The sensory organs of the inner ear possess resident populations of macrophages, but the function of those cells is poorly understood. In many tissues, macrophages participate in the removal of cellular debris after injury and can also promote tissue repair. The present study examined injury-evoked macrophage activity in the mouse utricle. Experiments used transgenic mice in which the gene for the human diphtheria toxin receptor (huDTR) was inserted under regulation of the Pou4f3 promoter. Hair cells in such mice can be selectively lesioned by systemic treatment with diphtheria toxin (DT). In order to visualize macrophages, Pou4f3-huDTR mice were crossed with a second transgenic line, in which one or both copies of the gene for the fractalkine receptor CX3CR1 were replaced with a gene for GFP. Such mice expressed GFP in all macrophages, and mice that were CX3CR1(GFP/GFP) lacked the necessary receptor for fractalkine signaling. Treatment with DT resulted in the death of ∼70% of utricular hair cells within 7 days, which was accompanied by increased numbers of macrophages within the utricular sensory epithelium. Many of these macrophages appeared to be actively engulfing hair cell debris, indicating that macrophages participate in the process of 'corpse removal' in the mammalian vestibular organs. However, we observed no apparent differences in injury-evoked macrophage numbers in the utricles of CX3CR1(+/GFP) mice vs. CX3CR1(GFP/GFP) mice, suggesting that fractalkine signaling is not necessary for macrophage recruitment in these sensory organs. Finally, we found that repair of sensory epithelia at short times after DT-induced hair cell lesions was mediated by relatively thin cables of F-actin. After 56 days recovery, however, all cell-cell junctions were characterized by very thick actin cables.

We applied a micro-cDNA-based subtraction method to identify genes expressed in the regenerating sensory epithelia (SE) of the chicken inner ear. Sensory hair cells in the avian utricle SE are in a constant state of turnover, where dying hair cells are replaced by new ones derived from supporting cells. In contrast, hair cells in the cochlea remain quiescent unless damaged. We used this difference to enrich for utricle-specific genes, using reiterative cDNA subtraction and demonstrate enrichment for utricle-specific sequences. A total of 1710 cDNA sequence reads revealed the presence of many cDNAs encoding known structural components of the SE (for example, Harmonin and beta-tectorin), proteins involved in cellular proliferation, such as P311, HIPK2, and SPALT1, among many others of unknown function. These libraries are the first of their kind and should prove useful for the discovery of candidate genes for hearing disorders, regenerative and apoptotic pathways, and novel chicken ESTs.

Hair cells in the auditory organ of the vertebrate inner ear are the sensory receptors that convert acoustic stimuli into electrical signals that are conveyed along the auditory nerve to the brainstem. Hair cells are highly susceptible to ototoxic drugs, infection, and acoustic trauma, which can cause cellular degeneration. In mammals, hair cells that are lost after damage are not replaced, leading to permanent hearing impairments. By contrast, supporting cells in birds and other non-mammalian vertebrates regenerate hair cells after damage, which restores hearing function. The cellular mechanisms that regulate hair cell regeneration are not well understood. We investigated the role of vascular endothelial growth factor (VEGF) during regeneration of auditory hair cells in chickens after ototoxic injury. Using RNA-Seq, immunolabeling, and in situ hybridization, we found that VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 were expressed in the auditory epithelium, with VEGFA expressed in hair cells and VEGFR1 and VEGFR2 expressed in supporting cells. Using organotypic cultures of the chicken cochlear duct, we found that blocking VEGF receptor activity during hair cell injury reduced supporting cell proliferation as well as the numbers of regenerated hair cells. By contrast, addition of recombinant human VEGFA to organ cultures caused an increase in both supporting cell division and hair cell regeneration. VEGF's effects on supporting cells were preserved in isolated supporting cell cultures, indicating that VEGF can act directly upon supporting cells. These observations demonstrate a heretofore uncharacterized function for VEGF signaling as a critical positive regulator of hair cell regeneration in the avian inner ear.

