Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration.

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

Dynamics of drying colloidal suspensions, measured by optical coherence tomography.

Colloidal suspensions are the basis of a wide variety of coatings, prepared as liquids and then dried into solid films. The processes at play during film formation, however, are difficult to observe directly. Here, we demonstrate that optical coherence tomography (OCT) can provide fast, non-contact, precise profiling of the dynamics within a drying suspension. Using a scanning Michelson interferometer with a broadband laser source, OCT creates cross-sectional images of the optical stratigraphy of a sample. With this method, we observed the drying of colloidal silica in Hele-Shaw cells with 10 µm transverse and 1.8 µm depth resolution, over a 1 cm scan line and a 15 s sampling period. The resulting images were calibrated to show how the concentration of colloidal particles varied with position and drying time. This gives access to important transport properties, for example, of how collective diffusion depends on particle concentration. Looking at early-time behaviours, we also show how a drying front initially develops, and how the induction time before the appearance of a solid film depends on the balance of diffusion and evaporation-driven motion. Pairing these results with optical microscopy and particle tracking techniques, we find that film formation can be significantly delayed by any density-driven circulation occurring near the drying front.

Time and spatially resolved VIS-NIR hyperspectral imaging as a novel monitoring tool for laser-based spectroscopy to mitigate radiation damage on paintings.

The increased adoption of non-invasive laser-based techniques for analysis of cultural assets has recently called into question the non-invasiveness of the techniques in practical operation. The methods to assess the occurrence of radiation-induced alteration on paintings are very limited and none of them can predict damage. Here we present a novel multimodal imaging approach to understand the time and spatial evolution and types of laser-induced surface alterations, through simultaneous monitoring using visible and near infrared (VIS-NIR) reflectance hyperspectral imaging (HSI) and thermal imaging during Raman spectroscopy. The resultant physical and chemical changes were examined in detail by optical coherence tomography and synchrotron based micro-X-ray powder diffraction. HSI was found to be the most sensitive in detecting laser induced alternations compared with conventional methods. It is orders of magnitude more sensitive than Raman spectroscopy and even synchrotron-based micro-X-ray powder diffraction. In cases of thermally driven alterations, transient and reversible reflectance changes were found to be the first indications of laser-induced modifications and can therefore be used as precursors to prevent damage. VIS-NIR reflectance spectroscopy should be used to monitor laser-based analysis and potentially other radiation-based techniques in situ to mitigate laser induced alteration.

Dissociating antibacterial from ototoxic effects of gentamicin C-subtypes.

Gentamicin is a potent broad-spectrum aminoglycoside antibiotic whose use is hampered by ototoxic side-effects. Hospital gentamicin is a mixture of five gentamicin C-subtypes and several impurities of various ranges of nonexact concentrations. We developed a purification strategy enabling assaying of individual C-subtypes and impurities for ototoxicity and antimicrobial activity. We found that C-subtypes displayed broad and potent in vitro antimicrobial activities comparable to the hospital gentamicin mixture. In contrast, they showed different degrees of ototoxicity in cochlear explants, with gentamicin C2b being the least and gentamicin C2 the most ototoxic. Structure-activity relationships identified sites in the C4'-C6' region on ring I that reduced ototoxicity while preserving antimicrobial activity, thus identifying targets for future drug design and mechanisms for hair cell toxicity. Structure-activity relationship data suggested and electrophysiological data showed that the C-subtypes both bind and permeate the hair cell mechanotransducer channel, with the stronger the binding the less ototoxic the compound. Finally, both individual and reformulated mixtures of C-subtypes demonstrated decreased ototoxicity while maintaining antimicrobial activity, thereby serving as a proof-of-concept of drug reformulation to minimizing ototoxicity of gentamicin in patients.

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.

Crash Performance of Rear-facing Restraints With a Fracture Type Spica Casted 1-Year-Old Dummy.

