RESUMO
Dysfunction of the endolymphatic sac (ES) is one of the etiologies of Meniere's disease (MD), the mechanism of which remains unclear. The aim of the present study was to explore the molecular pathological characteristics of ES during the development of MD. Metabolomic profiling of ES luminal fluid from patients with MD and patients with acoustic neuroma (AN) was performed. Diluted ES luminal fluid (ELF) samples were obtained from 10 patients who underwent endolymphatic duct blockage for the treatment of intractable MD and from 6 patients who underwent translabyrinthine surgery for AN. ELF analysis was performed using liquid chromatography-mass spectrometry before the raw data were normalized and subjected to subsequent statistical analysis by MetaboAnalyst. Using thresholds of P ≤ 0.05 and variable important in projection > 1, a total of 111 differential metabolites were screened in the ELF, including 52 metabolites in negative mode and 59 in positive mode. Furthermore, 15 differentially altered metabolites corresponding to 15 compound names were identified using a Student's t-test, including 7 significant increased metabolites and 8 significant decreased metabolites. Moreover, two differentially altered metabolites, hyaluronic acid (HA) and 4-hydroxynonenal (4-HNE), were validated to be upregulated in the epithelial lining of the ES, as well as in the subepithelial connective-tissue in patients with MD comparing with that in patients with AN. Among these differentially altered metabolites, an upregulated expression of HA detected in the ES lumen of the patients with MD was supposed to be associated with the increased endolymph in ES, while an increased level of 4-HNE found in the ELF of the patients with MD provided direct evidence to support that oxidative damage and inflammatory lesions underlie the mechanism of MD. Furthermore, citrate and ethylenediaminetetraacetic acid were detected to be decreased substantially in the ELF of the patients with MD, suggesting the elevated endolymphatic Ca2+ in the ears with chronic endolymphatic hydrops is likely to be associated with the reduction of these two chelators of Ca2+ in ES. The results in the present study indicate metabolomic analysis in the ELF of the patients with MD can potentially improve our understanding on the molecular pathophysiological mechanism in the ES during the development of MD.
Assuntos
Saco Endolinfático , Doença de Meniere , Humanos , Doença de Meniere/cirurgia , Saco Endolinfático/cirurgia , Saco Endolinfático/metabolismo , Saco Endolinfático/patologiaRESUMO
AIMS: Although inactivation of the von Hippel-Lindau gene (VHL) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (ELSTs), the genetic background of sporadic ELST is largely unknown. The aim of this study was to determine the prevalence of VHL mutations in sporadic ELSTs and compare their characteristics to VHL-disease-related tumours. METHODS: Genetic and epigenetic alterations were compared between 11 sporadic and 11 VHL-disease-related ELSTs by targeted sequencing and DNA methylation analysis. RESULTS: VHL mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic ELSTs (82%). No other cancer-related genetic pathway was altered except for TERT promoter mutations in two sporadic ELST and one VHL-disease-related ELST (15%). Loss of heterozygosity of chromosome 3 was found in 6/10 (60%) VHL-disease-related and 10/11 (91%) sporadic ELSTs resulting in biallelic VHL inactivation in 8/10 (73%) sporadic ELSTs. DNA methylation profiling did not reveal differences between sporadic and VHL-disease-related ELSTs but reliably distinguished ELST from morphological mimics of the cerebellopontine angle. VHL patients were significantly younger at disease onset compared to sporadic ELSTs (29 vs. 52 years, p < 0.0001, Fisher's exact test). VHL-disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression-free survival (p = 0.0002, log-rank test). CONCLUSION: Biallelic inactivation of VHL is the main mechanism underlying ELSTs, but unknown mechanisms beyond VHL may rarely be involved in the pathogenesis of sporadic ELSTs.
