MEF2C Hypofunction in GABAergic Cells Alters Sociability and Prefrontal Cortex Inhibitory Synaptic Transmission in a Sex-Dependent Manner.

Background: Heterozygous mutations or deletions of MEF2C cause a neurodevelopmental disorder termed MEF2C haploinsufficiency syndrome (MCHS), characterized by autism spectrum disorder and neurological symptoms. In mice, global Mef2c heterozygosity has produced multiple MCHS-like phenotypes. MEF2C is highly expressed in multiple cell types of the developing brain, including GABAergic (gamma-aminobutyric acidergic) inhibitory neurons, but the influence of MEF2C hypofunction in GABAergic neurons on MCHS-like phenotypes remains unclear. Methods: We employed GABAergic cell type-specific manipulations to study mouse Mef2c heterozygosity in a battery of MCHS-like behaviors. We also performed electroencephalography, single-cell transcriptomics, and patch-clamp electrophysiology and optogenetics to assess the impact of Mef2c haploinsufficiency on gene expression and prefrontal cortex microcircuits. Results: Mef2c heterozygosity in developing GABAergic cells produced female-specific deficits in social preference and altered approach-avoidance behavior. In female, but not male, mice, we observed that Mef2c heterozygosity in developing GABAergic cells produced 1) differentially expressed genes in multiple cell types, including parvalbumin-expressing GABAergic neurons, 2) baseline and social-related frontocortical network activity alterations, and 3) reductions in parvalbumin cell intrinsic excitability and inhibitory synaptic transmission onto deep-layer pyramidal neurons. Conclusions: MEF2C hypofunction in female, but not male, developing GABAergic cells is important for typical sociability and approach-avoidance behaviors and normal parvalbumin inhibitory neuron function in the prefrontal cortex of mice. While there is no apparent sex bias in autism spectrum disorder symptoms of MCHS, our findings suggest that GABAergic cell-specific dysfunction in females with MCHS may contribute disproportionately to sociability symptoms.

Complement factor B is essential for the proper function of the peripheral auditory system.

Sensorineural hearing loss is associated with dysfunction of cochlear cells. Although immune cells play a critical role in maintaining the inner ear microenvironment, the precise immune-related molecular mechanisms underlying the pathophysiology of hearing loss remain unclear. The complement cascade contributes to the regulation of immune cell activity. Additionally, activation of the complement cascade can lead to the cellular opsonization of cells and pathogens, resulting in their engulfment and elimination by phagocytes. Complement factor B (fB) is an essential activator protein in the alternative complement pathway, and variations in the fB gene are associated with age-related macular degeneration. Here we show that mice of both sexes deficient in fB functional alleles (fB-/-) demonstrate progressive hearing impairment. Transcriptomic analysis of auditory nerves from adult mice detected 706 genes that were significantly differentially expressed between fB-/- and wild-type control animals, including genes related to the extracellular matrix and neural development processes. Additionally, a subset of differentially expressed genes was related to myelin function and neural crest development. Histological and immunohistochemical investigations revealed pathological alterations in auditory nerve myelin sheathes of fB-/- mice. Pathological alterations were also seen in the stria vascularis of the cochlear lateral wall in these mice. Our results implicate fB as an integral regulator of myelin maintenance and stria vascularis integrity, underscoring the importance of understanding the involvement of immune signaling pathways in sensorineural hearing loss.

The Stria Vascularis in Mice and Humans Is an Early Site of Age-Related Cochlear Degeneration, Macrophage Dysfunction, and Inflammation.