The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 h. Consistent with previous work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed hyperpolarized mitochondrial membrane potentials (ΔΨm) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.

In amniotes, head movements are encoded by two types of vestibular hair cells (type I and type II) with unique morphology, physiology, and innervation. After hair cell destruction in mature rodents, supporting cells regenerate some type II hair cells, but no type I hair cells are replaced. The transcription factor Atoh1 is required for hair cell development, and Atoh1 is upregulated in supporting cells, the hair cell progenitors, in mature chickens and mice following hair cell damage. We investigated whether Atoh1 is required for type II hair cell regeneration in adult mice after genetic ablation of hair cells. First, we used a knock-in Atoh1 reporter to demonstrate that supporting cells in the utricle, a vestibular organ that detects linear acceleration of the head, upregulate Atoh1 expression by 7 days after hair cell destruction was initiated. Next, we labeled supporting cells prior to damage and fate-mapped them over time to test whether conditional deletion of Atoh1 from supporting cells prevented them from converting into hair cells after damage. In mice with normal Atoh1 expression, fate-mapped supporting cells in the adult utricle gave rise to hundreds of type II hair cells after hair cell destruction, but they did not form new type I hair cells. By contrast, mice with Atoh1 deletion prior to hair cell damage had only 10-20 fate-mapped type II hair cells per utricle at 3 weeks post-damage, and numbers did not change at 12 weeks after hair cell destruction. Supporting cells had normal cell shape and nuclear density up to 12 weeks after Atoh1 deletion. Similar observations were made in two other vestibular organs, the saccule and the lateral ampulla. Our findings demonstrate that Atoh1 is necessary in adult mouse supporting cells for regeneration of type II vestibular hair cells and that deletion of Atoh1 from supporting cells prior to damage does not appear to induce supporting cells to die or to proliferate.

The sensory organs of the inner ear contain resident populations of macrophages, which are recruited to sites of cellular injury. Such macrophages are known to phagocytose the debris of dying cells but the full role of macrophages in otic pathology is not understood. Lateral line neuromasts of zebrafish contain hair cells that are nearly identical to those in the inner ear, and the optical clarity of larval zebrafish permits direct imaging of cellular interactions. In this study, we used larval zebrafish to characterize the response of macrophages to ototoxic injury of lateral line hair cells. Macrophages migrated into neuromasts within 20 min of exposure to the ototoxic antibiotic neomycin. The number of macrophages in the near vicinity of injured neuromasts was similar to that observed near uninjured neuromasts, suggesting that this early inflammatory response was mediated by "local" macrophages. Upon entering injured neuromasts, macrophages actively phagocytosed hair cell debris. The injury-evoked migration of macrophages was significantly reduced by inhibition of Src-family kinases. Using chemical-genetic ablation of macrophages before the ototoxic injury, we also examined whether macrophages were essential for the initiation of hair cell regeneration. Results revealed only minor differences in hair cell recovery in macrophage-depleted vs. control fish, suggesting that macrophages are not essential for the regeneration of lateral line hair cells.

Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.

During the development of the inner ear, auditory and vestibular ganglion neurons are generated in a highly regulated sequential process. First, neuroblasts are specified, delaminate from the epithelium of the otocyst, and migrate to form the auditory-vestibular ganglion (AVG). These neuroblasts then undergo proliferation and differentiate into afferent neurons of the auditory and vestibular ganglia. The zinc finger transcription factor Gata3 has been shown to play a role in cell proliferation and differentiation in various regions of the inner ear. Here we profile the spatiotemporal expression pattern of Gata3 in the developing auditory and vestibular ganglia of the chick embryo. Gata3 is expressed in a distinct population of sensorineural precursor cells within the otic epithelium, but is not expressed in migrating or proliferating neuroblasts. Following terminal mitosis, Gata3 expression is restricted to very few cells in the auditory ganglion and is not expressed in any cells of the vestibular ganglion. Gata3 expression levels then increase in auditory neurons as they mature. The increase of Gata3 in auditory ganglion neurons is accompanied by decreased expression of NeuroD. Our results suggest that Gata3 may be specifically involved in the differentiation of auditory ganglion neurons.

