RESUMO
Foxg1 plays important roles in regeneration of hair cell (HC) in the cochlea of neonatal mouse. Here, we used Sox9-CreER to knock down Foxg1 in supporting cells (SCs) in the utricle in order to investigate the role of Foxg1 in HC regeneration in the utricle. We found Sox9 an ideal marker of utricle SCs and bred Sox9CreER/+Foxg1loxp/loxp mice to conditionally knock down Foxg1 in utricular SCs. Conditional knockdown (cKD) of Foxg1 in SCs at postnatal day one (P01) led to increased number of HCs at P08. These regenerated HCs had normal characteristics, and could survive to at least P30. Lineage tracing showed that a significant portion of newly regenerated HCs originated from SCs in Foxg1 cKD mice compared to the mice subjected to the same treatment, which suggested SCs trans-differentiate into HCs in the Foxg1 cKD mouse utricle. After neomycin treatment in vitro, more HCs were observed in Foxg1 cKD mice utricle compared to the control group. Together, these results suggest that Foxg1 cKD in utricular SCs may promote HC regeneration by inducing trans-differentiation of SCs. This research therefore provides theoretical basis for the effects of Foxg1 in trans-differentiation of SCs and regeneration of HCs in the mouse utricle.
Assuntos
Transdiferenciação Celular , Fatores de Transcrição Forkhead/deficiência , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/metabolismo , Proteínas do Tecido Nervoso/deficiência , Fatores de Transcrição SOX9/metabolismo , Sáculo e Utrículo/metabolismo , Animais , Animais Recém-Nascidos , Linhagem da Célula , Proliferação de Células , Feminino , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/patologia , Masculino , Camundongos Knockout , Neomicina/toxicidade , Proteínas do Tecido Nervoso/genética , Ototoxicidade , Fenótipo , Fatores de Transcrição SOX9/genética , Sáculo e Utrículo/efeitos dos fármacos , Sáculo e Utrículo/patologia , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas Aviárias/metabolismo , Proliferação de Células , Células Ciliadas Auditivas Internas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regeneração , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Proteínas Aviárias/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galinhas , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Mecanotransdução Celular , Regeneração/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Comunicação Celular , Proliferação de Células , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/metabolismo , Regeneração , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transdiferenciação Celular , Células Ciliadas Auditivas/patologia , Movimentos da Cabeça , Células Labirínticas de Suporte/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
In mammals, the cochlear sensory epithelium becomes quiescent early during development. After the first postnatal week, there is no cell replacement or proliferation, and severe damage leads to permanent deafness. Supporting cells' trans-differentiation has been suggested as a way to regenerate cochlear hair cells after damage. However, they are also needed for proper functionality. Cdkn1b (p27Kip1) participates in the cochlear terminal mitosis state achieved during development. Its expression is maintained in adult supporting cells and its postnatal deletion has induced cochlear proliferation in vitro and in vivo. Therefore, its manipulation has been proposed as a feasible way to induce proliferation of supporting cells after birth. Nevertheless, the literature is scarce regarding feasible methods to directly decrease p27Kip1 in the clinical domain. The effects of p27Kip1 knockdown using viral vectors are not completely elucidated and no pharmacological approaches to decrease p27Kip1 in the cochlea have been tested in vivo before. This study explores the ability of p27Kip1 messenger knockdown and pharmacological transcriptional inhibition to induce proliferation of supporting cells in the P0 neonatal rat cochlea in vivo. Respectively, lentiviral vectors transducing shRNA against p27Kip1 were administered into the scala media or Alsterpaullone 2-Cyanoethyl into the round window niche. Cell markers and gene expression were assessed through immunostaining and qRT-PCR. Despite both methods significantly decreasing p27Kip1 expression in vivo, signs of toxicity in the organ of Corti were not found; however, relevant proliferation was not found either. Finally, cochlear damage was added to increase the response in vitro, achieving only a mild to moderate proliferation induction. We conclude that our approaches were not able to stimulate the recall of supporting cell proliferation despite significantly decreased p27Kip1 levels in vivo. Considering the evaluation of the cochlea at a very responsive stage, we propose that the level of isolated modification of p27Kip1 expression in living mammals achievable through these approaches is insufficient to induce proliferation of supporting cells. Future proliferation induction experiments in the cochlea should study other methods and genes.