Age-related hearing loss, or presbyacusis, is a common degenerative disorder affecting communication and quality of life for millions of older adults. Multiple pathophysiologic manifestations, along with many cellular and molecular alterations, have been linked to presbyacusis; however, the initial events and causal factors have not been clearly established. Comparisons of the transcriptome in the lateral wall (LW) with other cochlear regions in a mouse model (of both sexes) of "normal" age-related hearing loss revealed that early pathophysiological alterations in the stria vascularis (SV) are associated with increased macrophage activation and a molecular signature indicative of inflammaging, a common form of immune dysfunction. Structure-function correlation analyses in mice across the lifespan showed that the age-dependent increase in macrophage activation in the stria vascularis is associated with a decline in auditory sensitivity. High-resolution imaging analysis of macrophage activation in middle-aged and aged mouse and human cochleas, along with transcriptomic analysis of age-dependent changes in mouse cochlear macrophage gene expression, support the hypothesis that aberrant macrophage activity is an important contributor to age-dependent strial dysfunction, cochlear pathology, and hearing loss. Thus, this study highlights the SV as a primary site of age-related cochlear degeneration and aberrant macrophage activity and dysregulation of the immune system as early indicators of age-related cochlear pathology and hearing loss. Importantly, novel new imaging methods described here now provide a means to analyze human temporal bones in a way that had not previously been feasible and thereby represent a significant new tool for otopathological evaluation.SIGNIFICANCE STATEMENT Age-related hearing loss is a common neurodegenerative disorder affecting communication and quality of life. Current interventions (primarily hearing aids and cochlear implants) offer imperfect and often unsuccessful therapeutic outcomes. Identification of early pathology and causal factors is crucial for the development of new treatments and early diagnostic tests. Here, we find that the SV, a nonsensory component of the cochlea, is an early site of structural and functional pathology in mice and humans that is characterized by aberrant immune cell activity. We also establish a new technique for evaluating cochleas from human temporal bones, an important but understudied area of research because of a lack of well-preserved human specimens and difficult tissue preparation and processing approaches.

Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism.

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language, and cognitive deficits. A mouse model of this syndromic ASD (Mef2c-Het) recapitulates many of the MEF2C haploinsufficiency syndrome-linked behaviors, including communication deficits. We show here that Mef2c-Het mice of both sexes exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types; and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments.SIGNIFICANCE STATEMENT This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD-related behaviors, including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, subcellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.

Age-related central gain with degraded neural synchrony in the auditory brainstem of mice and humans.

Aging is associated with auditory nerve (AN) functional deficits and decreased inhibition in the central auditory system, amplifying central responses in a process referred to here as central gain. Although central gain increases response amplitudes, central gain may not restore disrupted response timing. In this translational study, we measured responses putatively generated by the AN and auditory midbrain in younger and older mice and humans. We hypothesized that older mice and humans exhibit increased central gain without an improvement in inter-trial synchrony in the midbrain. Our data demonstrated greater age-related deficits in AN response amplitudes than auditory midbrain response amplitudes, as shown by significant interactions between inferred neural generator and age group, indicating increased central gain in auditory midbrain. However, synchrony decreases with age in both the AN and midbrain responses. These results reveal age-related increases in central gain without concomitant improvements in synchrony, consistent with those predictions based on decreases in inhibition. Persistent decreases in synchrony may contribute to auditory processing deficits in older mice and humans.

Two distinct types of nodes of Ranvier support auditory nerve function in the mouse cochlea.

The auditory nerve (AN) of the inner ear is the primary conveyor of acoustic information from sensory hair cells to the brainstem. Approximately 95% of peripheral AN fibers are myelinated by glial cells. The integrity of myelin and the glial-associated paranodal structures at the node of Ranvier is critical for normal AN activity and axonal survival and function in the central auditory nervous system. However, little is known about the node of Ranvier's spatiotemporal development in the AN, how the aging process (or injury) affects the activity of myelinating glial cells, and how downstream alterations in myelin and paranodal structure contribute to AN degeneration and sensorineural hearing loss. Here, we characterized two types of Ranvier nodes-the axonal node and the ganglion node-in the mouse peripheral AN, and found that they are distinct in several features of postnatal myelination and age-related degeneration. Cellular, molecular, and structure-function correlations revealed that the two node types are each critical for different aspects of peripheral AN function. Neural processing speed and synchrony is associated with the length of the axonal node, while stimulus level-dependent amplitude growth and action potentials are associated with the ganglion node. Moreover, our data indicate that dysregulation of glial cells (e.g., satellite cells) and degeneration of the ganglion node structure are an important new mechanism of age-related hearing loss.