BACKGROUND: Motor vehicle crashes represent a significant cause of mortality and morbidity for young children. Safely restraining a child is typically more complicated for special cases such as children treated with a hip spica cast. In the current study, hip spica casts typical for treatment of a femoral fracture were applied to a crash dummy representing the size and weight of a 1-year-old child. This spica casted dummy was used to study the performance of 4 rear-facing car seats in a series of simulated frontal impacts. METHODS: The restrained, rear-facing dummy was subjected to a frontal crash test at 30 mph (48 kph) per federal guidelines. Two of the tested car seats were specifically designed for transporting children with hip spica casts, while the other 2 were conventional seats capable of accommodating the cast. All seats were installed per the manufacturer's instructions. As a control, tests were performed without a cast using the conventional/standard seats. RESULTS: The lowest overall loading of the dummy's head, neck, and chest occurred during tests with the standard seats. While it was easier to seat the casted child in the spica-specific seats, these designs led to greater loading on the dummy's body. In a spica-specific seat, the chest acceleration values exceeded the federal limit in a test where the seat was installed in a reclined orientation that was within the manufacturer's described positioning. CONCLUSIONS: Spica-specific seats more easily accommodate the cast, but conventional seats can provide similar levels of protection in a crash. As cast and seat designs continue to evolve, hospitals might consider having a range of seats available for patient use. It is important to help caregivers make informed decisions on how and when to transport children with hip spica casts.

Modification of Stryker T5TM and Stryker Flyte® Personal Protection Surgical Helmets to Function as Powered Air-Purifying Respirators.

Since the SARS-CoV-2 (COVID-19) outbreak, health-care workers (HCWs) have had to create personal protective equipment (PPE) due to the worldwide demand and thus ensuing shortage. To address the dearth of available PPE, HCWs have quickly explored options to repurpose in-hospital equipment to provide alternative PPE to caregivers. We report the modification of a Stryker T5TM and Stryker Flyte® personal protection surgical helmets as a powered air-purifying respirator.

Frontal Crash Injury Metrics Are Below Mandated Limits for a Spica Casted Child Dummy in Currently Available Restraints.

BACKGROUND: There is a paucity of data defining safe transport protocols for children treated with hip spica casting. Although restraint devices for casted children are available, all federally mandated testing uses a noncasted anthropomorphic test device (ATD or crash dummy). The purpose of this study was to evaluate current restraint options in simulated frontal crash testing using a casted pediatric ATD to determine injury risk to the head, cervical spine, chest, and pelvis. METHODS: Using a 3-year-old ATD, dynamic crash sled tests simulating frontal crash were performed in accordance with government safety standards. The ATD was casted in a double-leg spica and the following restraint devices were tested: a seat designed for spica casted children, a restraint vest-harness, a traditional booster seat, and 2 traditional forward-facing car seats. RESULTS: Although the presence of the cast increased many of the injury metrics measured, all seats passed current federal guidelines for the head and chest. No single seat performed best in all metrics. The greatest magnitude of neck loading and second-highest head injury criterion values were observed for the booster seat. The vest-harness produced the highest head injury criterion and the chest compression exceeded proposed federal limits. CONCLUSIONS: The results suggest safe transport in commercially available seats is possible with the child properly restrained in a correctly fitting seat. However, parents should not assume a child restraint system is appropriate for use just based on fit as, for example, seats with harnesses outperformed an easy to fit booster seat. CLINICAL RELEVANCE: Each child and the position of the child's cast are unique and discharge planning involves consideration of safe transportation. Although this study suggests several seats used to transport spica casted children pass the federal head and chest injury prevention requirements, it is important to recognize that some children may still require emergency vehicle transport.

Sensory hair cell development and regeneration: similarities and differences.

Sensory hair cells are mechanoreceptors of the auditory and vestibular systems and are crucial for hearing and balance. In adult mammals, auditory hair cells are unable to regenerate, and damage to these cells results in permanent hearing loss. By contrast, hair cells in the chick cochlea and the zebrafish lateral line are able to regenerate, prompting studies into the signaling pathways, morphogen gradients and transcription factors that regulate hair cell development and regeneration in various species. Here, we review these findings and discuss how various signaling pathways and factors function to modulate sensory hair cell development and regeneration. By comparing and contrasting development and regeneration, we also highlight the utility and limitations of using defined developmental cues to drive mammalian hair cell regeneration.