Assuntos
Proliferação de Células , Cóclea/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Labirínticas de Suporte/metabolismo , Animais , Animais Recém-Nascidos , Benzazepinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/patologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação para Baixo , Indóis/farmacologia , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
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.
Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Células Labirínticas de Suporte/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Animais , Antibacterianos/toxicidade , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/agonistas , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/farmacologia , Comunicação Celular/efeitos dos fármacos , Transdiferenciação Celular , Galinhas , Feminino , Gentamicinas/toxicidade , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Paraquat (PQ), one of the most widely used herbicides, is extremely dangerous because it generates the highly toxic superoxide radical. When paraquat was applied to cochlear organotypic cultures, it not only damaged the outer hair cells (OHCs) and inner hair cells (IHCs), but also caused dislocation of the hair cell rows. We hypothesized that the dislocation arose from damage to the support cells (SCs) that anchors hair cells within the epithelium. To test this hypothesis, rat postnatal cochlear cultures were treated with PQ. Shortly after PQ treatment, the rows of OHCs separated from one another and migrated radially away from IHCs suggesting loss of cell-cell adhesion that hold the hair cells in proper alignment. Hair cells dislocation was associated with extensive loss of SCs in the organ of Corti, loss of tympanic border cells (TBCs) beneath the basilar membrane, the early appearance of superoxide staining and caspase-8 labeling in SCs below the OHCs and disintegration of E-cadherin and ß-catenin in the organ of Corti. Damage to the TBCs and SCs occurred prior to loss of OHC or IHC loss suggesting a form of detachment-induced apoptosis referred to as anoikis.
Assuntos
Anoikis/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Herbicidas/toxicidade , Células Labirínticas de Suporte/efeitos dos fármacos , Paraquat/toxicidade , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Caspase 8/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cóclea/metabolismo , Cóclea/patologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos , beta Catenina/metabolismoRESUMO
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.
Assuntos
Apoptose , Orelha Interna/patologia , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/patologia , Macrófagos/patologia , Fagocitose , Animais , Movimento Celular , Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Humanos , Células Labirínticas de Suporte/metabolismo , Macrófagos/metabolismo , Camundongos , Modelos Animais , Fenótipo , Transdução de Sinais , Fatores de TempoRESUMO
Ear is a sensitive organ involved in hearing and balance function. The complex signaling network in the auditory system plays a crucial role in maintaining normal physiological function of the ear. The inner ear comprises a variety of host signaling pathways working in synergy to deliver clear sensory messages. Any disruption, as minor as it can be, has the potential to affect this finely tuned system with temporary or permanent sequelae including vestibular deficits and hearing loss. Mutations linked to auditory symptoms, whether inherited or acquired, are being actively researched for ways to reverse, silence, or suppress them. In this article, we discuss recent advancements in understanding the pathways involved in auditory system signaling, from hair cell development through transmission to cortical centers. Our review discusses Notch and Wnt signaling, cell to cell communication through connexin and pannexin channels, and the detrimental effects of reactive oxygen species on the auditory system. There has been an increased interest in the auditory community to explore the signaling system in the ear for hair cell regeneration. Understanding signaling pathways in the auditory system will pave the way for the novel avenues to regenerate sensory hair cells and restore hearing function. J. Cell. Physiol. 232: 2710-2721, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Ciliadas Auditivas/metabolismo , Audição , Receptores Notch/metabolismo , Regeneração , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Vias Auditivas/metabolismo , Vias Auditivas/patologia , Conexinas/metabolismo , Células Ciliadas Auditivas/patologia , Humanos , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , NADPH Oxidases/metabolismo , FenótipoRESUMO