Cochlear Immune Response in Presbyacusis: a Focus on Dysregulation of Macrophage Activity.

Age-related hearing loss, or presbyacusis, is a prominent chronic degenerative disorder that affects many older people. Based on presbyacusis pathology, the degeneration occurs in both sensory and non-sensory cells, along with changes in the cochlear microenvironment. The progression of age-related neurodegenerative diseases is associated with an altered microenvironment that reflects chronic inflammatory signaling. Under these conditions, resident and recruited immune cells, such as microglia/macrophages, have aberrant activity that contributes to chronic neuroinflammation and neural cell degeneration. Recently, researchers identified and characterized macrophages in human cochleae (including those from older donors). Along with the age-related changes in cochlear macrophages in animal models, these studies revealed that macrophages, an underappreciated group of immune cells, may play a critical role in maintaining the functional integrity of the cochlea. Although several studies deciphered the molecular mechanisms that regulate microglia/macrophage dysfunction in multiple neurodegenerative diseases, limited studies have assessed the mechanisms underlying macrophage dysfunction in aged cochleae. In this review, we highlight the age-related changes in cochlear macrophage activities in mouse and human temporal bones. We focus on how complement dysregulation and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 inflammasome could affect macrophage activity in the aged peripheral auditory system. By understanding the molecular mechanisms that underlie these regulatory systems, we may uncover therapeutic strategies to treat presbyacusis and other forms of sensorineural hearing loss.

Translational and interdisciplinary insights into presbyacusis: A multidimensional disease.

There are multiple etiologies and phenotypes of age-related hearing loss or presbyacusis. In this review we summarize findings from animal and human studies of presbyacusis, including those that provide the theoretical framework for distinct metabolic, sensory, and neural presbyacusis phenotypes. A key finding in quiet-aged animals is a decline in the endocochlear potential (EP) that results in elevated pure-tone thresholds across frequencies with greater losses at higher frequencies. In contrast, sensory presbyacusis appears to derive, in part, from acute and cumulative effects on hair cells of a lifetime of environmental exposures (e.g., noise), which often result in pronounced high frequency hearing loss. These patterns of hearing loss in animals are recognizable in the human audiogram and can be classified into metabolic and sensory presbyacusis phenotypes, as well as a mixed metabolic+sensory phenotype. However, the audiogram does not fully characterize age-related changes in auditory function. Along with the effects of peripheral auditory system declines on the auditory nerve, primary degeneration in the spiral ganglion also appears to contribute to central auditory system aging. These inner ear alterations often correlate with structural and functional changes throughout the central nervous system and may explain suprathreshold speech communication difficulties in older adults with hearing loss. Throughout this review we highlight potential methods and research directions, with the goal of advancing our understanding, prevention, diagnosis, and treatment of presbyacusis.

A multi-metric approach to characterizing mouse peripheral auditory nerve function using the auditory brainstem response.

BACKGROUND: The auditory brainstem response (ABR), specifically wave I, is widely used to noninvasively measure auditory nerve (AN) function. Recent work in humans has introduced novel electrocochleographic measures to comprehensively characterize AN function that emphasize suprathreshold processing and estimate neural synchrony. NEW METHOD: This study establishes new tools for evaluating AN function in vivo in adult mice using tone-evoked ABRs obtained from young-adult CBA/CaJ mice, adapting the approach previously introduced in humans. Six metrics are obtained from ABR wave I at suprathreshold stimulus levels. RESULTS: Change-point analyses show that the metrics' rate of change with stimulus level differs between moderate and high suprathreshold levels, suggesting that this approach can potentially characterize the presence of heterogeneous AN fiber types. COMPARISON WITH EXISTING METHODS: Traditional ABR approaches focus on response thresholds and averaged amplitudes/latencies. In contrast, our multi-metric approach, which uses single-trial data and suprathreshold stimuli, provides novel information and identifies evidence of neural synchrony deficits and changes in the heterogeneity of AN fibers underlying AN behavior. CONCLUSION: The techniques reported here provide a novel tool to assess changes in AN function in vivo in a commonly used animal model. A benchmark of most current hearing research is the transition from animal to human studies. Here we established a translational objective approach, applying methods that were first developed in humans to animals. This approach enables researchers to identify changes in AN function arising from the animal models with well-characterized pathology, and predict similar pathological changes in human AN dysfunction and hearing loss.

