Combinatorial Atoh1, Gfi1, Pou4f3, and Six1 gene transfer induces hair cell regeneration in the flat epithelium of mature guinea pigs.

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE.

Role of auditory feedback for vocal production learning in the Egyptian fruit-bat.

Some species have evolved the ability to use the sense of hearing to modify existing vocalizations, or even create new ones. This ability corresponds to various forms of vocal production learning that are all possessed by humans, and independently displayed by distantly related vertebrates. Among mammals, a few species, including the Egyptian fruit-bat, would possess such vocal production learning abilities. Yet the necessity of an intact auditory system for the development of the Egyptian fruit-bat typical vocal repertoire has not been tested. Furthermore, a systematic causal examination of learned and innate aspects of the entire repertoire has never been performed in any vocal learner. Here we addressed these gaps by eliminating pups' sense of hearing at birth and assessing its effects on vocal production in adulthood. The deafening treatment enabled us to both causally test these bats vocal learning ability and discern learned from innate aspects of their vocalizations. Leveraging wireless individual audio recordings from freely interacting adults, we show that a subset of the Egyptian fruit-bat vocal repertoire necessitates auditory feedback. Intriguingly, these affected vocalizations belong to different acoustic groups in the vocal repertoire of males and females. These findings open the possibilities for targeted studies of the mammalian neural circuits that enable sexually dimorphic forms of vocal learning.

Diet versatility and functional trade-offs shape tooth morphology in squirrels.

Identifying the drivers of adaptation is key to understanding the origin and evolution of diversity. Here we study the morphological evolution of tooth morphology, a classic example of a conserved structure, to gain insights into the conditions that can overcome resistance to evolutionary change. We use geometric morphometrics of the occlusal surface outline of the fourth lower premolar (p4) of squirrels, a paradigm of a stable tooth morphology, to explore morphological adaptations to diet. Although a versatile generalist dental morphology favors the retention of the ancestral shape, the acquisition of diets that require strong mechanical processing drives morphological change. In particular, species that eat both grass and dry fruits evolved disparate tooth shape morphologies, related to trade-offs between feeding performance that lead to a more or less pronounced change depending on the proportion of those items in their diet. Also, some folivores develop relatively large p4s, and most bark gleaners have relatively small p4s. Ultimately, despite the role of diet shaping these patterns, we showed that diet is not the only factor driving the evolution of tooth morphology.

Cochlear Health and Cochlear-implant Function.

The cochlear implant (CI) is widely considered to be one of the most innovative and successful neuroprosthetic treatments developed to date. Although outcomes vary, CIs are able to effectively improve hearing in nearly all recipients and can substantially improve speech understanding and quality of life for patients with significant hearing loss. A wealth of research has focused on underlying factors that contribute to success with a CI, and recent evidence suggests that the overall health of the cochlea could potentially play a larger role than previously recognized. This article defines and reviews attributes of cochlear health and describes procedures to evaluate cochlear health in humans and animal models in order to examine the effects of cochlear health on performance with a CI. Lastly, we describe how future biologic approaches can be used to preserve and/or enhance cochlear health in order to maximize performance for individual CI recipients.

Components of impedance in a cochlear implant animal model with TGFß1-accelerated fibrosis.

Outcomes of cochlear implantation are likely influenced by the biological state of the cochlea. Fibrosis is a pathological change frequently seen in implanted ears. The goal of this work was to investigate the relationship between fibrosis and impedance. To that end, we employed an animal model of extensive fibrosis and tested whether aspects of impedance differed from controls. Specifically, an adenovirus with a TGF-ß1 gene insert (Ad.TGF-ß1) was injected into guinea pig scala tympani to elicit rapid onset fibrosis and investigate the relation between fibrosis and impedance. We found a significant correlation between treatment and rate of impedance increase. A physical circuit model of impedance was used to separate the effect of fibrosis from other confounding factors. Supported by preliminary, yet nonconclusive, electron microscopy data, this modeling suggested that deposits on the electrode surface are an important contributor to impedance change over time.

Complex adaptive landscape for a "Simple" structure: The role of trade-offs in the evolutionary dynamics of mandibular shape in ground squirrels.