Reverse oblique end screws in nonlocking plates decrease construct strength in synthetic osteoporotic bone medium.

Fracture stability can be challenging for osteoporotic individuals. The end screw of nonlocked plates is subjected to the greatest loading and is typically the site of construct failure. To enhance fixation, the end screw can be angled away from the fracture. The current study biomechanically evaluated screws angled the other direction: toward the fracture using 3.5-mm dynamic compression plates in an osteoporotic bone model. Three different plate lengths (6-, 8-, 12-hole) were tested in three-point bending with an oblique, perpendicular, or reverse oblique end screw. The peak load for loss of screw fixation for the reverse oblique end screw constructs was significantly less than the other screw orientations for all plate lengths. The 12-hole peak load, energy, and displacement magnitudes for all three screw orientations were significantly greater than all 6- and 8-hole constructs. The use of a reverse oblique end screw is inferior to both perpendicular and oblique end screws.

Angle stable nails provide improved healing for a complex fracture model in the femur.

BACKGROUND: Conventional nails are being used for an expanding range of fractures from simple to more complex. Angle stable designs are a relatively new innovation; however, it is unknown if they will improve healing for complex fractures. QUESTIONS/PURPOSES: When comparing traditional and angle stable nails to treat complex open canine femur fractures, the current study addressed the following questions: do the two constructs differ in (1) radiographic evidence of bone union across the cortices; (2) stability as determined by toggle (torsional motion with little accompanying torque) and angular deformation; (3) biomechanical properties, including stiffness in bending, axial compression, and torsional loading, and construct failure properties in torsion; and (4) degree of bone tissue mineralization? METHODS: Ten hounds with a 1-cm femoral defect and periosteal stripping were treated with a reamed titanium angle stable or nonangle stable nail after the creation of a long soft tissue wound. Before the study, the animals were randomly assigned to receive one of the nails and to be evaluated with biomechanical testing or histology. After euthanasia at 16 weeks, all operative femora were assessed radiographically. Histological or biomechanical evaluation was conducted of the operative bones with nails left in situ compared with the nonoperative contralateral femora. RESULTS: Radiographic and gross inspection demonstrated hypertrophic nonunion in all 10 animals treated with the nonangle stable nail, whereas six of 10 animals treated with the angle stable nail bridged at least one cortex (p = 0.023). The angle stable nail construct demonstrated no toggle in nine of 10 animals, whereas all control femora exhibited toggle. The angle stable nail demonstrated less angular deformation and toggle (p ≤ 0.005) and increased compressive stiffness (p = 0.001) compared with the conventional nonangle stable nail. Histology demonstrated more nonmineralized tissue in the limbs treated with the conventional nail (p = 0.005). CONCLUSIONS: Angle stable nails that eliminate toggle lead to enhanced yet incomplete fracture healing in a complex canine fracture model. CLINICAL RELEVANCE: Care should be taken in tailoring the nail design features to the characteristics of the fracture and the patient.

Should a low starting point be abandoned for cannulated screw fixation of femoral neck fractures?

A validated femoral neck fracture model stabilized with three inverted cannulated screws was used to consider different intraoperative scenarios when the inferior screw hole is inadvertently started too inferiorly. These scenarios were to: (1) abandon the misplaced inferior screw hole and restart this hole more proximally, or (2) accept the mispositioned placement of the inferior screw and insert the remaining superior screws parallel or convergent to the inferior screw. Utilizing the second option and accepting the errant hole was associated with the greatest interfragmentary motion and stresses in the bone and hardware. In contrast, the first option created an improved mechanical environment for healing.

Loss of Pax3 causes reduction of melanocytes in the developing mouse cochlea.

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of skin and retinal melanocytes, manifested as congenital hearing loss (~ 70%) and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. Cochlear melanocytes in the stria vascularis originated from Pax3-traced melanoblasts and Plp1-traced Schwann cell precursors, both of which derive from neural crest cells. Here, using a Pax3-Cre knock-in mouse that allows lineage tracing of Pax3-expressing cells and disruption of Pax3, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3+ derivatives contribute to S100+, Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all of which are significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in humans.

Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.

Difference in Bioimpedance Across the Knee in Un-Injured Young Adults.

Background: Knee injuries induce swelling and resolution of swelling may be a useful factor in identifying states of healing and time to return to sports activities. Recent work has suggested that bioimpedance can provide an objective measure of swelling following total knee arthroplasty (TKA) and therefore may also provide guidance for clinical decision-making following knee injury. This study measures knee bioimpedance in young, active people to help define baseline variability and factors that influence limb to limb differences. Methods: Bioimpedance was measured via sensors placed at the foot/ankle and thigh, in positions similar to those suggested for monitoring post-TKA swelling. Initial tests were performed to verify method repeatability, then bioimpedance was measured in a convenience sample of 78 subjects (median age 21yrs). The influence of age, BMI, thigh circumference, and knee function (KOOS-JR) on the impedance measures and difference in impedance between the subject's knees were examined using a generalized multivariable linear regression. Results: The repeatability study measurements were highly consistent with a COV of 1.5% for resistance and an ICC of 97.9%. Women exhibited significantly larger dominant limb impedance and larger limb to limb difference in impedance than men. Regression analysis indicated that subject sex and BMI significantly influenced bioimpedance but joint score and age did not. The limb to limb differences in impedance were small on average (<5%), with larger magnitudes of difference associated with female sex, lower knee function scores, and larger limb to limb differences in thigh circumference. Conclusion: Bioimpedance measurements across right and left knees of healthy young people were similar, supporting use of bioimpedance measures from a patient's uninjured knee as a benchmark to monitor healing of a contralateral injured knee. Future work should focus on understanding how knee function scores and bioimpedance are related, and further explore how sex and side to side anatomic differences impact the measurement. Level of Evidence: IV.

Pax3 deficiency diminishes melanocytes in the developing mouse cochlea.

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of melanocytes, manifested as congenital hearing loss and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. During development, cochlear melanocytes in the stria vascularis are dually derived from Pax3-Cre+ melanoblasts migrating from neuroepithelial cells including neural crest cells and Plp1+ Schwann cell precursors originated from also neural crest cells, differentiating in a basal-apical manner. Here, using a Pax3-Cre mouse line, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3-Cre derivatives contribute to S100+ , Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in human.

Selection of viral capsids and promoters affects the efficacy of rescue of Tmprss3-deficient cochlea.

Adeno-associated virus (AAV)-mediated gene transfer has shown promise in rescuing mouse models of genetic hearing loss, but how viral capsid and promoter selection affects efficacy is poorly characterized. Here, we tested combinations of AAVs and promoters to deliver Tmprss3, mutations in which are associated with hearing loss in humans. Tmprss3tm1/tm1 mice display severe cochlear hair cell degeneration, loss of auditory brainstem responses, and delayed loss of spiral ganglion neurons. Under the ubiquitous CAG promoter and AAV-KP1 capsid, Tmprss3 overexpression caused striking cytotoxicity in vitro and in vivo and failed to rescue degeneration or dysfunction of the Tmprss3tm1/tm1 cochlea. Reducing the dosage or using AAV-DJ-CAG-Tmprss3 diminished cytotoxicity without rescue of the Tmprss3tm1/tm1 cochlea. Finally, the combination of AAV-KP1 capsid and the EF1α promoter prevented cytotoxicity and reduced hair cell degeneration, loss of spiral ganglion neurons, and improved hearing thresholds in Tmprss3tm1/tm1 mice. Together, our study illustrates toxicity of exogenous genes and factors governing rescue efficiency, and suggests that cochlear gene therapy likely requires precisely targeted transgene expression.

A modified intramedullary nail interlocking design yields improved stability for fatigue cycling in a canine femur fracture model.