From our permanent collection of plastic-embedded flat preparations of chinchilla cochleae, 22 controls and 199 ears from noise-exposed animals were used to determine when, postexposure, hair cell (HC) and supporting cell (SC) degeneration were completed. The exposed ears were divided into four groups based on exposure parameters: 0.5- or 4-kHz octave band of noise at moderate (M) or high (H) intensities. Postexposure survival ranged from <1 h to 2.5 y. Ears fixed ≤ 0-12 h postexposure were called 'acute'. For 'chronic' ears, postexposure survival was ≥7 d for groups 0.5M and 4M, ≥ 1 mo for the 4H group and ≥7 mo for the 0.5H group. The time course of inner-ear degeneration after noise exposure was determined from data in the 0.5H and 4H groups because these groups contained ears with intermediate survival times. Outer hair cells (OHCs) began dying during the exposure. OHC loss slowed down beyond 1 mo but was still present. Conversely, much inner hair cell loss was delayed until 1-3 wk postexposure. Outer pillar and inner pillar losses were present at a low level in acute ears but increased exponentially thereafter. These results are the first to demonstrate quantitatively that hair cells (HCs) and supporting cells (SCs) may continue to degenerate for months postexposure. With short postexposure survivals, the remaining SCs often had pathological changes, including: buckled pillar bodies, shifted Deiters' cell (DC) nuclei, detachment of DCs from the basilar membrane and/or splitting of the reticular lamina. These pathological changes appeared to allow endolymph and perilymph to intermix in the fluid spaces of the organ of Corti, damaging additional HCs, SCs and nerve fibers. This mechanism may account for some postexposure degeneration. In ears exposed to moderate noise, some of these SC changes appeared to be reversible. In ears exposed to high-level noise, these changes appeared to indicate impending degeneration.
Assuntos
Perda Auditiva Provocada por Ruído/patologia , Ruído/efeitos adversos , Órgão Espiral/patologia , Estimulação Acústica , Animais , Morte Celular , Chinchila , Modelos Animais de Doenças , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/patologia , Perda Auditiva Provocada por Ruído/etiologia , Células Labirínticas de Suporte/patologia , Degeneração Neural , Fatores de TempoRESUMO
Noise-induced hearing loss (NIHL) affects a large number of military personnel and civilians. Regenerating inner-ear cochlear hair cells (HCs) is a promising strategy to restore hearing after NIHL. In this review, we first summarize recent transcriptome profile analysis of zebrafish lateral lines and chick utricles where spontaneous HC regeneration occurs after HC damage. We then discuss recent studies in other mammalian regenerative systems such as pancreas, heart and central nervous system. Both spontaneous and forced HC regeneration occurs in mammalian cochleae in vivo involving proliferation and direct lineage conversion. However, both processes are inefficient and incomplete, and decline with age. For direct lineage conversion in vivo in cochleae and in other systems, further improvement requires multiple factors, including transcription, epigenetic and trophic factors, with appropriate stoichiometry in appropriate architectural niche. Increasing evidence from other systems indicates that the molecular paths of direct lineage conversion may be different from those of normal developmental lineages. We therefore hypothesize that HC regeneration does not have to follow HC development and that epigenetic memory of supporting cells influences the HC regeneration, which may be a key to successful cochlear HC regeneration. Finally, we discuss recent efforts in viral gene therapy and drug discovery for HC regeneration. We hope that combination therapy targeting multiple factors and epigenetic signaling pathways will provide promising avenues for HC regeneration in humans with NIHL and other types of hearing loss.
