RESUMO
In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.
Assuntos
Dependovirus , Orelha Interna , Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Dependovirus/genética , Camundongos , Orelha Interna/metabolismo , Orelha Interna/embriologia , Orelha Interna/citologia , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Feminino , Transdução Genética/métodos , Gravidez , Terapia Genética/métodos , HumanosRESUMO
Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.
Assuntos
Regeneração , Análise de Célula Única , Transcriptoma , Humanos , Animais , Regeneração/genética , Camundongos , Sáculo e Utrículo/metabolismo , Sáculo e Utrículo/citologia , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Orelha Interna/metabolismo , Orelha Interna/citologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/genética , Masculino , Células Ciliadas Vestibulares/metabolismo , Feminino , Perfilação da Expressão GênicaRESUMO
Nucleotide excision repair (NER) is a multistep biochemical process that maintains the integrity of the genome. Unlike other mechanisms that maintain genomic integrity, NER is distinguished by two irreversible nucleolytic events that are executed by the xeroderma pigmentosum group G (XPG) and xeroderma pigmentosum group F (XPF) structure-specific endonucleases. Beyond nucleolysis, XPG and XPF regulate the overall efficiency of NER through various protein-protein interactions. The current experiments evaluated whether an environmental stressor could negatively affect the expression of Xpg (Ercc5: excision repair cross-complementing 5) or Xpf (Ercc4: excision repair cross-complementing 4) in the mammalian cochlea. Ubiquitous background noise was used as an environmental stressor. Gene expression levels for Xpg and Xpf were quantified from the cochlear neurosensory epithelium after noise exposure. Further, nonlinear cochlear signal processing was investigated as a functional consequence of changes in endonuclease expression levels. Exposure to stressful background noise abrogated the expression of both Xpg and Xpf, and these effects were associated with pathological nonlinear signal processing from receptor cells within the mammalian inner ear. Given that exposure to environmental sounds (noise, music, etc.) is ubiquitous in daily life, sound-induced limitations to structure-specific endonucleases might represent an overlooked genomic threat.
Assuntos
Orelha Interna , Xeroderma Pigmentoso , Animais , Endonucleases/genética , Endonucleases/metabolismo , Orelha Interna/metabolismo , Reparo do DNA , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3-5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.
Assuntos
Orelha Interna , Síndromes de Usher , Animais , Camundongos , Caderinas/metabolismo , Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição/genética , Síndromes de Usher/genética , Síndromes de Usher/terapia , Proteínas Relacionadas a Caderinas/metabolismoRESUMO
The EYA1 gene is essential for normal inner ear development and affects the development and function of the inner ear in a dose-dependent manner. However, the mechanisms regulating EYA1 gene expression are not well understood. Recently, miRNAs have become recognized as important regulators of gene expression. In this study, we identified miR-124-3p through a microRNA (miRNA) target prediction website and found that miR-124-3p and its target site in the EYA1 3' untranslated region (3'UTR) are conserved in most vertebrates. Both in vivo and in vitro, the interaction of miR-124-3p with the EYA1 3'UTR exerts a negative regulatory effect. Microinjection of agomiR-124-3p into zebrafish embryos resulted in a phenotype of reduced auricular area, suggesting inner ear dysplasia. In addition, injection of agomiR-124-3p or antagomiR-124-3p caused abnormal hearing function in zebrafish. In conclusion, our results suggest that miR-124-3p can affect inner ear development and hearing function in zebrafish by regulating EYA1.