Macrophages are the primary effector cells of the innate immune system and are also activated in response to tissue injury. The avian cochlea contains a population of resident macrophages, but the precise function of those cells is not known. The present study characterized the behavior of cochlear macrophages after aminoglycoside ototoxicity and also examined the possible role of macrophages in sensory regeneration. We found that the undamaged chick cochlea contains a large resting population of macrophages that reside in the hyaline cell region, immediately outside the abneural (inferior) border of the sensory epithelium. Following ototoxic injury, macrophages appear to migrate out of the hyaline cell region and towards the basilar membrane, congregating immediately below the lesioned sensory epithelium. In order to determine whether recruited macrophages contribute to the regeneration of sensory receptors, we quantified supporting cell proliferation and hair cell recovery after the elimination of most resident macrophages via application of liposomally-encapsulated clodronate. Examination of macrophage-depleted specimens at two days following ototoxic injury revealed no deficits in hair cell clearance, when compared to normal controls. In addition, we found that elimination of macrophages did not affect either regenerative proliferation of supporting cells or the production of replacement hair cells. However, we did find that macrophage-depleted cochleae contained reduced numbers of proliferative mesothelial cells below the basilar membrane. Our data suggest that macrophages are not required for normal debris clearance and regeneration, but that they may play a role in the maintenance of the basilar membrane.

We carried out an analysis of the expression of Prox1, a homeo-domain transcription factor, during mouse inner ear development with particular emphasis on the auditory system. Prox1 is expressed in the otocyst beginning at embryonic day (E)11, in the developing vestibular sensory patches. Expression is down regulated in maturing (myosin VIIA immunoreactive) vestibular hair cells and subsequently in the underlying support cell layer by E16.5. In the auditory sensory epithelium, Prox1 is initially expressed at embryonic day 14.5 in a narrow stripe of cells at the base of the cochlea. This stripe encompasses the full thickness of the sensory epithelium, including developing hair cells and support cells. Over the next several days the stripe of expression extends to the apex, and as the sensory epithelium differentiates Prox1 becomes restricted to a subset of support cells. Double labeling for Prox1 and cell-type-specific markers revealed that the outer hair cells transiently express Prox1. After E18, Prox1 protein is no longer detectable in hair cells, but it continues to be expressed in support cells for the rest of embryogenesis and into the second postnatal week. During this time, Prox1 is not expressed in all support cell types in the organ of Corti, but is restricted to developing Deiters' and pillar cells. The expression is maintained in these cells into the second week of postnatal life, at which time Prox1 is dynamically down regulated. These studies form a baseline from which we can analyze the role of Prox1 in vertebrate sensory development.

Sensory receptors in the vestibular organs of birds can regenerate after ototoxic injury. Notably, this regenerative process leads to the restoration of the correct patterning of hair cell phenotype and afferent innervation within the repaired sensory epithelium. The molecular signals that specify cell phenotype and regulate neuronal guidance during sensory regeneration are not known, but they are likely to be similar to the signals that direct these processes during embryonic development. The present study examined the recovery of hair cell phenotype during regeneration in the avian utricle, a vestibular organ that detects linear acceleration and head orientation. First, we show that Type I hair cells in the avian vestibular maculae are immunoreactive for the extracellular matrix molecule tenascin and that treatment with the ototoxic antibiotic streptomycin results in a nearly complete elimination of tenascin immunoreactivity. Cells that express tenascin begin to recover after about 2 weeks and are then contacted by calyx terminals of vestibular neurons. In addition, our previous work had shown that the zinc finger transcription factor GATA3 is uniquely expressed within the striolar reversal zone of the utricle (Hawkins et al. [2003] Hum Mol Genet 12:1261-1272), and we show here that this regionalized expression of GATA3 is maintained after severe hair cell lesions and after transplantation of the sensory epithelium onto a chemically defined substrate. In contrast, the expression of three other supporting cell markers--alpha- and beta-tectorin and SCA--is reduced following ototoxic injury. These observations suggest that GATA3 expression may maintain positional information in the maculae during sensory regeneration.