Assuntos
Proliferação de Células , Cóclea/patologia , Células Ciliadas Auditivas Internas/patologia , Perda Auditiva Provocada por Ruído/patologia , Regeneração Nervosa , Neurogênese , Ruído/efeitos adversos , Animais , Linhagem da Célula , Cóclea/metabolismo , Cóclea/fisiopatologia , Epigênese Genética , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/metabolismo , Perda Auditiva Provocada por Ruído/genética , Perda Auditiva Provocada por Ruído/fisiopatologia , Perda Auditiva Provocada por Ruído/terapia , Humanos , Células Labirínticas de Suporte/patologia , Militares , Exposição Ocupacional/efeitos adversosRESUMO
Experimental and human investigations have raised the level of concern about the potential ototoxicity of organic solvents and their interaction with noise. The main objective of this study was to characterize the effects of the combined noise and styrene exposure on hearing focusing on the mechanism of damage on the sensorineural cells and supporting cells of the organ of Corti and neurons of the ganglion of Corti. The impact of single and combined exposures on hearing was evaluated by auditory functional testing and histological analyses of cochlear specimens. The mechanism of damage was studied by analyzing superoxide anion and lipid peroxidation expression and by computational analyses of immunofluorescence data to evaluate and compare the oxidative stress pattern in outer hair cells versus the supporting epithelial cells of the organ of Corti. The oxidative stress hypothesis was further analyzed by evaluating the protective effect of a Coenzyme Q10 analogue, the water soluble Qter, molecule known to have protective antioxidant properties against noise induced hearing loss and by the analysis of the expression of the endogenous defense enzymes. This study provides evidence of a reciprocal noise-styrene synergism based on a redox imbalance mechanism affecting, although with a different intensity of damage, the outer hair cell (OHC) sensory epithelium. Moreover, these two damaging agents address preferentially different cochlear targets: noise mainly the sensory epithelium, styrene the supporting epithelial cells. Namely, the increase pattern of lipid peroxidation in the organ of Corti matched the cell damage distribution, involving predominantly OHC layer in noise exposed cochleae and both OHC and Deiters' cell layers in the styrene or combined exposed cochleae. The antioxidant treatment reduced the lipid peroxidation increase, potentiated the endogenous antioxidant defense system at OHC level in both exposures but it failed to ameliorate the oxidative imbalance and cell death of Deiters' cells in the styrene and combined exposures. Current antioxidant therapeutic approaches to preventing sensory loss focus on hair cells alone. It remains to be seen whether targeting supporting cells, in addition to hair cells, might be an effective approach to protecting exposed subjects.
Assuntos
Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/metabolismo , Células Labirínticas de Suporte/efeitos dos fármacos , Ruído/efeitos adversos , Estireno/toxicidade , Animais , Antioxidantes/farmacologia , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patologia , Perda Auditiva Provocada por Ruído/patologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Perda Auditiva Provocada por Ruído/prevenção & controle , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Oxirredução , Estresse Oxidativo , Ratos , Ratos Wistar , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
Hair cells in posthatch chickens regenerate spontaneously through mitosis or the transdifferentiation of supporting cells in response to antibiotic injury. However, how embryonic chicken cochleae respond to antibiotic treatment remains unknown. This study is the first to indicate that unlike hair cells in posthatch chickens, the auditory epithelium was free from antibiotic injury (25-250 mg gentamicin/kg) in embryonic chickens, although FITC-conjugated gentamicin actually reached embryonic hair cells. Next, we examined and counted the cells and performed labeling for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) (triple or double labeling) in the injured cochlea ducts after gentamicin treatment at 2 h (h), 15 h, 24 h, 2 days (d), 3 d and 7 d after BrdU treatment in posthatch chickens. Our results indicated that following gentamicin administration, proliferating cells (BrdU+) were labeled for Atoh1/Math1 in the damaged areas 3d after gentamicin administration, whereas hair cells (PV+) renewed through mitosis (BrdU+) or direct transdifferentiation (BrdU-) were evident only after 5 d of gentamicin administration. In addition, Sox2 expression was up-regulated in triggered supporting cells at an early stage of regeneration, but stopped at the advent of mature hair cells. Our study also indicated that p27(kip1) was expressed in both hair cells and supporting cells but was down-regulated in a subgroup of the supporting cells that gave rise to hair cells. These data and the obtained dynamic changes of the cells labeled for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) are useful for understanding supporting cell behaviors and their fate specification during hair cell regeneration.