Neurotrophin gene therapy to promote survival of spiral ganglion neurons after deafness.

Hearing impairment is a major health and economic concern worldwide. Currently, the cochlear implant (CI) is the standard of care for remediation of severe to profound hearing loss, and in general, contemporary CIs are highly successful. But there is great variability in outcomes among individuals, especially in children, with many CI users deriving much less or even marginal benefit. Much of this variability is related to differences in auditory nerve survival, and there has been substantial interest in recent years in exploring potential therapies to improve survival of the cochlear spiral ganglion neurons (SGN) after deafness. Preclinical studies using osmotic pumps and other approaches in deafened animal models to deliver neurotrophic factors (NTs) directly to the cochlea have shown promising results, especially with Brain-Derived Neurotrophic Factor (BDNF). More recent studies have focused on the use of NT gene therapy to force expression of NTs by target cells within the cochlea. This could provide the means for a one-time treatment to promote long-term NT expression and improve neural survival after deafness. This review summarizes the evidence for the efficacy of exogenous NTs in preventing SGN degeneration after hearing loss and reviews the animal research to date suggesting that NT gene therapy can elicit long-term NT expression in the cochlea, resulting in significantly improved SGN and radial nerve fiber survival after deafness. In addition, we discuss NT gene therapy in other non-auditory applications and consider some of the remaining issues with regard to selecting optimal vectors, timing of treatment, and place/method of delivery, etc. that must be resolved prior to considering clinical application.

Age-Related Changes in Immune Cells of the Human Cochlea.

Age-related hearing loss is a chronic degenerative disorder affecting one in two individuals above the age of 75. Current population projections predict a steady climb in the number of older individuals making the search for interventions to prevent or reverse this disorder even more critical. There is growing acceptance that aberrant activity of resident or infiltrating immune cells, such as macrophages, is a major factor contributing to the onset and progression of age-related degenerative diseases. However, how macrophage populations and their functionally-driven morphological characteristics change with age in the human cochlea remains largely unknown. In this study, we employed immunohistochemical approaches along with confocal and super-resolution imaging, three-dimensional reconstructions, and quantitative analysis to determine age-related changes in macrophage numbers and morphology as well as interactions with other cell-types and structures of the auditory nerve and lateral wall in the human cochlea. In the cochlea of human ears from young and middle aged adults those macrophages in the auditory nerve assumed a worm-like structure in contrast to those in the spiral ligament or associated with the dense microvascular network in the stria vascularis which exhibited a highly ramified morphology. Macrophages in both the auditory nerve and cochlear lateral wall showed morphological alterations with age. The population of activated macrophages in the auditory nerve increased in cochleas obtained from older donors. Dual-immunohistochemical staining with macrophage, myelin, and neuronal markers revealed increased interactions of macrophages with the glial and neuronal components of the aged auditory nerve. These findings implicate the involvement of abnormal macrophage-glia interactions in age-related physiological and pathological alterations in the human cochlea. There is clearly a need to further investigate the contribution of macrophage-associated inflammatory dysregulation in human presbyacusis.

Age-dependent alterations of Kir4.1 expression in neural crest-derived cells of the mouse and human cochlea.