Loss of inner ear sensory hair cells (HC) is a leading cause of human hearing loss and balance disorders. Unlike mammals, many lower vertebrates can regenerate these cells. We used cross-species microarrays to examine this process in the avian inner ear. Specifically, changes in expression of over 1700 transcription factor (TF) genes were investigated in hair cells of auditory and vestibular organs following treatment with two different damaging agents and regeneration in vitro. Multiple components of seven distinct known signaling pathways were clearly identifiable: TGFbeta, PAX, NOTCH, WNT, NFKappaB, INSULIN/IGF1 and AP1. Numerous components of apoptotic and cell cycle control pathways were differentially expressed, including p27(KIP) and TFs that regulate its expression. A comparison of expression trends across tissues and treatments revealed identical patterns of expression that occurred at identical times during regenerative proliferation. Network analysis of the patterns of gene expression in this large dataset also revealed the additional presence of many components (and possible network interactions) of estrogen receptor signaling, circadian rhythm genes and parts of the polycomb complex (among others). Equal numbers of differentially expressed genes were identified that have not yet been placed into any known pathway. Specific time points and tissues also exhibited interesting differences: For example, 45 zinc finger genes were specifically up-regulated at later stages of cochlear regeneration. These results are the first of their kind and should provide the starting point for more detailed investigations of the role of these many pathways in HC recovery, and for a description of their possible interactions.

The cochlea and the vestibular organs are populated by resident macrophages, but their role in inner ear maintenance and pathology is not entirely clear. Resident macrophages in other organs are responsible for phagocytosis of injured or infected cells, and it is likely that macrophages in the inner ear serve a similar role. Hair cell injury causes macrophages to accumulate within proximity of damaged regions of the inner ear, either by exiting the vasculature and entering the labyrinth or by the resident macrophages reorganizing themselves through local movement to the areas of injury. Direct evidence for macrophage engulfment of apoptotic hair cells has been observed in several conditions. Here, we review evidence for phagocytosis of damaged hair cells in the sensory epithelium by tissue macrophages in the published literature and in some new experiments that are presented here as original work. Several studies also suggest that macrophages are not the only phaogocytic cells in the inner ear, but that supporting cells of the sensory epithelium also play an important role in debris clearance. We describe the various ways in which the sensory epithelia of the inner ear are adapted to eliminate damaged and dying cells. A collaborative effort between resident and migratory macrophages as well as neighboring supporting cells results in the rapid and efficient clearance of cellular debris, even in cases where hair cell loss is rapid and complete.

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin-1β, when compared to mice with intact CX3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX3 CR1 may contribute to inflammation-mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long-term survival of target-deprived afferent neurons.