Assuntos
Antibacterianos/toxicidade , Linhagem da Célula/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Ducto Coclear/efeitos dos fármacos , Gentamicinas/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Células Labirínticas de Suporte/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Embrião de Galinha , Galinhas , Ducto Coclear/embriologia , Ducto Coclear/metabolismo , Ducto Coclear/patologia , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Mitose/efeitos dos fármacos , Fatores de TempoRESUMO
Cochlear hair cell loss results in secondary regression of peripheral auditory fibers (PAFs) and loss of spiral ganglion neurons (SGNs). The performance of cochlear implants (CI) in rehabilitating hearing depends on survival of SGNs. Here we compare the effects of adeno-associated virus vectors with neurotrophin gene inserts, AAV.BDNF and AAV.Ntf3, on guinea pig ears deafened systemically (kanamycin and furosemide) or locally (neomycin). AAV.BDNF or AAV.Ntf3 was delivered to the guinea pig cochlea one week following deafening and ears were assessed morphologically 3 months later. At that time, neurotrophins levels were not significantly elevated in the cochlear fluids, even though in vitro and shorter term in vivo experiments demonstrate robust elevation of neurotrophins with these viral vectors. Nevertheless, animals receiving these vectors exhibited considerable re-growth of PAFs in the basilar membrane area. In systemically deafened animals there was a negative correlation between the presence of differentiated supporting cells and PAFs, suggesting that supporting cells influence the outcome of neurotrophin over-expression aimed at enhancing the cochlear neural substrate. Counts of SGN in Rosenthal's canal indicate that BDNF was more effective than NT-3 in preserving SGNs. The results demonstrate that a transient elevation in neurotrophin levels can sustain the cochlear neural substrate in the long term.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Surdez/terapia , Dependovirus/metabolismo , Orelha/patologia , Neurotrofina 3/uso terapêutico , Envelhecimento , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Sobrevivência Celular , Meios de Cultura , Surdez/patologia , Epitélio/metabolismo , Epitélio/patologia , Feminino , Vetores Genéticos , Cobaias , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Masculino , Neomicina , Neurotrofina 3/genética , Perilinfa/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/patologia , Resultado do TratamentoRESUMO
CONCLUSION: Programmed cell death (PCD) initially starts in the support cells (SCs) after electrode insertion trauma (EIT), followed by PCD in hair cells (HCs). Activation of caspase-3 was observed only in SCs. Protecting both SCs and HCs with selective otoprotective drugs at an early stage post implantation may help to preserve residual hearing. OBJECTIVES: Cochlear implant EIT can initiate sensory cell losses via necrosis and PCD within the organ of Corti, which can lead to a loss of residual hearing. PCD appears to be a major factor in HC loss post-EIT. The current study aimed to: (1) determine the onset of PCD in both SCs and HCs within the traumatized organ of Corti; and (2) identify the molecular mechanisms active within the HCs and SCs that are undergoing PCD. METHODS: Adult guinea pigs were assigned to one of two groups: (1) EIT and (2) unoperated contralateral ears as controls. Immunostaining of dissected organ of Corti surface preparations for phosphorylated-Jun, cleaved caspase-3, and 4-hydroxy-2,3-nonenal (HNE) were performed at 6, 12, and 24 h post-EIT and for contralateral control ears. RESULTS: At 6 h post-EIT the SCs immunolabeled for the presence of phosphorylated-Jun and activated caspase-3. Phosphorylated p-Jun labeling was observed at 12 h in both the HCs and SCs of middle and basal cochlear turns. Cleaved caspase-3 was not observed in HCs of any cochlear turn at up to 24 h post-EIT. Lipid peroxidation (HNE immunostaining) was first observed at 12 h post-EIT in both the HCs and SCs of the basal turn, and reached the apical turn by 24 h post-EIT.