Assuntos
Neoplasias da Orelha/patologia , Saco Endolinfático/patologia , Proteínas Supressoras de Tumor/metabolismo , Doença de von Hippel-Lindau/patologia , Adulto , Neoplasias da Orelha/complicações , Neoplasias da Orelha/genética , Saco Endolinfático/metabolismo , Humanos , Pessoa de Meia-Idade , Mutação/genética , Risco , Proteínas Supressoras de Tumor/genética , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/genéticaRESUMO
The endolymphatic sac (ES) is the third part of the inner ear, along with the cochlea and vestibular apparatus. A refined sampling technique was developed to analyse the proteomics of ES endolymph. With a tailored solid phase micro-extraction probe, five ES endolymph samples were collected, and six sac tissue biopsies were obtained in patients undergoing trans-labyrinthine surgery for sporadic vestibular schwannoma. The samples were analysed using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) to identify the total number of proteins. Pathway identification regarding molecular function and protein class was presented. A total of 1656 non-redundant proteins were identified, with 1211 proteins detected in the ES endolymph. A total of 110 proteins were unique to the ES endolymph. The results from the study both validate a strategy for in vivo and in situ human sampling during surgery and may also form a platform for further investigations to better understand the function of this intriguing part of the inner ear.
Assuntos
Endolinfa/metabolismo , Saco Endolinfático/metabolismo , Neuroma Acústico/metabolismo , Proteoma/metabolismo , Adulto , Idoso , Animais , Biópsia , Cromatografia Líquida , Cóclea , Orelha Interna/fisiologia , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem , Vestíbulo do Labirinto , Microtomografia por Raio-X , Adulto JovemRESUMO
After dedicated CT and MRI, Ga-DOTATATE PET/CT was performed in a patient with a temporal bone mass with primary diagnostic considerations of an endolymphatic sac tumor versus a glomus jugulotympanicum paraganglioma. The Ga-DOTATATE PET showed mild radiotracer uptake in the mass (SUVmax, 10.9). After surgical resection, pathology revealed an endolymphatic sac tumor. Immunohistochemical staining demonstrated somatostatin receptor type 2A expression in the vasculature of the mass, but not in the tumor cells.
Assuntos
Neoplasias da Orelha/diagnóstico por imagem , Neoplasias da Orelha/patologia , Saco Endolinfático/diagnóstico por imagem , Saco Endolinfático/patologia , Compostos Organometálicos/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Adulto , Idoso , Diagnóstico Diferencial , Neoplasias da Orelha/metabolismo , Saco Endolinfático/metabolismo , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Paraganglioma/diagnóstico , Receptores de Somatostatina/metabolismoRESUMO
Rationale: Mutations of SLC26A4 that abrogate pendrin, expressed in endolymphatic sac, cochlea and vestibule, are known to cause autosomal recessive sensorineural hearing loss with enlargement of the membranous labyrinth. This is the first study to demonstrate the feasibility of gene therapy for pendrin-related hearing loss. Methods: We used a recombinant viral vector to transfect Slc26a4 cDNA into embryonic day 12.5 otocysts of pendrin-deficient knock-out (Slc26a4∆/∆ ) and pendrin-deficient knock-in (Slc26a4tm1Dontuh/tm1Dontuh ) mice. Results: Local gene-delivery resulted in spatially and temporally limited pendrin expression, prevented enlargement, failed to restore vestibular function, but succeeded in the restoration of hearing. Restored hearing phenotypes included normal hearing as well as sudden, fluctuating, and progressive hearing loss. Conclusion: Our study illustrates the feasibility of gene therapy for pendrin-related hearing loss, suggests differences in the requirement of pendrin between the cochlea and the vestibular labyrinth, and documents that insufficient pendrin expression during late embryonal and early postnatal development of the inner ear can cause sudden, fluctuating and progressive hearing loss without obligatory enlargement of the membranous labyrinth.