Aging, disease, and trauma can lead to loss of vestibular hair cells and permanent vestibular dysfunction. Previous work showed that, following acute destruction of ∼95% of vestibular hair cells in adult mice, ∼20% regenerate naturally (without exogenous factors) through supporting cell transdifferentiation. There is, however, no evidence for the recovery of vestibular function. To gain insight into the lack of functional recovery, we assessed functional differentiation in regenerated hair cells for up to 15 months, focusing on key stages in stimulus transduction and transmission: hair bundles, voltage-gated conductances, and synaptic contacts. Regenerated hair cells had many features of mature type II vestibular hair cells, including polarized mechanosensitive hair bundles with zone-appropriate stereocilia heights, large voltage-gated potassium currents, basolateral processes, and afferent and efferent synapses. Regeneration failed, however, to recapture the full range of properties of normal populations, and many regenerated hair cells had some properties of immature hair cells, including small transduction currents, voltage-gated sodium currents, and small or absent HCN (hyperpolarization-activated cyclic nucleotide-gated) currents. Furthermore, although mouse vestibular epithelia normally have slightly more type I hair cells than type II hair cells, regenerated hair cells acquired neither the low-voltage-activated potassium channels nor the afferent synaptic calyces that distinguish mature type I hair cells from type II hair cells and confer distinctive physiology. Thus, natural regeneration of vestibular hair cells in adult mice is limited in total cell number, cell type diversity, and extent of cellular differentiation, suggesting that manipulations are needed to promote full regeneration with the potential for recovery of vestibular function.SIGNIFICANCE STATEMENT Death of inner ear hair cells in adult mammals causes permanent loss of hearing and balance. In adult mice, the sudden death of most vestibular hair cells stimulates the production of new hair cells but does not restore balance. We investigated whether the lack of systems-level function reflects functional deficiencies in the regenerated hair cells. The regenerated population acquired mechanosensitivity, voltage-gated channels, and afferent synapses, but did not reproduce the full range of hair cell types. Notably, no regenerated cells acquired the distinctive properties of type I hair cells, a major functional class in amniote vestibular organs. To recover vestibular system function in adults, we may need to solve how to regenerate the normal variety of mature hair cells.

The Hippo signaling pathway is a key regulator of tissue development and regeneration. Activation of the Hippo pathway leads to nuclear translocation of the YAP1 transcriptional coactivator, resulting in changes in gene expression and cell cycle entry. Recent studies have demonstrated the nuclear translocation of YAP1 during the development of the sensory organs of the inner ear, but the possible role of YAP1 in sensory regeneration of the inner ear is unclear. The present study characterized the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after HC injury. During neonatal development, YAP1 expression was observed in the cytoplasm of supporting cells, and was transiently expressed in the cytoplasm of some differentiating hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle after short periods in organotypic culture. However, little or no nuclear translocation of YAP1 was observed in the utricles of neonatal or mature mice after ototoxic injury. In contrast, substantial YAP1 nuclear translocation was observed in the chicken utricle after streptomycin treatment in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may partially account for the differing regenerative abilities of the avian vs. mammalian inner ear.

Resident cochlear macrophages rapidly migrate into the inner hair cell synaptic region and directly contact the damaged synaptic connections after noise-induced synaptopathy. Eventually, such damaged synapses are spontaneously repaired, but the precise role of macrophages in synaptic degeneration and repair remains unknown. To address this, cochlear macrophages were eliminated using colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Sustained treatment with PLX5622 in CX3CR1 GFP/+ mice of both sexes led to robust elimination of resident macrophages (∼94%) without significant adverse effects on peripheral leukocytes, cochlear function, and structure. At 1 day (d) post noise exposure of 93 or 90 dB SPL for 2 hours, the degree of hearing loss and synapse loss were comparable in the presence and absence of macrophages. At 30 d after exposure, damaged synapses appeared repaired in the presence of macrophages. However, in the absence of macrophages, such synaptic repair was significantly reduced. Remarkably, on cessation of PLX5622 treatment, macrophages repopulated the cochlea, leading to enhanced synaptic repair. Elevated auditory brainstem response thresholds and reduced auditory brainstem response Peak 1 amplitudes showed limited recovery in the absence of macrophages but recovered similarly with resident and repopulated macrophages. Cochlear neuron loss was augmented in the absence of macrophages but showed preservation with resident and repopulated macrophages after noise exposure. While the central auditory effects of PLX5622 treatment and microglia depletion remain to be investigated, these data demonstrate that macrophages do not affect synaptic degeneration but are necessary and sufficient to restore cochlear synapses and function after noise-induced synaptopathy.SIGNIFICANCE STATEMENT The synaptic connections between cochlear inner hair cells and spiral ganglion neurons can be lost because of noise over exposure or biological aging. This loss may represent the most common causes of sensorineural hearing loss also known as hidden hearing loss. Synaptic loss results in degradation of auditory information, leading to difficulty in listening in noisy environments and other auditory perceptual disorders. We demonstrate that resident macrophages of the cochlea are necessary and sufficient to restore synapses and function following synaptopathic noise exposure. Our work reveals a novel role for innate-immune cells, such as macrophages in synaptic repair, that could be harnessed to regenerate lost ribbon synapses in noise- or age-linked cochlear synaptopathy, hidden hearing loss, and associated perceptual anomalies.

