid couplings influence the OHCs' radial forces and that this is a key factor in the excitation of ion channels that enables their activity in helping the brain to detect sound.

Pneumococcal Meningitis Induces Hearing Loss and Cochlear Ossification Modulated by Chemokine Receptors CX3CR1 and CCR2.

PURPOSE: Pneumococcal meningitis is a major cause of hearing loss and permanent neurological impairment despite widely available antimicrobial therapies to control infection. Methods to improve hearing outcomes for those who survive bacterial meningitis remains elusive. We used a mouse model of pneumococcal meningitis to evaluate the impact of mononuclear phagocytes on hearing outcomes and cochlear ossification by altering the expression of CX3CR1 and CCR2 in these infected mice. METHODS: We induced pneumococcal meningitis in approximately 500 C57Bl6 adult mice using live Streptococcus pneumoniae (serotype 3, 1 × 105 colony forming units (cfu) in 10 µl) injected directly into the cisterna magna of anesthetized mice and treated these mice with ceftriaxone daily until recovered. We evaluated hearing thresholds over time, characterized the cochlear inflammatory response, and quantified the amount of new bone formation during meningitis recovery. We used microcomputed tomography (microCT) scans to quantify cochlear volume loss caused by neo-ossification. We also performed perilymph sampling in live mice to assess the integrity of the blood-perilymph barrier during various time intervals after meningitis. We then evaluated the effect of CX3CR1 or CCR2 deletion in meningitis symptoms, hearing loss, macrophage/monocyte recruitment, neo-ossification, and blood labyrinth barrier function. RESULTS: Sixty percent of mice with pneumococcal meningitis developed hearing loss. Cochlear fibrosis could be detected within 4 days of infection, and neo-ossification by 14 days. Loss of spiral ganglion neurons was common, and inner ear anatomy was distorted by scarring caused by new soft tissue and bone deposited within the scalae. The blood-perilymph barrier was disrupted at 3 days post infection (DPI) and was restored by seven DPI. Both CCR2 and CX3CR1 monocytes and macrophages were present in the cochlea in large numbers after infection. Neither chemokine receptor was necessary for the induction of hearing loss, cochlear fibrosis, ossification, or disruption of the blood-perilymph barrier. CCR2 knockout (KO) mice suffered the most severe hearing loss. CX3CR1 KO mice demonstrated an intermediate phenotype with greater susceptibility to hearing loss compared to control mice. Elimination of CX3CR1 mononuclear phagocytes during the first 2 weeks after meningitis in CX3CR1-DTR transgenic mice did not protect mice from any of the systemic or hearing sequelae of pneumococcal meningitis. CONCLUSIONS: Pneumococcal meningitis can have devastating effects on cochlear structure and function, although not all mice experienced hearing loss or cochlear damage. Meningitis can result in rapid progression of hearing loss with fibrosis starting at four DPI and ossification within 2 weeks of infection detectable by light microscopy. The inflammatory response to bacterial meningitis is robust and can affect all three scalae. Our results suggest that CCR2 may assist in controlling infection and maintaining cochlear patency, as CCR2 knockout mice experienced more severe disease, more rapid hearing loss, and more advanced cochlear ossification after pneumococcal meningitis. CX3CR1 also may play an important role in the maintenance of cochlear patency.

The crucial role of diverse animal models to investigate cochlear aging and hearing loss.

Age-related hearing loss affects a large and growing segment of the population, with profound impacts on quality of life. Age-related pathology of the cochlea-the mammalian hearing organ-underlies age-related hearing loss. Because investigating age-related changes in the cochlea in humans is challenging and often impossible, animal models are indispensable to investigate these mechanisms as well as the complex consequences of age-related hearing loss on the brain and behavior. In this review, we advocate for a comparative and interdisciplinary approach while also addressing the challenges of comparing age-related hearing loss across species with varying lifespans. We describe the experimental advantages and limitations as well as areas for future research in well-established models of age-related hearing loss, including mice, rats, gerbils, chinchillas, and birds. We also indicate the need to expand characterization of age-related hearing loss in other established animal models, especially guinea pigs, cats, and non-human primates, in which auditory function is well characterized but age-related cochlear pathology is understudied. Finally, we highlight the potential of emerging animal models for advancing our understanding of age-related hearing loss, including deer mice, with their notably extended lifespans and preserved hearing, naked mole rats, with their exceptional longevity and extensive vocal communications, as well as zebrafish, which offer genetic tractability and suitability for drug screening. Ultimately, a comparative and interdisciplinary approach in auditory research, combining insights from various animal models with human studies, is key to robust and reliable research outcomes that better advance our understanding and treatment of age-related hearing loss.