Assuntos
Orelha Interna , MicroRNAs , Animais , Peixe-Zebra/metabolismo , Regulação para Baixo , Regiões 3' não Traduzidas , Orelha Interna/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Audição , Expressão GênicaRESUMO
Sensorineural hearing loss is the most common type of hearing loss in adults and occurs due to damage of the inner ear caused by a range of factors including ageing, excessive noise, toxins, and cancer. Auto-inflammatory disease is also a cause of hearing loss and there is evidence that inflammation could contribute to hearing loss in other conditions. Within the inner ear there are resident macrophage cells that respond to insults and whose activation correlates with damage. The NLRP3 inflammasome is a multi-molecular pro-inflammatory protein complex that forms in activated macrophages and may contribute to hearing loss. The aim of this article is to discuss the evidence for the NLRP3 inflammasome and associated cytokines as potential therapeutic targets for sensorineural hearing loss in conditions ranging from auto-inflammatory disease to tumour-induced hearing loss in vestibular schwannoma.
Assuntos
Orelha Interna , Perda Auditiva Neurossensorial , Perda Auditiva , Adulto , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/metabolismo , Orelha Interna/metabolismo , Perda Auditiva/complicaçõesRESUMO
Sensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown that Lgr5+ supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno-associated virus-inner ear system to upregulate Rps14 expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over-expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5+ progenitors. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.
Assuntos
Orelha Interna , Células Ciliadas Auditivas , Proteínas Ribossômicas , Animais , Camundongos , Animais Recém-Nascidos , Diferenciação Celular , Proliferação de Células , Orelha Interna/metabolismo , Mamíferos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima , Proteínas Ribossômicas/metabolismoRESUMO
NOD-like receptor protein 3 (NLRP3) inflammasomes trigger the inflammatory cascades and participate in various inflammatory diseases, including noise-induced hearing loss (NIHL) caused by oxidative stress. Recently, the anti-inflammatory traditional medicine oridonin (Ori) has been reported to provide hearing protection in mice after noise exposure by blocking the NLRP3-never in mitosis gene A-related kinase 7 (NEK7)-inflammasome complex assembly. Using RNA sequencing analysis, we further elucidated that interleukin 1 receptor type 2 (IL1R2) may be another crucial factor regulated by Ori to protect NIHL. We observed that IL1R2 expression was localized in spiral ganglion neurons, inner and outer hair cells, in Ori-treated mouse cochleae. Additionally, we confirmed that ectopic overexpression of IL1R2 in the inner ears of healthy mice using an adeno-associated virus delivery system significantly reduced noise-induced ribbon synapse lesions and hearing loss by blocking the "cytokine storm" in the inner ear. This study provides a novel theoretical foundation for guiding the clinical treatment of NIHL.
Assuntos
Orelha Interna , Perda Auditiva Provocada por Ruído , Otite , Camundongos , Animais , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Perda Auditiva Provocada por Ruído/etiologia , Perda Auditiva Provocada por Ruído/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Orelha Interna/metabolismo , Orelha Interna/patologia , Inflamação/complicações , Anti-Inflamatórios/farmacologia , Otite/complicações , Receptores de Interleucina-1RESUMO
BACKGROUND: Otosclerosis is a common ear disease that causes fixation of the stapes and conductive hearing impairment. However, the pathogenesis of otosclerosis is still unknown. Otosclerosis could be associated with the unique bony environment found in the otic capsule. Normal bone remodelling is almost completely absent around the inner ear after birth allowing degenerative changes and dead osteocytes to accumulate. High levels of inner ear anti resorptive osteoprotegerin (OPG) is most likely responsible for this capsular configuration. Studies have demonstrated how osteocyte lifespan variation creates occasional clusters of dead osteocytes, so-called cellular voids, at otosclerotic predilection sites in the human otic capsule. These cellular voids have been suggested as possible starting points of otosclerosis. AIM: To describe the cellular viability in otosclerotic lesions and compare it to that of cellular voids. MATERIALS AND METHODS: The study was based on unbiased stereological quantifications in undecalcified human temporal bones with otosclerosis. RESULTS: Osteocyte viability was found to vary within the otosclerotic lesions. Furthermore, the results presented here illustrate that inactive otosclerotic lesions consist of mainly dead interstitial bone, much like cellular voids. CONCLUSIONS AND SIGNIFICANCE: Focal degeneration in the otic capsule may play an important role in the pathogenesis of otosclerosis.