Cisplatin is a widely used and highly effective anti-cancer drug with significant side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced ototoxicity and nephrotoxicity, we used PLX3397, an FDA-approved inhibitor of the colony-stimulating factor 1 receptor (CSF1R), to eliminate tissue-resident macrophages during the course of cisplatin administration. Mice treated with cisplatin alone (cisplatin/vehicle) had significant hearing loss (ototoxicity) as well as kidney injury (nephrotoxicity). Macrophage ablation using PLX3397 resulted in significantly reduced hearing loss measured by auditory brainstem responses (ABR) and distortion-product otoacoustic emissions (DPOAE). Sensory hair cells in the cochlea were protected against cisplatin-induced death in mice treated with PLX3397. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis as well as reduced plasma blood urea nitrogen (BUN) and neutrophil gelatinase-associated lipocalin (NGAL) levels. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together our data indicate that ablation of tissue-resident macrophages represents a novel strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

Permanent hearing loss is often a result of damage to cochlear hair cells, which mammals are unable to regenerate. Non-mammalian vertebrates such as birds replace damaged hair cells and restore hearing function, but mechanisms controlling regeneration are not understood. The secreted protein bone morphogenetic protein 4 (BMP4) regulates inner ear morphogenesis and hair cell development. To investigate mechanisms controlling hair cell regeneration in birds, we examined expression and function of BMP4 in the auditory epithelia (basilar papillae) of chickens of either sex after hair cell destruction by ototoxic antibiotics. In mature basilar papillae, BMP4 mRNA is highly expressed in hair cells, but not in hair cell progenitors (supporting cells). Supporting cells transcribe genes encoding receptors for BMP4 (BMPR1A, BMPR1B, and BMPR2) and effectors of BMP4 signaling (ID transcription factors). Following hair cell destruction, BMP4 transcripts are lost from the sensory epithelium. Using organotypic cultures, we demonstrate that treatments with BMP4 during hair cell destruction prevent supporting cells from upregulating expression of the pro-hair cell transcription factor ATOH1, entering the cell cycle, and fully transdifferentiating into hair cells, but they do not induce cell death. By contrast, noggin, a BMP4 inhibitor, increases numbers of regenerated hair cells. These findings demonstrate that BMP4 antagonizes hair cell regeneration in the chicken basilar papilla, at least in part by preventing accumulation of ATOH1 in hair cell precursors.

Early postnatal mouse cochlear cultures were treated with a small panel of kinase inhibitors to elucidate the mechanisms underlying the maintenance of hair-bundle structure in the developing inner ear. At low concentrations (1-10 nM), staurosporine causes the collapse and loss of hair bundles without provoking hair-cell death, as judged by lack of terminal transferase dUTP nick end labeling (TUNEL) labeling or reactivity to anti-activated caspase-3. Staurosporine exposure results in the fusion of the hair bundle's stereocilia, a resorption of the parallel actin bundles of the stereocilia into the cytoplasm of the hair cell, a detachment of the apical, non-stereociliary membrane of the hair cell from the underlying cuticular plate, and a severing of the hair-bundle's rootlets from the actin cores of the stereocilia. It does not block membrane retrieval at the apical pole of the hair cells, nor does it elicit the externalization of phosphatidylserine. Staurosporine treatment causes a reduction in levels of the phosphorylated forms of ezrin, radixin, and moesin in cochlear cultures during the period of hair-bundle loss, indicating the integrity of the hair bundle may be actively maintained by the phosphorylation status of these proteins.