Intramedullary nailing has evolved to become the standard of care for most diaphyseal femoral and tibial fractures, as well as an expanding number of metaphyseal fractures. Owing to the unstable nature of some fractures, the intramedullary device may be subjected to significant stresses owing to a lack of solid cortical contact after nailing. In such cases, excessive interfragmentary motion (due to construct toggle) has been shown to occur. Such motion increases the likelihood of a non- or delayed-union. In the current study, two versions of a modified, angle stable interlocking design were subjected to fatigue testing in a segmental defect fracture model representing a canine femur. As a control, a third group of constructs were stabilized with a traditional nail that allowed a small amount of toggle. All constructs were subjected to 50,000 fatigue cycles representing 12 weeks of cage activity at physiologic levels of combined axial-torsional loading. Torsional testing pre- and post-fatigue revealed 4.6 +/- 1.3 degrees of toggle in the traditional nail and no toggle with the angle stable nail designs. The stable nails were also significantly stiffer in axial compression and torsion before and after cycling. These data indicate that the enhanced stability of the modified interlocking designs can be maintained throughout fatigue cycling in a challenging fracture model.

Development of an interlocked nail for segmental defects in the rabbit tibia.

Previous animal models have been developed to study intramedullary nailing for challenging segmental defects in the tibia. In large animals, interlocked nail fixation created a stable environment suitable to study new bone growth technologies placed in the defect. To our knowledge, there are no comparable interlocked tibial defect models for the rabbit in which new technologies could be evaluated. Such a model would be helpful since the rabbit is a popular initial model for orthopedic research studies owing to its wide availability and low cost. While numerous studies have nailed the rabbit tibia, all were non-locked implants that allowed some degree of instability between the fracture fragments. In addition, the non-locked nails were constructed of stainless steel, whereas human nails are increasingly made from titanium alloy. In the current study, an interlocked titanium nail was developed for the rabbit tibia. It was implanted in cadaver tibiae and subjected to fatigue cycling in combined compression and bending at physiologic levels to 21,061 cycles. This duration is estimated to represent 12 weeks of gait by the animal. Before and after fatigue cycling, monotonic testing was performed in compression and bending at physiologic levels. The intact contralateral limbs served as controls. All limbs completed the cycling; the instrumented limbs exhibited interfragmentary cyclic strain amplitudes during fatigue (616 +/- 139 micro-strain), which was significantly greater than the control limbs (136 +/- 35 microstrain). Monotonic strain amplitudes for the test limbs in bending and compression were 4839 +/- 1028 and 542 +/- 122 microstrain, respectively; corresponding values for the control bones were 407 +/- 118 and 95 +/- 38 microstrain, respectively. These data are similar to those presented in prior studies in larger bone models. The current study presents one method for interlocked nail fixation for this complex tibial shaft fracture in a small animal.

Effects of an enhanced acoustic environment on residual hearing following chronic cochlear implantation and electrical stimulation in the partially deafened cat.

There is an increasing trend to provide cochlear implants for people with useful residual hearing, typically in the low frequency range (<2 kHz). These recipients typically use both electrical stimulation from their implant and acoustic stimulation that has been amplified with a hearing aid to access their residual hearing, so called electro-acoustic stimulation (EAS). However, a significant problem is the loss of residual hearing following implantation that can occur immediately following surgery or delayed over many months. One potential cause of the loss of residual hearing is the over stimulation of remaining hair cells due to the combination of an amplified acoustic input and direct electrical activation. This paper aims to test this hypothesis. Here, we have used a neonatal aminoglycoside-induced partial hearing cat model that resulted in a high frequency hearing loss (>4 kHz). Two separate cohorts of animals were implanted and received unilateral chronic electrical stimulation using clinical stimulators and speech processors over 5 months. To simulate potential over stimulation via a hearing aid, one cohort of animals were also exposed to an enhanced acoustic environment consisting of 80 dB SPL 4-talker babble presented 14 h per day. Hearing thresholds for both stimulated and unstimulated ears were measured throughout the implantation period. Cochleae were collected for histology to measure spiral ganglion neuron survival, hair cell survival and tissue response to chronic implantation and electrical stimulation. Consistent with clinical observations, cochlear implantation and stimulation resulted in an increase in threshold across the population. There was no significant effect of the enhanced acoustic environment on auditory thresholds or histological measures (hair cell survival, neuronal survival) of hearing, indicating that hair cell overstimulation was not a significant driver of loss of residual hearing.