Trade-offs are inherent features of many biomechanical systems and are often seen as evolutionary constraints. Structural decoupling may provide a way to escape those limits in some systems but not for structures that transmit large forces, such as mammalian mandibles. For such structures to evolve in multiple directions on a complex adaptive landscape, different regions must change shape while maintaining structural integrity. We evaluated the complexity of the adaptive landscape for mandibular shape in Marmotini, a lineage of ground squirrels that varies in the proportions of seeds and foliage in their diets, by comparing the fit of models based on traits that predict changes in mandibular loading. The adaptive landscape was more complex than predicted by a two-peak model with a single dietary shift. The large number of adaptive peaks reflects a high diversity of directions of shape evolution. The number of adaptive peaks also reflects a multiplicity of functional trade-offs posed by the conflicting demands of processing foods with various combinations of material properties. The ability to balance trade-offs for diets with different proportions of the same foods may account for diversification and disparity of lineages in heterogeneous environments. Rather than constraints, trade-offs may be the impetus of evolutionary change.

Estimating health of the implanted cochlea using psychophysical strength-duration functions and electrode configuration.

It is generally believed that the efficacy of cochlear implants is partly dependent on the condition of the stimulated neural population. Cochlear pathology is likely to affect the manner in which neurons respond to electrical stimulation, potentially resulting in differences in perception of electrical stimuli across cochlear implant recipients and across the electrode array in individual cochlear implant users. Several psychophysical and electrophysiological measures have been shown to predict cochlear health in animals and were used to assess conditions near individual stimulation sites in humans. In this study, we examined the relationship between psychophysical strength-duration functions and spiral ganglion neuron density in two groups of guinea pigs with cochlear implants who had minimally-overlapping cochlear health profiles. One group was implanted in a hearing ear (N = 10) and the other group was deafened by cochlear perfusion of neomycin, inoculated with an adeno-associated viral vector with an Ntf3-gene insert (AAV.Ntf3) and implanted (N = 14). Psychophysically measured strength-duration functions for both monopolar and tripolar electrode configurations were then compared for the two treatment groups. Results were also compared to their histological outcomes. Overall, there were considerable differences between the two treatment groups in terms of their psychophysical performance as well as the relation between their functional performance and histological data. Animals in the neomycin-deafened, neurotrophin-treated, and implanted group (NNI) exhibited steeper strength-duration function slopes; slopes were positively correlated with SGN density (steeper slopes in animals that had higher SGN densities). In comparison, the implanted hearing (IH) group had shallower slopes and there was no relation between slopes and spiral ganglion density. Across all animals, slopes were negatively correlated with ensemble spontaneous activity levels (shallower slopes with higher ensemble spontaneous activity levels). We hypothesize that differences in strength-duration function slopes between the two treatment groups were related to the condition of the inner hair cells, which generate spontaneous activity that could affect the across-fiber synchrony and/or the size of the population of neural elements responding to electrical stimulation. In addition, it is likely that spiral ganglion neuron peripheral processes were present in the IH group, which could affect membrane properties of the stimulated neurons. Results suggest that the two treatment groups exhibited distinct patterns of variation in conditions near the stimulating electrodes that altered detection thresholds. Overall, the results of this study suggest a complex relationship between psychophysical detection thresholds for cochlear implant stimulation and nerve survival in the implanted cochlea. This relationship seems to depend on the characteristics of the electrical stimulus, the electrode configuration, and other biological features of the implanted cochlea such as the condition of the inner hair cells and the peripheral processes.

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function.

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

Development of a chronically-implanted mouse model for studies of cochlear health and implant function.