Assuntos
Apoptose/fisiologia , Implante Coclear/efeitos adversos , Implantes Cocleares/efeitos adversos , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/patologia , Transdução de Sinais/fisiologia , Aldeídos/metabolismo , Animais , Caspase 3/metabolismo , Implante Coclear/instrumentação , Modelos Animais de Doenças , Cobaias , Células Ciliadas Auditivas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Labirínticas de Suporte/metabolismo , Estresse Oxidativo/fisiologia , Fatores de TempoRESUMO
Reactive oxygen species (ROS) accumulation are involved in noise- and ototoxic drug-induced hair cell loss, which is the major cause of hearing loss. Bmi1 is a member of the Polycomb protein family and has been reported to regulate mitochondrial function and ROS level in thymocytes and neurons. In this study, we reported the expression of Bmi1 in mouse cochlea and investigated the role of Bmi1 in hair cell survival. Bmi1 expressed in hair cells and supporting cells in mouse cochlea. Bmi1(-/-) mice displayed severe hearing loss and patched outer hair cell loss from postnatal day 22. Ototoxic drug-induced hair cells loss dramatically increased in Bmi1(-/-) mice compared with that in wild-type controls both in vivo and in vitro, indicating Bmi1(-/-) hair cells were significantly more sensitive to ototoxic drug-induced damage. Cleaved caspase-3 and TUNEL staining demonstrated that apoptosis was involved in the increased hair cell loss of Bmi1(-/-) mice. Aminophenyl fluorescein and MitoSOX Red staining showed the level of free radicals and mitochondrial ROS increased in Bmi1(-/-) hair cells due to the aggravated disequilibrium of antioxidant-prooxidant balance. Furthermore, the antioxidant N-acetylcysteine rescued Bmi1(-/-) hair cells from neomycin injury both in vitro and in vivo, suggesting that ROS accumulation was mainly responsible for the increased aminoglycosides sensitivity in Bmi1(-/-) hair cells. Our findings demonstrate that Bmi1 has an important role in hair cell survival by controlling redox balance and ROS level, thus suggesting that Bmi1 may work as a new therapeutic target for the prevention of hair cell death.
Assuntos
Células Ciliadas Auditivas/patologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Perda Auditiva/induzido quimicamente , Perda Auditiva/patologia , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neomicina/efeitos adversos , Oxidantes/metabolismo , Oxirredução/efeitos dos fármacos , Complexo Repressor Polycomb 1/deficiência , Proteínas Proto-Oncogênicas/deficiência , Espécies Reativas de Oxigênio/metabolismoRESUMO
The organ of Corti consists of sensory hair cells (HCs) interdigitated with nonsensory supporting cells (SCs) to form a checkerboard-like cellular pattern. HCs are equipped with hair bundles on their apical surfaces. We previously reported that cell-adhesive nectins regulate the checkerboard-like cellular patterning of HCs and SCs in the mouse auditory epithelium. Nectin-1 and -3 are differentially expressed in normal HCs and SCs, respectively, and in Nectin-3-deficient mice a number of HCs are aberrantly attached to each other. We show here that these aberrantly attached HCs in Nectin-3-deficient mice, but not unattached ones, show disturbances of the orientation and morphology of the hair bundles and the positioning of the kinocilium, with additional abnormal localisation of cadherin-catenin complexes and the apical-basal polarity proteins Pals1 and Par-3. These results indicate that, owing to the loss of Nectin-3, hair cells contact each other inappropriately and form abnormal junctions, ultimately resulting in abnormal hair bundle orientation and morphology.
Assuntos
Moléculas de Adesão Celular/deficiência , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Órgão Espiral/anormalidades , Órgão Espiral/metabolismo , Animais , Proteínas de Transporte/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Feminino , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Junções Intercelulares/metabolismo , Junções Intercelulares/patologia , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nectinas , Órgão Espiral/embriologia , GravidezRESUMO
In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.
Assuntos
Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Diferenciação Celular , Sobrevivência Celular , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Gentamicinas , Proteínas de Homeodomínio/metabolismo , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Camundongos , Fenótipo , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Regeneração , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Mammalian hearing relies upon active cochlear mechanics, which arises from outer hair cell electromotility and hair bundle movement, to amplify acoustic stimulations increasing hearing sensitivity and frequency selectivity. Here we describe the novel finding that gap junctions between cochlear supporting cells also have a critical role in active cochlear amplification in vivo. We find that targeted-deletion of connexin 26 in Deiters cells and outer pillar cells, which constrain outer hair cells standing on the basilar membrane, causes a leftward shift in outer hair cell electromotility towards hyperpolarization, and reduces active cochlear amplification with hearing loss. Coincident with large reduction in distortion product otoacoustic emission and severe hearing loss at high frequencies, the shift is larger in shorter outer hair cells. Our study demonstrates that active cochlear amplification in vivo is dependent on supporting cell gap junctions. These new findings also show that connexin 26 deficiency can reduce active cochlear amplification to induce hearing loss.