Assuntos
Terapia Genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/terapia , Audição/genética , Mutação/genética , Transportadores de Sulfato/genética , Animais , Cóclea/metabolismo , Dependovirus , Orelha Interna/metabolismo , Saco Endolinfático/embriologia , Saco Endolinfático/metabolismo , Células Epiteliais/metabolismo , Células Ciliadas Auditivas/metabolismo , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membrana dos Otólitos/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estria Vascular/metabolismo , Transportadores de Sulfato/metabolismo , Transcrição GênicaRESUMO
OBJECTIVES: Tricellulin is a tight junction (TJ)-forming protein that participates in the sealing function of tricellular TJs. Tricellulin-knockout (Tric-/-) mice show progressive hearing loss with degeneration of hair cells in the cochlea without physiological or physical disorders. In the present study, we investigated the tricellulin expression and its deletion effects in the endolymphatic sac (ES) using Tric-/- mice. MATERIALS AND METHODS: The ES epithelia from wild-type (WT) mice were laser-microdissected, and RT-PCR was performed. The ES sections from Tric-/- and WT mice were immunostained with an anti-tricellulin antibody. Hematoxylin and eosin staining was performed for morphological examination. The inner ear of Tric-/- mice was perfused with biotinylation reagents, and the ES sections were observed for tracer permeability assay after applying streptavidin-Alexa Fluor 488 conjugate. RESULTS: The tricellulin expression was confirmed by RT-PCR and by immunohistochemistry in the WT ES. The ES in Tric-/- mice showed normal morphology and revealed no biotin leakage from the lumen. CONCLUSION: The ES in Tric-/- mice showed no changes in morphology or disruption in macromolecular barrier function. The effects of solute leakages in the ES of Tric-/- mice may be very limited and compensatable, or that the ES epithelia may have other sealing system covering the lack of tricellulin.
Assuntos
Saco Endolinfático/metabolismo , Perda Auditiva/metabolismo , Proteína 2 com Domínio MARVEL/metabolismo , Animais , Saco Endolinfático/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva/patologia , Junções Intercelulares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout/metabolismo , Permeabilidade , Junções Íntimas/metabolismoRESUMO
Objectives Endolymphatic sac (ELS) pathophysiology in Ménière's disease (MD) remains poorly understood. We identified from the literature a group of proteins expressed on the ELS and involved in endolymph volume regulation: aquaporin-2 (AQP2), vasopressin receptor V2R, sodium potassium chloride cotransporter 2 (NKCC2), and transient receptor potential cation channel V4 (TRPV4). Our objective was to determine whether their ELS expression was altered in MD, to better understand the pathophysiology of endolymphatic hydrops. Study Design Prospective case-control study. Setting Tertiary care center. Subjects Twenty-four patients with definite MD undergoing endolymphatic duct blockage surgery were recruited, as well as 23 controls with no history of MD undergoing surgery for vestibular schwannoma (VS). Methods ELS biopsies and blood samples for plasma arginine vasopressin (AVP) were obtained. Immunohistochemistry for AQP2, V2R, NKCC2, and TRPV4 was performed. Slides were scanned digitally for highly sensitive pixel density analysis by specialized software (VIS; Visiopharm). Results Global scores generated by the software represent total and relative protein expression density of 3 staining intensity levels, exclusively on ELS epithelium. AQP2 expression density was significantly elevated in MD compared to VS ( P = .003). There was no significant difference in plasma AVP, V2R, NKCC2, and TRPV4 expression. Conclusion This original study evaluates simultaneous in situ expression of AQP2, V2R, NKCC2, and TRPV4 on the human ELS in MD, with a control group. Our results show only AQP2 upregulation on the ELS of patients with MD. We suggest a constitutively increased expression of AQP2 in MD, independent of its regulatory axis (AVP-V2R). Acquired regulator sequence mutations could support this model.