Assuntos
Orelha Interna , Osteócitos , Osteoprotegerina , Otosclerose , Humanos , Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Orelha Interna/metabolismo , Orelha Interna/patologia , Osteócitos/metabolismo , Osteócitos/patologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Otosclerose/etiologia , Otosclerose/genética , Otosclerose/metabolismo , Otosclerose/patologia , Estribo/metabolismo , Estribo/patologia , Osso Temporal/metabolismo , Osso Temporal/patologiaRESUMO
The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects â¼0.3% of newborns, and other syndromic disorders.
Assuntos
Surdez , Orelha Interna , Fatores de Transcrição SOX9 , Fatores de Transcrição SOXE , Animais , Camundongos , Surdez/metabolismo , Orelha Interna/metabolismo , Audição/genética , Homeostase , Camundongos Knockout , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismoRESUMO
RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.
Assuntos
Orelha Interna , Animais , Humanos , Orelha Interna/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Cabelo/metabolismoRESUMO
Background: Human inner ear contains macrophages whose functional role in early development is yet unclear. Recent studies describe inner ear macrophages act as effector cells of the innate immune system and are often activated following acoustic trauma or exposure to ototoxic drugs. Few or limited literature describing the role of macrophages during inner ear development and organogenesis. Material and Methods: We performed a study combining immunohistochemistry and immunofluorescence using antibodies against IBA1, CX3CL1, CD168, CD68, CD45 and CollagenIV. Immune staining and quantification was performed on human embryonic inner ear sections from gestational week 09 to 17. Results: The study showed IBA1 and CD45 positive cells in the mesenchymal tissue at GW 09 to GW17. No IBA1 positive macrophages were detected in the sensory epithelium of the cochlea and vestibulum. Fractalkine (CX3CL1) signalling was initiated GW10 and parallel chemotactic attraction and migration of macrophages into the inner ear. Macrophages also migrated into the spiral ganglion, cochlear nerve, and peripheral nerve fibers and tissue-expressing CX3CL1. The mesenchymal tissue at all gestational weeks expressed CD163 and CD68. Conclusion: Expressions of markers for resident and non-resident macrophages (IBA1, CD45, CD68, and CD163) were identified in the human fetal inner ear. We speculate that these cells play a role for the development of human inner ear tissue including shaping of the gracile structures.
Assuntos
Quimiocina CX3CL1 , Orelha Interna , Quimiocina CX3CL1/metabolismo , Cóclea , Orelha Interna/metabolismo , Humanos , Macrófagos , Gânglio Espiral da CócleaRESUMO
Objective: The mechanism of ototoxicity caused by cisplatin is currently unclear, and the induced apoptosis may play an important role in inner ear injury. Melatonin has high antioxidant and antiapoptotic effects. This study is aimed at clarifying the protective effect on the inner ear and the underlying mechanism of melatonin. Design: The mice and HEI-OC1 cells were randomly separated into four groups: control group, cisplatin group, melatonin group, and cisplatin exposure after melatonin pretreatment group. Place and Duration of the Study. From September 2018 to September 2021, all experiments were completed at the Second Hospital of Shandong University. And the study was approved by the Ethics Committee of the Second Hospital of Shandong University (KYLL-2020 (KJ) A-0191). Methodology. Mice were pretreated with peritoneal injection of melatonin prior to the application of cisplatin. Auditory Brainstem Response (ABR) test was performed before and after treatment, then the temporal bones were collected for histology investigation. HEI-OC1 cells were pretreated with melatonin before adding cisplatin. The apoptosis of HEI-OC1 cells was observed by MTS, TUNEL, and flow cytometry, respectively. Moreover, the mRNA expression of apoptosis-related factors was detected by qRT-PCR. Results: ABR and morphological analysis showed that cisplatin caused damage to the function and structure of the inner ear. MTS, TUNEL, and flow cytometry showed that the application of cisplatin caused a significant increase in the apoptosis level of HEI-OC1 cells, and melatonin pretreatment reduced this damage. Moreover, melatonin pretreatment reversed the mRNA expression changes of apoptosis-related factors induced by cisplatin. Conclusions: Apoptosis is involved in the inner ear dysfunction caused by cisplatin. Melatonin reduces the ototoxicity of cisplatin by regulating the induced apoptosis response.