Assuntos
Junções Comunicantes/metabolismo , Células Labirínticas de Suporte/metabolismo , Animais , Limiar Auditivo , Potenciais Microfônicos da Cóclea , Conexina 26 , Conexinas/deficiência , Conexinas/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico , Deleção de Genes , Marcação de Genes , Células Ciliadas Auditivas Externas/patologia , Perda Auditiva/patologia , Perda Auditiva/fisiopatologia , Células Labirínticas de Suporte/patologia , Camundongos , Camundongos Knockout , Proteínas Motores Moleculares/metabolismo , Dinâmica não Linear , Emissões Otoacústicas Espontâneas , Gânglio Espiral da Cóclea/patologia , Gânglio Espiral da Cóclea/fisiopatologiaRESUMO
Loss of hair cells in humans leads to irreversible hearing deficits, since auditory hair cells are not replaced. In contrast, hair cells are regenerated in the auditory epithelium of mature birds after damage by non-sensory supporting cells that transdifferentiate into hair cells by mitotic and/or non-mitotic mechanisms. Factors controlling these processes are poorly understood. The basic helix-loop-helix transcription factor ATOH1 is both necessary and sufficient for developmental hair cell differentiation, but it is unclear if it plays the same role in the mitotic and non-mitotic pathways in hair cell regeneration. We examined Atoh1 expression and function during hair cell regeneration in chickens. Atoh1 transcripts were increased in many supporting cells in the damaged auditory epithelium shortly after ototoxin administration and later became restricted to differentiating hair cells. Fate-mapping in vitro using an Atoh1 enhancer reporter demonstrated that only 56% of the supporting cells that spontaneously upregulate Atoh1 enhancer activity after damage acquired the hair cell fate. Inhibition of notch signaling using a gamma secretase antagonist stimulated an increase in Atoh1 reporter activity and induced a higher proportion of supporting cells with Atoh1 activity (73%) to differentiate as hair cells. Forced overexpression of Atoh1 in supporting cells triggered 66% of them to acquire the hair cell fate and nearly tripled their likelihood of cell cycle entry. These findings demonstrate that Atoh1 is broadly upregulated in supporting cells after damage, but a substantial proportion of supporting cells with Atoh1 activation fails to acquire hair cell features, in part due to gamma secretase-dependent activities.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/metabolismo , Regeneração , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ciclo Celular , Transdiferenciação Celular , Galinhas , Dipeptídeos/farmacologia , Eletroporação , Elementos Facilitadores Genéticos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/patologia , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo , Receptores Notch/metabolismo , Regeneração/efeitos dos fármacos , Estreptomicina/toxicidade , Fatores de Tempo , Ativação TranscricionalRESUMO
When inner ear hair cells die, humans and other mammals experience permanent hearing and balance deficits, but non-mammalian vertebrates quickly recover these senses after epithelial supporting cells give rise to replacement hair cells. A postnatal decline in cellular plasticity appears to limit regeneration in mammalian balance organs, where declining proliferation responses are correlated with decreased spreading of supporting cells on artificial and native substrates. By culturing balance epithelia on substrates that differed in flexibility, we assessed spreading effects independent of age, showing a strong correlation between shape change and supporting cell proliferation. Then we made excision wounds in utricles cultured from young and old chickens and mice and compared quantified levels of spreading and proliferation. In utricles from young mice, and both young and old chickens, wounds re-epithelialized in <24 hours, while those in utricles from mature mice took three times longer. More cells changed shape in the fastest healing wounds, which accounted for some differences in the levels of proliferation, but inter-species and age-related differences in shape-sensitive restriction points, i.e., the cellular thresholds for shape changes that promote S-phase, were evident and may be particularly influential in the responses to hair cell losses in vivo.