Assuntos
Aquaporina 2/metabolismo , Saco Endolinfático/metabolismo , Doença de Meniere/metabolismo , Doença de Meniere/fisiopatologia , Receptores de Vasopressinas/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Canais de Cátion TRPV/metabolismo , Biópsia , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Imuno-Histoquímica , Masculino , Doença de Meniere/cirurgia , Pessoa de Meia-Idade , Estudos Prospectivos , Quebeque , SoftwareRESUMO
Hearing and balance functions of the inner ear rely on the homeostasis of the endolymphatic fluid. When disturbed, pathologic endolymphatic hydrops evolves as observed in Menière's disease. The molecular basis of inner ear fluid regulation across the endolymphatic epithelium is largely unknown. In this study we identified the specific expression of the tight junction (TJ) molecules Claudin 3, 4, 6, 7, 8, 10, and 16 in epithelial preparations of the rat inner ear endolymphatic duct (ED) and endolymphatic sac (ES) by high-throughput qPCR and immunofluorescence confocal microscopy. Further we showed that Claudin 4 in the ES is a target of arginine-vasopressin (AVP), a hormone elevated in Menière's disease. Moreover, our transmission-electron microscopy (TEM) analysis revealed that the TJs of the ED were shallow and shorter compared to the TJ of the ES indicating facilitation of a paracellular fluid transport across the ED epithelium. The significant differences in the subcellular localization of the barrier-forming protein Claudin 3 between the ED and ES epithelium further support the TEM observations. Our results indicate a high relevance of Claudin 3 and Claudin 4 as important paracellular barrier molecules in the ED and ES epithelium with potential involvement in the pathophysiology of Menière's disease.
Assuntos
Arginina Vasopressina/farmacologia , Transporte Biológico/efeitos dos fármacos , Claudinas/metabolismo , Ducto Endolinfático/metabolismo , Saco Endolinfático/metabolismo , Células Epiteliais/metabolismo , Animais , Claudinas/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos , Doença de Meniere/metabolismo , Doença de Meniere/patologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Junções Íntimas/fisiologia , Junções Íntimas/ultraestruturaRESUMO
INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression, using adjacent dura mater as control. The expression of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.
Assuntos
Saco Endolinfático/inervação , Saco Endolinfático/metabolismo , Saco Endolinfático/ultraestrutura , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Fibras Nervosas/ultraestrutura , Receptores de Neurotransmissores/biossíntese , TranscriptomaRESUMO
OBJECTIVES/HYPOTHESIS: The function of the human endolymphatic sac (ES) has been enigmatic for decades. Hypotheses include controlling endolymphatic fluid homeostasis and inner ear immunological defense. Additionally, several studies indicate a possible endocrine capacity and a yet undefined role in intracranial pressure homeostasis. However, no direct evidence of such capacity exists. This study aims to explore and identify the hypothesized endocrine capacity of the human ES. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: Twelve tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression. Genes specific for an endocrine function were determined, and results were verified by immunohistochemistry. RESULTS: Several natriuretic peptides were found expressed significantly in the ES, including uroguanylin and brain natriuretic peptide, but also peptides regulating vascular tone, including adrenomedullin 2. In addition, both neurophysin and oxytocin (OXT) were found significantly expressed. All peptides were verified by immunohistochemistry. CONCLUSION: The present data support the hypothesis that the human ES may have an endocrine/paracrine capacity through expression of several peptides with potent natriuretic activity. Furthermore, the ES may influence the hypothalamo-pituitary-adrenal axis and may regulate vasopressin receptors and aquaporin-2 channels in the inner ear via OXT expression. We hypothesize that the ES is likely to regulate inner ear endolymphatic homeostasis, possibly through secretion of several peptides, but it may also influence systemic and/or intracranial blood pressure through direct and indirect action on the vascular system and the kidney. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E201-E208, 2017.