Assuntos
Antineoplásicos , Orelha Interna , Melatonina , Ototoxicidade , Animais , Antineoplásicos/toxicidade , Apoptose , Sobrevivência Celular , Cisplatino/metabolismo , Cisplatino/toxicidade , Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Humanos , Melatonina/metabolismo , Melatonina/farmacologia , Camundongos , Ototoxicidade/tratamento farmacológico , Ototoxicidade/etiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologiaRESUMO
Meniere's disease (MD) is a disorder of the inner ear characterized by episodes of spontaneous vertigo, fluctuating hearing loss, and tinnitus. Recent studies have demonstrated that IgE may play a role in the pathogenesis of MD. Patients with MD (n = 103), acoustic neuroma (n = 5), and healthy subjects (n = 72) were recruited into the study. Serum from the participants was analyzed for IgE and type 2-related cytokines. IgE and CD23 expression levels in vestibular end organs of patients, C57BL/6 mice, or mouse HEI-OC1 cells were analyzed. Finally, the role of CD23 in IgE transcytosis was assessed using HEI-OC1 cells. Serum IgE was elevated in patients with MD and positively correlated with clinical symptoms. IL-4, IL-5, IL-10, IL-13, and CD23 levels were increased in patients with MD compared with the control group. In the transcytosis assay, mouse IgE was found to be bidirectionally transported across the HEI-OC1 cell monolayer. Additionally, CD23 downregulation using a small interfering RNA approach significantly reduced the efficiency of IgE transcytosis, suggesting that IgE is transported by CD23. Furthermore, exposure to IL-4 increased CD23 expression and enhanced IgE transcytosis in the HEI-OC1 cells and primary vestibular end organs. Our study indicated that IgE may play a role in the pathophysiology of MD. In addition, CD23-mediated IgE transcytosis in the hair cells may play a critical role in initiating inflammation in the inner ear. Thus, reducing the level of IgE may be a potentially effective approach for MD treatment.
Assuntos
Orelha Interna/imunologia , Orelha Interna/metabolismo , Imunoglobulina E/imunologia , Lectinas Tipo C/metabolismo , Doença de Meniere/etiologia , Doença de Meniere/metabolismo , Receptores de IgE/metabolismo , Adulto , Idoso , Animais , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Imunofluorescência , Humanos , Imunoglobulina E/metabolismo , Lectinas Tipo C/genética , Masculino , Doença de Meniere/diagnóstico , Camundongos , Pessoa de Meia-Idade , Imagem Molecular , Fenótipo , Ligação Proteica , Transporte Proteico , Receptores de IgE/genética , Transcitose/imunologia , Vestíbulo do Labirinto/imunologia , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/patologiaRESUMO
Spiral ligament fibrocytes of the cochlea play homoeostatic roles in hearing and their degeneration contributes to hearing loss. Culturing fibrocytes in vitro provides a way to evaluate their functional characteristics and study possible therapies for hearing loss. We investigated whether in vivo characteristics of fibrocytes could be recapitulated in vitro by modifying the culture substrates and carried out proof of concept studies for potential transplantation of culture cells into the inner ear. Fibrocytes cultured from 4 to 5-week old CD/1 mice were grown on 2D substrates coated with collagen I, II, V or IX and, after harvesting, onto or into 3D substrates (hydrogels) of collagen I alone or mixed collagen I and II at a 1:1 ratio. We also assessed magnetic nanoparticle (MNP) uptake. Cell counts, immunohistochemical and ultrastructural studies showed that fibrocytes grown on 2D substrates proliferated, formed both small spindle-shaped and large flat cells that avidly took up MNPs. Of the different collagen coatings, only collagen II had an effect, causing a reduced size of the larger cells. On hydrogels, the cells were plump/rounded with extended processes, resembling native cells. They formed networks over the surface and became incorporated into the gel. In all culture formats, the majority co-expressed caldesmon, aquaporin 1, S-100 and sodium potassium ATPase, indicating a mixed or uncharacterised phenotype. Time-course experiments showed a decrease to â¼50% of the starting population by 4d after seeding on collagen I hydrogels, but better survival (â¼60%) was found on collagen I + II gels, whilst TEM revealed the presence of apoptotic cells. Cells grown within gels additionally showed necrosis. These results demonstrate that fibrocytes grown in 3D recapitulate in vivo morphology of native fibrocytes, but have poorer survival, compared with 2D. Therefore hydrogel cultures could be used to study fibrocyte function and might also offer avenues for cell-replacement therapies, but need more optimization for therapeutic use. Fibrocyte function could be modified using MNPs in combination, for example, with gene transfection.