Mice with chronic cochlear implants can significantly contribute to our understanding of the relationship between cochlear health and implant function because of the availability of molecular tools for controlling conditions in the cochlea and transgenic lines modeling human disease. To date, research in implanted mice has mainly consisted of short-term studies, but since there are large changes in implant function following implant insertion trauma, and subsequent recovery in many cases, longer-term studies are needed to evaluate function and perception under stable conditions. Because frequent anesthetic administration can be especially problematic in mice, a chronic model that can be tested in the awake condition is desirable. Electrically-evoked compound action potentials (ECAPs) recorded with multichannel cochlear implants are useful functional measures because they can be obtained daily without anesthesia. In this study, we assessed changes and stability of ECAPs, electrically-evoked auditory brainstem responses (EABRs), ensemble spontaneous activity (ESA), and impedance data over time after implanting mice with multichannel implants. We then compared these data to histological findings in these implanted cochleae, and compared results from this chronic mouse model to data previously obtained in a well-established chronically-implanted guinea pig model. We determined that mice can be chronically implanted with cochlear implants, and ECAP recordings can be obtained frequently in an awake state for up to at least 42 days after implantation. These recordings can effectively monitor changes or stability in cochlear function over time. ECAP and EABR amplitude-growth functions (AGFs), AGF slopes, ESA levels and impedances in mice with multichannel implants appear similar to those found in guinea pigs with long-term multichannel implants. Animals with better survival of spiral ganglion neurons (SGNs) and inner hair cells (IHCs) have steeper AGF slopes, and larger ESA responses. The time course of post-surgical ear recovery may be quicker in mice and can show different patterns of recovery which seem to be dependent on the degree of insertion trauma and subsequent histological conditions. Histology showed varying degrees of cochlear damage with fibrosis present in all implanted mouse ears and small amounts of new bone in a few ears. Impedance changes over time varied within and across animals and may represent changes over time in multiple variables in the cochlear environment post-implantation. Due to the small size of the mouse, susceptibility to stress, and the higher potential for implant failure, chronic implantation in mice can be challenging, but overall is feasible and useful for cochlear implant research.

Scar Formation and Debris Elimination during Hair Cell Degeneration in the Adult DTR Mouse.

The auditory sensory epithelium of the mammalian inner ear is a highly organized structure that contains sensory hair cells (HCs) and non-sensory supporting cells (SCs). Following the partial loss of HCs after cochlear insults such as overstimulation or ototoxic drugs, SCs seal the luminal epithelial surface (reticular lamina) and reorganize its cellular pattern. Here we investigated the changes in the sensory epithelium following a rapid and severe cochlear insult in the diphtheria toxin receptor (DTR) mouse, where diphtheria toxin (DT) injection leads to a HC-specific lesion resulting in a complete HC loss. We found that DT-induced selective HC ablation could lead to a pattern of scar formation and apical cell-cell adherens and tight junction reorganization similar to that occurring after other types of cochlear insult. Prestin, an outer HC-specific protein, was present in amorphous clumps at the sites where SCs had expanded to fill the spaces vacated by the dead HCs for up to 2 months after the DT induced lesion. Many of the prestin clumps appeared to occupy spaces within SCs, suggesting that SCs participate in the removal process of HC corpses in the DTR deafness model. Prestin clumps could be seen in different areas all along the length of the SCs, and appeared to be inside the SCs as well as in the inter-cellular spaces between SCs. The findings suggest that HC elimination in the DTR deafness model follows a mechanism similar to that in overstimulation or ototoxicity models, making the DTR mouse useful for understanding the process underlying HC elimination and the role of SCs in this process.

How electrically evoked compound action potentials in chronically implanted guinea pigs relate to auditory nerve health and electrode impedance.

This study examined how multiple measures based on the electrically evoked compound action potential (ECAP) amplitude-growth functions (AGFs) were related to estimates of neural [spiral ganglion neuron (SGN) density and cell size] and electrode impedance measures in 34 specific pathogen free pigmented guinea pigs that were chronically implanted (4.9-15.4 months) with a cochlear implant electrode array. Two interphase gaps (IPGs) were used for the biphasic pulses and the effect of the IPG on each ECAP measure was measured ("IPG effect"). When using a stimulus with a constant IPG, SGN density was related to the across-subject variance in ECAP AGF linear slope, peak amplitude, and N1 latency. The SGN density values also help to explain a significant proportion of variance in the IPG effect for AGF linear slope and peak amplitude measures. Regression modeling revealed that SGN density was the primary dependent variable contributing to across-subject variance for ECAP measures; SGN cell size did not significantly improve the fitting of the model. Results showed that simple impedance measures were weakly related to most ECAP measures but did not typically improve the fit of the regression model.

Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea.

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Relationships between Intrascalar Tissue, Neuron Survival, and Cochlear Implant Function.