Assuntos
Endolinfa/metabolismo , Saco Endolinfático/metabolismo , Expressão Gênica , Peptídeos Natriuréticos/metabolismo , Orelha Interna/cirurgia , Saco Endolinfático/patologia , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Imuno-Histoquímica , Peptídeo Natriurético Encefálico/metabolismo , Neuroma Acústico/patologia , Neuroma Acústico/cirurgia , Neurofisinas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ocitocina/metabolismo , Hormônios Peptídicos/metabolismo , Sistema Hipófise-Suprarrenal/metabolismoRESUMO
The endolymphatic sac (ES) is a cystic structure of the inner ear connected to the cochlea and vestibule, which plays a role in regulating ion homeostasis in inner ear fluid. Disruption of ion homeostasis can cause inner ear disorders with hearing loss and dizziness, such as Meniere's disease. Herein, we found, for the first time, functional evidence for the involvement of ß1- and ß2-adrenergic receptors in apical electrogenic ion transport by human ES epithelium by using electrophysiological/pharmacological and molecular biological methods, which were dependent on K+ and Cl- ion transport. The apical electrogenic transport was absent or very weak in ES epithelia of patients with Meniere's disease. These results suggested that adrenergic stimulation via ß1- and ß2-adrenergic receptors in the human ES was involved in regulation of inner ear fluid ion homeostasis and impairment of this response could be a pathological mechanism of Meniere's disease.
Assuntos
Saco Endolinfático/metabolismo , Epitélio/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Bário/farmacologia , Transporte Biológico/efeitos dos fármacos , Saco Endolinfático/efeitos dos fármacos , Saco Endolinfático/patologia , Epitélio/efeitos dos fármacos , Epitélio/patologia , Feminino , Humanos , Isoproterenol/farmacologia , Masculino , Doença de Meniere/patologia , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genéticaRESUMO
The human endolymphatic sac has been shown recently to have immunological capacities and has thus been proposed as the main entity protecting the inner ear from pathogen invasion, equivalent to mucosa-associated lymphoid tissue (MALT). Although the sac expresses molecules of the innate immune system, the potential expression of members of the important mucin family has not been detailed. Thus, this paper explores endolymphatic sac expression of a number of mucins and mucin precursors. Twelve fresh tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery. The expression of Mucin 1, 2, 5B/AC and 16, as well as the core structure elements (mucin precursors) T-antigen, Tn-antigen and Sialyl-Tn-antigen was investigated by immunohistochemistry. The endolymphatic sac epithelium expressed MUC1 (both apically towards the endolymphatic sac (ES) lumen and basally towards the capillary network), MUC 16 and Tn-antigen. There was no labeling after incubation with antibodies against T-antigen, sialyl-Tn-antigen, MUC2 and MUC5B/AC. We conclude that the human endolymphatic sac epithelium expresses a number of mucin molecules, which supports the hypothesis of the sac as the primary immunological tissue structure of the inner ear, equivalent to MALT in other organs. The mucins may also play a role in the formation and continuous homeostasis of the inner ear fluids, as well as the pathogenesis of Meniere's disease.
Assuntos
Saco Endolinfático/química , Saco Endolinfático/imunologia , Imunidade Inata/fisiologia , Mucina-1/análise , Mucina-1/imunologia , Orelha Interna/química , Orelha Interna/imunologia , Orelha Interna/metabolismo , Saco Endolinfático/metabolismo , Expressão Gênica , Humanos , Mucina-1/biossínteseRESUMO
OBJECTIVE: Against recent reports concerning cytokine or chemokine in mouse or rat inner ear cells, it is almost unknown whether human inner ear cells would produce cytokine or chemokine. We have for the first time established the human inner-ear-derived fibroblasts from endolymphatic sac. METHODS: The expression levels of Toll-like receptors (TLRs) in human endolymphatic sac fibroblasts, and the effect on cytokine or chemokine production of the TLR ligands have been examined. To demonstrate the intracellular pathways involved in the regulation of cytokine-production, we used specific inhibitors of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK)-signaling and N-acetyl-l-cysteine (NAC). RESULTS: TLR 2, 3, 4 and 9 were highly expressed in human endolymphatic sac fibroblasts. The TLR 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)) significantly enhanced the secretion of thymic stromal lymphopoietin (TSLP), B lymphocyte stimulator (BLyS), IFNγ-inducible protein 10 (IP-10), and macrophage inflammatory protein 1 alpha (MIP-1α) from the cells. The inhibitor of JNK strongly reduced the poly(I:C)-induced TSLP-production. The antioxidant drug, NAC also reduced the TSLP-production in fibroblasts stimulated with poly(I:C). CONCLUSION: Our findings suggest human inner-ear-endolymphatic sac derived fibroblasts can produce the cytokine and chemokine in response to TLR ligands and play a certain role during the initiation of an immune response.