Age-related hearing loss (or presbyacusis) is a progressive pathophysiological process. This study addressed the hypothesis that degeneration/dysfunction of multiple nonsensory cell types contributes to presbyacusis by evaluating tissues obtained from young and aged CBA/CaJ mouse ears and human temporal bones. Ultrastructural examination and transcriptomic analysis of mouse cochleas revealed age-dependent pathophysiological alterations in 3 types of neural crest-derived cells, namely intermediate cells in the stria vascularis, outer sulcus cells in the cochlear lateral wall, and satellite cells in the spiral ganglion. A significant decline in immunoreactivity for Kir4.1, an inwardly rectifying potassium channel, was seen in strial intermediate cells and outer sulcus cells in the ears of older mice. Age-dependent alterations in Kir4.1 immunostaining also were observed in satellite cells ensheathing spiral ganglion neurons. Expression alterations of Kir4.1 were observed in these same cell populations in the aged human cochlea. These results suggest that degeneration/dysfunction of neural crest-derived cells maybe an important contributing factor to both metabolic and neural forms of presbyacusis.

Use of Proteomic Imaging Coupled With Transcriptomic Analysis to Identify Biomolecules Responsive to Cochlear Injury.

Exposure to noise or ototoxic agents can result in degeneration of cells in the sensory epithelium and auditory nerve, as well as non-sensory cells of the cochlear lateral wall. However, the molecular mechanisms underlying this pathology remain unclear. The purpose of this study was to localize and identify proteins in the cochlea that are responsive to noise or ototoxic exposure using a complementary proteo-transcriptomic approach. MALDI imaging of cochlear sections revealed numerous protein signals with distinct cochlear localization patterns in both cochlear injury models, of which six were chosen for further investigation. A query of proteomic databases identified 709 candidates corresponding to m/z values for the six proteins. An evaluation of mRNA expression data from our previous studies of these injured models indicated that 208 of the candidates were affected in both injury models. Downstream validation analyses yielded proteins with confirmatory distributions and responses to injury. The combined analysis of MALDI imaging with gene expression data provides a new strategy to identify molecular regulators responsive to cochlear injury. This study demonstrates the applicability of MALDI imaging for investigating protein localization and abundance in frozen sections from animals modeling cochlear pathology.

Noise-Induced Dysregulation of Quaking RNA Binding Proteins Contributes to Auditory Nerve Demyelination and Hearing Loss.

Noise exposure causes auditory nerve (AN) degeneration and hearing deficiency, though the proximal biological consequences are not entirely understood. Most AN fibers and spiral ganglion neurons are ensheathed by myelinating glia that provide insulation and ensure rapid transmission of nerve impulses from the cochlea to the brain. Here we show that noise exposure administered to mice of either sex rapidly affects myelinating glial cells, causing molecular and cellular consequences that precede nerve degeneration. This response is characterized by demyelination, inflammation, and widespread expression changes in myelin-related genes, including the RNA splicing regulator Quaking (QKI) and numerous QKI target genes. Analysis of mice deficient in QKI revealed that QKI production in cochlear glial cells is essential for proper myelination of spiral ganglion neurons and AN fibers, and for normal hearing. Our findings implicate QKI dysregulation as a critical early component in the noise response, influencing cochlear glia function that leads to AN demyelination and, ultimately, to hearing deficiency.SIGNIFICANCE STATEMENT Auditory glia cells ensheath a majority of spiral ganglion neurons with myelin, protect auditory neurons, and allow for fast conduction of electrical impulses along the auditory nerve. Here we show that noise exposure causes glial dysfunction leading to myelin abnormality and altered expression of numerous genes in the auditory nerve, including QKI, a gene implicated in regulating myelination. Study of a conditional mouse model that specifically depleted QKI in glia showed that QKI deficiency alone was sufficient to elicit myelin-related abnormality and auditory functional declines. These results establish QKI as a key molecular target in the noise response and a causative agent in hearing loss.

Macrophage-Mediated Glial Cell Elimination in the Postnatal Mouse Cochlea.