Fibrous tissue and/or new bone are often found surrounding a cochlear implant in the cochlear scalae. This new intrascalar tissue could potentially limit cochlear implant function by increasing impedance and altering signaling pathways between the implant and the auditory nerve. In this study, we investigated the relationship between intrascalar tissue and 5 measures of implant function in guinea pigs. Variation in both spiral ganglion neuron (SGN) survival and intrascalar tissue was produced by implanting hearing ears, ears deafened with neomycin, and neomycin-deafened ears treated with a neurotrophin. We found significant effects of SGN density on 4 functional measures but adding intrascalar tissue level to the analysis did not explain more variation in any measure than was explained by SGN density alone. These results suggest that effects of intrascalar tissue on electrical hearing are relatively unimportant in comparison to degeneration of the auditory nerve, although additional studies in human implant recipients are still needed to assess the effects of this tissue on complex hearing tasks like speech perception. The results also suggest that efforts to minimize the trauma that aggravates both tissue development and SGN loss could be beneficial.

Stasis of functionally versatile specialists.

A classic hypothesis posits that lineages exhibiting long-term stasis are broadly adapted generalists that remain well-adapted despite environmental change. However, lacking constraints that steepen adaptive peaks and stabilize the optimum, generalists' phenotypes might drift around a broad adaptive plateau. We propose that stasis would be likely for morphological specialists that behave as ecological generalists much of the time because specialists' functional constraints stabilize the optimum, but those with a broad niche, such as generalists, can persist despite environmental change. Tree squirrels (Callosciurinae and Sciurini) exemplify ecologically versatile specialists, being extreme in adaptations for forceful biting that expand rather than limit niche breadth. Here, we examine the structure of disparity and the evolutionary dynamics of their trophic morphology (mandible size and shape) to determine if they exhibit stasis. In both lineages, a few dietary specialists disproportionately account for disparity; excluding them, we find compelling evidence for stasis of jaw shape but not size. The primary optima of these lineages diverge little, if at all over approximately 30 million years. Once their trophic apparatus was assembled, their morphological specialization steepened the slopes of their adaptive peak and constrained the position of the optima without limiting niche breadth.

Changes over time in the electrically evoked compound action potential (ECAP) interphase gap (IPG) effect following cochlear implantation in Guinea pigs.

The electrically-evoked compound action potential (ECAP) is correlated with spiral ganglion neuron (SGN) density in cochlear implanted animals. In a previous study, we showed that ECAP amplitude growth function (AGF) linear slopes for stimuli with a constant interphase gap (IPG) changed significantly over time following implantation. Related studies have also shown that 1) IPG sensitivity for ECAP measures ("IPG Effect") is related to SGN density in animals and 2) the ECAP IPG Effect is related to speech recognition performance in humans with cochlear implants. The current study examined how the ECAP IPG Effect changed following cochlear implantation in four non-deafened guinea pigs with residual inner hair cells (IHCs) and 5 deafened, neurotrophin-treated guinea pigs. Simple impedances were measured on the same days as the ECAP measures. Generally, non-deafened implanted animals with higher SGN survival demonstrated higher ECAP AGF linear slope and peak amplitude values than the deafened, implanted guinea pigs. The ECAP IPG Effect for the AGF slopes and peak amplitudes was also larger in the hearing animals. The N1 latencies for a constant IPG were not different between groups, but the N1 latency IPG Effect was smaller in the non-deafened, implanted animals. Similar to previously reported results, ECAP measures using a fixed or changing IPG required as many as three months after implantation before a stable point could be calculated, but this was dependent on the animal and condition. For all ECAP measures most animals showed greater variance in the first 30 days post-implantation. Post-implantation changes in ECAPs and impedances were not correlated with one another. Results from this study are helpful for estimating the mechanisms underlying ECAP characteristics and have implications for clinical application of the ECAP measures in long-term human cochlear implant recipients. Specifically, these measures could help to monitor neural health over a period of time, or during a time of stability these measures could be used to help select electrode sites for activation in clinical programming.

Sclerostin Antibody-Induced Changes in Bone Mass Are Site Specific in Developing Crania.