Assuntos
Citocinas/metabolismo , Saco Endolinfático/metabolismo , Fibroblastos/metabolismo , Receptores Toll-Like/metabolismo , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator Ativador de Células B/efeitos dos fármacos , Fator Ativador de Células B/metabolismo , Quimiocina CXCL10/efeitos dos fármacos , Quimiocina CXCL10/metabolismo , Quimiocinas/metabolismo , Citocinas/efeitos dos fármacos , Saco Endolinfático/citologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Sequestradores de Radicais Livres/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Poli I-C/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Linfopoietina do Estroma do TimoRESUMO
Ion transport and its regulation in the endolymphatic sac (ES) are reviewed on the basis of recent lines of evidence. The morphological and physiological findings demonstrate that epithelial cells in the intermediate portion of the ES are more functional in ion transport than those in the other portions. Several ion channels, ion transporters, ion exchangers, and so on have been reported to be present in epithelial cells of ES intermediate portion. An imaging study has shown that mitochondria-rich cells in the ES intermediate portion have a higher activity of Na+, K+-ATPase and a higher Na+ permeability than other type of cells, implying that molecules related to Na+ transport, such as epithelial sodium channel (ENaC), Na+-K+-2Cl- cotransporter 2 (NKCC2) and thiazide-sensitive Na+-Cl- cotransporter (NCC), may be present in mitochondria-rich cells. Accumulated lines of evidence suggests that Na+ transport is most important in the ES, and that mitochondria-rich cells play crucial roles in Na+ transport in the ES. Several lines of evidence support the hypothesis that aldosterone may regulate Na+ transport in ES, resulting in endolymph volume regulation. The presence of molecules related to acid/base transport, such as H+-ATPase, Na+-H+ exchanger (NHE), pendrin (SLC26A4), Cl--HCO3- exchanger (SLC4A2), and carbonic anhydrase in ES epithelial cells, suggests that acid/base transport is another important one in the ES. Recent basic and clinical studies suggest that aldosterone may be involved in the effect of salt-reduced diet treatment in Meniere's disease.
Assuntos
Saco Endolinfático/metabolismo , Transporte de Íons , Doença de Meniere/metabolismo , Sódio/metabolismo , Aldosterona/fisiologia , Animais , Endolinfa/metabolismo , Canais Epiteliais de Sódio , Humanos , Canais Iônicos/metabolismo , Mitocôndrias/metabolismoRESUMO
We investigated the influence of vasopressin type 2 receptor antagonist (OPC-41061; Tolvaptan) on experimentally induced endolymphatic hydrops (EH) in guinea pigs. In the first series, the endolymphatic sac (ES) of the left ear of all animals was electrocauterized. Four weeks after surgery, the animals were allocated to four groups: three systemic applications groups (saline, OPC 10 and 100 mg/kg) and a local round window (RW) OPC 1 mg/body application group. We examined the histopathology of the temporal bones and assessed volumetric changes of the endolymphatic space in the cochlea and saccule. In the second series, we investigated the effects of systemic and topical applications of OPC on plasma vasopressin (p-VP) concentrations and plasma osmolality (p-OSM). In the first series, we found that EH was reduced in the OPC 10 mg/kg systemic and OPC RW application groups. In contrast, EH increased in the OPC 100 mg/kg systemic application group. In the second series, neither p-VP levels nor p-OSM were significantly different among the non-OPC, OPC 10 mg/kg systemic, and OPC RW application groups. However, in the OPC 100 mg/kg systemic application group, the p-VP level was significantly higher than that in other groups, and p-OSM was higher than that in the non-OPC group. The systemic application of a low dose of OPC and topical application of OPC resulted in reduced EH in the face of minimal systemic effects (p-VP and p-OSM). These findings suggest that OPC-41061 may be one useful treatment option for EH.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Benzazepinas/farmacologia , Hidropisia Endolinfática/tratamento farmacológico , Saco Endolinfático/efeitos dos fármacos , Receptores de Vasopressinas/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Administração Oral , Administração Tópica , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/administração & dosagem , Benzazepinas/administração & dosagem , Modelos Animais de Doenças , Hidropisia Endolinfática/sangue , Hidropisia Endolinfática/fisiopatologia , Saco Endolinfático/metabolismo , Saco Endolinfático/fisiopatologia , Feminino , Cobaias , Doença de Meniere/sangue , Doença de Meniere/tratamento farmacológico , Doença de Meniere/fisiopatologia , Concentração Osmolar , Receptores de Vasopressinas/metabolismo , Tolvaptan , Vasopressinas/sangueRESUMO
The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K(+) channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K(+) channels in the electrogenic transport of human ES epithelium. The identified K(+) channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K(+) transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid.