Assuntos
Cóclea , Orelha Interna , Animais , Técnicas de Cultura de Células , Cóclea/metabolismo , Orelha Interna/metabolismo , Audição , Camundongos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Objective: To observe the effects of estradiol on expression of plasma membrane Ca2+-ATPase isoform 2 in inner ear of rats. Methods: Twenty-five Three-months-old female Sprague-Dawley rats were randomly and equally divided into five groups by random number table mathod,with five rats in each group. Animals in Sham group were sham-operated while others were bilateral ovariactmized. One month after modeling, the OVX groups were supplemented with estradiol (E2 group), progesterone (P group), estradiol and progesterone (E2+P group)and vehicle sesame oil (Veh group), while the Sham operation group (Sham group) was supplemented with vehicle sesame oil.All rats were sacrificed and otocysts were obtained immediately. Enzyme-linked immunosorbent assay was used to detect the changes in serum estradiol and progesterone levels of each group of rats before operation, before treatment and before sacrifice. Western blot and quantitative real-time polymerase chain reaction were used to detect the expression of total PMCA2 protein and mRNA in the inner ear of each group. Results: There was no significant difference in serum estradiol and progesterone levels among the five groups before operated(P>0.05). Before treatment, the serum estradiol and progesterone levels of rats in each group were significantly lower than those in Sham group (P<0.05). The serum estradiol level in E2 group and E2+P group was not significantly different from that in Sham group (P>0.05), while the serum estradiol level in P group and Veh group was significantly different from that in Sham group (P<0.05). The level of progesterone in P group and E2+P group was higher than that in Sham group (P<0.05), while the level of progesterone in Veh group and E2 group was lower than that in Sham group (P<0.05). Protein and mRNA expression of PMCA2 in P and Veh groups were significantly decreased compared with that of Sham group (P<0.05) while the expression levels underwent no significantly change in E2 and E2+P groups (P<0.05). Conclusion: The decrease of serum estradiol level can reduce the expression of otolith regulatory protein PMCA2 in rats, and then affect otolith metabolism, which may be an important link of estrogen affecting otolith metabolism.
Assuntos
Orelha Interna , Estradiol , Animais , Feminino , Ratos , Adenosina Trifosfatases , Orelha Interna/metabolismo , Estradiol/farmacologia , Progesterona/farmacologia , Isoformas de Proteínas , Ratos Sprague-Dawley , RNA Mensageiro/metabolismo , Óleo de GergelimRESUMO
Several studies have shown that type IV fibrocytes, located in the spiral ligament, degenerate first after noise exposure. Interestingly, this is the region where Coch expression is most abundant. As it is suggested that cochlin plays a role in our innate immune system, our goal is to investigate hearing thresholds and inner ear inflammation after noise exposure in Coch knockout (Coch-/-) mice compared to Coch wildtype (Coch+/+) mice. Animals were randomly allocated to a noise exposure group and a control group. Vestibular and auditory testing was performed at 48 h and one week after noise exposure. Whole mount staining and cryosectioning of the cochlea was performed in order to investigate hair cells, spiral ganglion neurons, inner ear inflammation, Coch expression and fibrocyte degeneration. Hearing assessment revealed that Coch+/+ mice had significantly larger threshold shifts than Coch-/- mice after noise exposure. We were unable to identify any differences in hair cells, neurons, fibrocytes and influx of macrophages in the inner ear between both groups. Interestingly, Coch expression was significantly lower in the group exposed to noise. Our results indicate that the absence of Coch has a protective influence on hearing thresholds after noise exposure, but this is not related to reduced inner ear inflammation in the knockout.