Sclerostin antibody (Scl-Ab) is an anabolic bone agent that has been shown to increase bone mass in clinical trials of adult diseases of low bone mass, such as osteoporosis and osteogenesis imperfecta (OI). Its use to decrease bone fragility in pediatric OI has shown efficacy in several growing mouse models, suggesting translational potential to pediatric disorders of low bone mass. However, the effects of pharmacologic inhibition of sclerostin during periods of rapid growth and development have not yet been described with respect to the cranium, where lifelong deficiency of functioning sclerostin leads to patterns of excessive bone growth, cranial compression, and facial palsy. In the present study, we undertook dimensional and volumetric measurements in the skulls of growing Brtl/+ OI mice treated with Scl-Ab to examine whether therapy-induced phenotypic changes were similar to those observed clinically in patients with sclerosteosis or Van Buchem disorder. Mice treated between 3 and 14 weeks of age with high doses of Scl-Ab show significant calvarial thickening capable of rescuing OI-induced deficiencies in skull thickness. Other changes in cranial morphology, such as lengths and distances between anatomic landmarks, intracranial volume, and suture interdigitation, showed minimal effects of Scl-Ab when compared with growth-induced differences over the treatment duration. Treatment-induced narrowing of foramina was limited to sites of vascular but not neural passage, suggesting patterns of local regulation. Together, these findings reveal a site specificity of Scl-Ab action in the calvaria with no measurable cranial nerve impingement or brainstem compression. This differentiation from the observed outcomes of lifelong sclerostin deficiency complements reports of Scl-Ab treatment efficacy at other skeletal sites with the prospect of minimal cranial secondary complications. © 2019 American Society for Bone and Mineral Research. © 2019 American Society for Bone and Mineral Research.

ROS Scavenger, Ebselen, Has No Preventive Effect in New Hearing Loss Model Using a Cholesterol-Chelating Agent.

BACKGROUND AND OBJECTIVES: The antioxidant ebselen will be able to limit or prevent the ototoxicity arising from 2-hydroxypropyl-ß-cyclodextrin (HPßCD). Niemann-Pick Type C (NPC) disease is a disorder of lysosomal storage manifested in sphingolipidosis. Recently, it was noted that experimental use of HPßCD could partially resolve the symptoms in both animals and human patients. Despite its desirable effect, HPßCD can induce hearing loss, which is the only major side effect noted to date. Understanding of the pathophysiology of hearing impairment after administration of HPßCD and further development of preventive methods are essential to reduce the ototoxic side effect. The mechanisms of HPßCD-induced ototoxicity remain unknown, but the resulting pathology bears some resemblance to other ototoxic agents, which involves oxidative stress pathways. To indirectly determine the involvement of oxidative stress in HPßCD-induced ototoxicity, we tested the efficacy of an antioxidant reagent, ebselen, on the extent of inner ear side effects caused by HPßCD. MATERIALS AND METHODS: Ebselen was applied prior to administration of HPßCD in mice. Auditory brainstem response thresholds and otopathology were assessed one week later. Bilateral effects of the drug treatments also were examined. RESULTS: HPßCD-alone resulted in bilateral, severe, and selective loss of outer hair cells from base to apex with an abrupt transition between lesions and intact areas. Ebselen co-treatment did not ameliorate HPßCD-induced hearing loss or alter the resulting histopathology. CONCLUSIONS: The results indirectly suggest that cochlear damage by HPßCD is unrelated to reactive oxygen species formation. However, further research into the mechanism(s) of HPßCD otopathology is necessary.

CHD7 represses the retinoic acid synthesis enzyme ALDH1A3 during inner ear development.

CHD7, an ATP-dependent chromatin remodeler, is disrupted in CHARGE syndrome, an autosomal dominant disorder characterized by variably penetrant abnormalities in craniofacial, cardiac, and nervous system tissues. The inner ear is uniquely sensitive to CHD7 levels and is the most commonly affected organ in individuals with CHARGE. Interestingly, upregulation or downregulation of retinoic acid (RA) signaling during embryogenesis also leads to developmental defects similar to those in CHARGE syndrome, suggesting that CHD7 and RA may have common target genes or signaling pathways. Here, we tested three separate potential mechanisms for CHD7 and RA interaction: (a) direct binding of CHD7 with RA receptors, (b) regulation of CHD7 levels by RA, and (c) CHD7 binding and regulation of RA-related genes. We show that CHD7 directly regulates expression of Aldh1a3, the gene encoding the RA synthetic enzyme ALDH1A3 and that loss of Aldh1a3 partially rescues Chd7 mutant mouse inner ear defects. Together, these studies indicate that ALDH1A3 acts with CHD7 in a common genetic pathway to regulate inner ear development, providing insights into how CHD7 and RA regulate gene expression and morphogenesis in the developing embryo.