Hearing relies on the transmission of auditory information from sensory hair cells (HCs) to the brain through the auditory nerve. This relay of information requires HCs to be innervated by spiral ganglion neurons (SGNs) in an exclusive manner and SGNs to be ensheathed by myelinating and non-myelinating glial cells. In the developing auditory nerve, mistargeted SGN axons are retracted or pruned and excessive cells are cleared in a process referred to as nerve refinement. Whether auditory glial cells are eliminated during auditory nerve refinement is unknown. Using early postnatal mice of either sex, we show that glial cell numbers decrease after the first postnatal week, corresponding temporally with nerve refinement in the developing auditory nerve. Additionally, expression of immune-related genes was upregulated and macrophage numbers increase in a manner coinciding with the reduction of glial cell numbers. Transient depletion of macrophages during early auditory nerve development, using transgenic CD11bDTR/EGFP mice, resulted in the appearance of excessive glial cells. Macrophage depletion caused abnormalities in myelin formation and transient edema of the stria vascularis. Macrophage-depleted mice also showed auditory function impairment that partially recovered in adulthood. These findings demonstrate that macrophages contribute to the regulation of glial cell number during postnatal development of the cochlea and that glial cells play a critical role in hearing onset and auditory nerve maturation.

Contributions of Mouse and Human Hematopoietic Cells to Remodeling of the Adult Auditory Nerve After Neuron Loss.

The peripheral auditory nerve (AN) carries sound information from sensory hair cells to the brain. The present study investigated the contribution of mouse and human hematopoietic stem cells (HSCs) to cellular diversity in the AN following the destruction of neuron cell bodies, also known as spiral ganglion neurons (SGNs). Exposure of the adult mouse cochlea to ouabain selectively killed type I SGNs and disrupted the blood-labyrinth barrier. This procedure also resulted in the upregulation of genes associated with hematopoietic cell homing and differentiation, and provided an environment conducive to the tissue engraftment of circulating stem/progenitor cells into the AN. Experiments were performed using both a mouse-mouse bone marrow transplantation model and a severely immune-incompetent mouse model transplanted with human CD34+ cord blood cells. Quantitative immunohistochemical analysis of recipient mice demonstrated that ouabain injury promoted an increase in the number of both HSC-derived macrophages and HSC-derived nonmacrophages in the AN. Although rare, a few HSC-derived cells in the injured AN exhibited glial-like qualities. These results suggest that human hematopoietic cells participate in remodeling of the AN after neuron cell body loss and that hematopoietic cells can be an important resource for promoting AN repair/regeneration in the adult inner ear.

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve.

The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration.

Sox10 expressing cells in the lateral wall of the aged mouse and human cochlea.

Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1-3 month-old) and aged (2-2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10(+) cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10(+) marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10(+) cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10(+) cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10(+) cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear.

Heptanol application to the mouse round window: a model for studying cochlear lateral wall regeneration.

OBJECTIVE: Identify cells supporting cochlear lateral wall regeneration. STUDY DESIGN: Prospective controlled trial. SETTING: Laboratory. Human presbyacusis occurs, in part, secondary to age-related degeneration of cochlear lateral wall structures such as the stria vascularis and spiral ligament fibrocytes. This degeneration is likely linked to the diminished regenerative capacity of lateral wall cells with age. While lateral wall regeneration is known to occur after an acute insult, this process remains poorly understood and the cells capable of self-replication unidentified. We hypothesized that spiral ligament fibrocytes constitute these proliferative cells. SUBJECTS AND METHODS: To test the hypothesis, an acute ototoxic insult was created in 65 normal-hearing, young adult mice via cochlear exposure to heptanol. Sacrifice occurred at 1 to 60 days posttreatment. Auditory brainstem responses, 5-ethynyl-2'-deoxyuridine assay, and immunostaining were used to assess regeneration. RESULTS: Posttreatment hearing thresholds were elevated in nearly all treated mice. Selective fibrocyte apoptosis and strial injury were observed at the time of peak hearing loss around 1 to 7 days posttreatment. Cellular proliferation was detected in the region of type II fibrocytes during this time. Hearing thresholds plateaued at 7 days posttreatment followed by a significant recovery of both hearing and morphologic appearance. Permanent outer hair cell degeneration was observed. CONCLUSIONS: Heptanol application to the round window of young adult mice is a rapid, selective, and reliable technique for investigating proliferation in the cochlear lateral wall. The data indirectly showed that spiral ligament fibrocytes may be the proliferative cells of the cochlear lateral wall. Further studies of this process are needed.