Assuntos
Envelhecimento/metabolismo , Proteínas da Matriz Extracelular/deficiência , Perda Auditiva Provocada por Ruído/metabolismo , Animais , Cóclea/metabolismo , Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição/fisiologia , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Neurônios/metabolismo , Ruído/efeitos adversosRESUMO
Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR.
Assuntos
Desenvolvimento Ósseo , Proteínas de Homeodomínio/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Síndrome Brânquio-Otorrenal/embriologia , Síndrome Brânquio-Otorrenal/genética , Núcleo Celular/metabolismo , Orelha Interna/embriologia , Orelha Interna/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Larva/crescimento & desenvolvimento , Metaloproteínas/genética , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Ativação Transcricional , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Melanocytes are pigmented cells residing mostly in the skin and hair follicles of vertebrates, where they contribute to colouration and protection against UV-B radiation. However, the spectrum of their functions reaches far beyond that. For instance, these pigment-producing cells are found inside the inner ear, where they contribute to the hearing function, and in the heart, where they are involved in the electrical conductivity and support the stiffness of cardiac valves. The embryonic origin of such extracutaneous melanocytes is not clear. We took advantage of lineage-tracing experiments combined with 3D visualizations and gene knockout strategies to address this long-standing question. We revealed that Schwann cell precursors are recruited from the local innervation during embryonic development and give rise to extracutaneous melanocytes in the heart, brain meninges, inner ear, and other locations. In embryos with a knockout of the EdnrB receptor, a condition imitating Waardenburg syndrome, we observed only nerve-associated melanoblasts, which failed to detach from the nerves and to enter the inner ear. Finally, we looked into the evolutionary aspects of extracutaneous melanocytes and found that pigment cells are associated mainly with nerves and blood vessels in amphibians and fish. This new knowledge of the nerve-dependent origin of extracutaneous pigment cells might be directly relevant to the formation of extracutaneous melanoma in humans.
Assuntos
Encéfalo/fisiologia , Orelha Interna/fisiologia , Coração/fisiologia , Meninges/fisiologia , Sistema Nervoso/fisiopatologia , Células de Schwann/fisiologia , Anfíbios/metabolismo , Anfíbios/fisiologia , Animais , Encéfalo/metabolismo , Linhagem da Célula/fisiologia , Orelha Interna/metabolismo , Desenvolvimento Embrionário/fisiologia , Feminino , Peixes/metabolismo , Peixes/fisiologia , Melanócitos/metabolismo , Melanócitos/fisiologia , Meninges/metabolismo , Camundongos , Sistema Nervoso/metabolismo , Gravidez , Receptor de Endotelina B/metabolismo , Células de Schwann/metabolismoRESUMO
In the inner ear, the neural crest gives rise to the glia of the VIII ganglion and two types of melanocytic cells: The pigmented cells of the vestibular system and intermediate cells of the stria vascularis. We analyzed the transcriptome of neonatal intermediate cells in an effort to better understand the development of the stria vascularis. We found that the expression of endothelin receptor B, which is essential for melanocyte development, persists in intermediate cells long after birth. In contrast, skin melanocytes rapidly downregulate the expression of EdnrB. Our findings suggest that endothelins might have co-opted new functions in the inner ear during evolution of the auditory organ.