Severe streptomycin ototoxicity in the mouse utricle leads to a flat epithelium but the peripheral neural degeneration is delayed.

The damaged vestibular sensory epithelium of mammals has a limited capacity for spontaneous hair cell regeneration, which largely depends on the transdifferentiation of surviving supporting cells. Little is known about the response of vestibular supporting cells to a severe insult. In the present study, we evaluated the impact of a severe ototoxic insult on the histology of utricular supporting cells and the changes in innervation that ensued. We infused a high dose of streptomycin into the mouse posterior semicircular canal to induce a severe lesion in the utricle. Both scanning electron microscopy and light microscopy of plastic sections showed replacement of the normal cytoarchitecture of the epithelial layer with a flat layer of cells in most of the samples. Immunofluorescence staining showed numerous cells in the severely damaged epithelial layer that were negative for hair cell and supporting cell markers. Nerve fibers under the flat epithelium had high density at the 1 month time point but very low density by 3 months. Similarly, the number of vestibular ganglion neurons was unchanged at 1 month after the lesion, but was significantly lower at 3 months. We therefore determined that the mouse utricular epithelium turns into a flat epithelium after a severe lesion, but the degeneration of neural components is slow, suggesting that treatments to restore balance by hair cell regeneration, stem cell therapy or vestibular prosthesis implantation will likely benefit from the short term preservation of the neural substrate.

Neurotrophin Gene Therapy in Deafened Ears with Cochlear Implants: Long-term Effects on Nerve Survival and Functional Measures.

Because cochlear implants function by stimulating the auditory nerve, it is assumed that the condition of the nerve plays an important role in the efficacy of the prosthesis. Thus, considerable research has been devoted to methods of preserving the nerve following deafness. Neurotrophins have been identified as a potential contributor to neural health, but most of the research to date has been done in young animals and for short periods (less than 3 to 6 months) after the onset of treatment. The first objective of the current experiment was to examine the effects of a neurotrophin gene therapy delivery method on spiral ganglion neuron (SGN) preservation and function in the long term (5 to 14 months) in mature guinea pigs with cochlear implants. The second objective was to examine several potential non-invasive monitors of auditory nerve health following the neurotrophin gene therapy procedure. Eighteen mature adult male guinea pigs were deafened by cochlear perfusion of neomycin and then one ear was inoculated with an adeno-associated viral vector with an Nft3-gene insert (AAV.Ntf3) and implanted with a cochlear implant electrode array. Five control animals were deafened and inoculated with an empty AAV and implanted. Data from 43 other guinea pig ears from this and previous experiments were used for comparison: 24 animals implanted in a hearing ear, nine animals deafened and implanted with no inoculation, and ten normal-hearing non-implanted ears. After 4 to 21 months of psychophysical and electrophysiological testing, the animals were prepared for histological examination of SGN densities and inner hair cell (IHC) survival. Seventy-eight percent of the ears deafened and inoculated with AAV.Ntf3 showed better SGN survival than the 14 deafened-control ears. The degree of SGN preservation following the gene therapy procedure was variable across animals and across cochlear turns. Slopes of psychophysical multipulse integration (MPI) functions were predictive of SGN density, but only in animals with preserved IHCs. MPI was not affected by the AAV.Ntf3 treatment, but there was a minor improvement in temporal integration (TI). AAV.Ntf3 treatment had significant effects on ECAP and EABR amplitude growth func-tion (AGF) slopes; the reduction in slope in deafened ears was ameliorated by the AAV.Ntf3 treatment. Slopes of the ECAP and EABR AGFs were predictive of SGN density in a broad area near and just apical to the implant. The highest ensemble spontaneous activity (ESA) values were seen in animals with surviving IHCs, but AAV.Ntf3 treatment in deafened ears resulted in slightly higher ESA values compared to deafened untreated ears. Overall, a combination of the psychophysical and electrophysiological measures can be useful for monitoring the health of the implanted cochlea in guinea pigs. These measures should be applicable for assessing cochlear health in human subjects.