Hyaluronic acid-ibuprofen conjugation: a novel ototherapeutic approach protecting inner ear cells from inflammation-mediated damage.

There is a substantial need of effective drugs for the treatment of hearing loss, which affects nearly 500 million individuals globally. Hearing loss can be the result of intense or prolonged noise exposure, ototoxic drugs, infections, and trauma, which trigger inflammatory signaling cascades that lead to irreversible damage to cochlear structures. To address this, we developed and characterized a series of covalent conjugates of anti-inflammatory drugs to hyaluronic acid (HA), for potential use as topical ototherapeutics. These conjugates were tested in in vitro assays designed to mirror physiological processes typically observed with acoustic trauma. Intense noise exposure leads to macrophage recruitment to the cochlea and subsequent inflammatory damage to sensory cells. We therefore first tested our conjugates' ability to reduce the release of inflammatory cytokines in macrophages. This anti-inflammatory effect on macrophages also translated to increased cochlear cell viability. In our initial screening, one conjugate, ibuprofen-HA, demonstrated significantly higher anti-inflammatory potential than its counterparts. Subsequent cytokine release profiling of ibuprofen-HA further confirmed its ability to reduce a wider range of inflammatory markers, to a greater extent than its equivalent unconjugated drug. The conjugate's potential as a topical therapeutic was then assessed in previously developed tympanic and round window membrane tissue permeation models. As expected, our data indicate that the conjugate has limited tympanic membrane model permeability; however, it readily permeated the round window membrane model and to a greater extent than the unconjugated drug. Interestingly, our data also revealed that ibuprofen-HA was well tolerated in cellular and tissue cytocompatibility assays, whereas the unconjugated drug displayed significant cytotoxicity at equivalent concentrations. Moreover, our data highlighted the importance of chemical conjugation of ibuprofen to HA; the conjugate had improved anti-inflammatory effects, significantly reduced cytotoxicity, and is more suitable for therapeutic formulation. Overall, this work suggests that ibuprofen-HA could be a promising safe and effective topical ototherapeutic for inflammation-mediated cochlear damage.

Corrigendum: Precision medicine: a new era for inner ear diseases.

[This corrects the article DOI: 10.3389/fphar.2024.1328460.].

Cochlear Ribbon Synapses in Aged Gerbils.

In mammalian hearing, type-I afferent auditory nerve fibers comprise the basis of the afferent auditory pathway. They are connected to inner hair cells of the cochlea via specialized ribbon synapses. Auditory nerve fibers of different physiological types differ subtly in their synaptic location and morphology. Low-spontaneous-rate auditory nerve fibers typically connect on the modiolar side of the inner hair cell, while high-spontaneous-rate fibers are typically found on the pillar side. In aging and noise-damaged ears, this fine-tuned balance between auditory nerve fiber populations can be disrupted and the functional consequences are currently unclear. Here, using immunofluorescent labeling of presynaptic ribbons and postsynaptic glutamate receptor patches, we investigated changes in synaptic morphology at three different tonotopic locations along the cochlea of aging gerbils compared to those of young adults. Quiet-aged gerbils showed about 20% loss of afferent ribbon synapses. While the loss was random at apical, low-frequency cochlear locations, at the basal, high-frequency location it almost exclusively affected the modiolar-located synapses. The subtle differences in volumes of pre- and postsynaptic elements located on the inner hair cell's modiolar versus pillar side were unaffected by age. This is consistent with known physiology and suggests a predominant, age-related loss in the low-spontaneous-rate auditory nerve population in the cochlear base, but not the apex.