Assuntos
Saco Endolinfático/fisiologia , Epitélio/fisiologia , Canais de Potássio/fisiologia , Potássio/metabolismo , Cromatografia Líquida , Cóclea/metabolismo , Cóclea/fisiologia , Fenômenos Eletrofisiológicos , Saco Endolinfático/metabolismo , Epitélio/metabolismo , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Transporte de Íons/fisiologia , Masculino , Pessoa de Meia-Idade , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Espectrometria de Massas em TandemRESUMO
OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the innate immune system in the human endolymphatic sac. It is hypothesized that the endolymphatic sac has a significant immunological function in the human inner ear. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic-sac tissue samples. METHODS: Twelve tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression using adjacent dura mater as control. The expression of genes specific for the innate immune system was determined and results for selected key molecules verified by immunohistochemistry. RESULTS: A comprehensive overview of expressed genes of the innate immune system was obtained. Multiple key elements of both the cellular and humoral innate immune system were expressed, including Toll-like receptors 4 and 7, as well as beta-defensin and lactoferrin. CONCLUSIONS: The present data provides the first direct evidence of an immunological capacity of the human endolymphatic sac. At the molecular level, the endolymphatic sac is capable of antigen recognition and processing for initiation of an immune response. In addition, potent molecules directly toxic to invading pathogens are expressed by the sac epithelium. This evidence strongly supports the endolymphatic sac as a significant immunological entity of the inner ear. LEVEL OF EVIDENCE: N/A.
Assuntos
Biomarcadores Tumorais/genética , DNA de Neoplasias/genética , Saco Endolinfático/imunologia , Regulação Neoplásica da Expressão Gênica , Imunidade Inata/genética , Adulto , Idoso , Biomarcadores Tumorais/biossíntese , Neoplasias da Orelha/genética , Neoplasias da Orelha/metabolismo , Neoplasias da Orelha/patologia , Saco Endolinfático/metabolismo , Saco Endolinfático/patologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neurilemoma/genética , Neurilemoma/metabolismo , Neurilemoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.
Assuntos
Saco Endolinfático/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Imageamento Tridimensional , Proteínas Supressoras de Tumor/metabolismo , Envelhecimento/metabolismo , Animais , Saco Endolinfático/citologia , Células Epiteliais/metabolismo , Fluorescência , Camundongos TransgênicosRESUMO
OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic sac tissue samples. METHODS: Twelve tissue samples of the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample expression of solute carrier family genes, using adjacent dura mater as control. Immunohistochemistry was used for verification of translation of selected genes, as well as localization of the specific protein within the sac. RESULTS: An extensive representation of the SLC family genes were upregulated in the human endolymphatic sac, including SLC26a4 Pendrin, SLC4a1 sodium-bicarbonate transporter, SLC9a2 sodium-hydrogen transporter, SLC12a3 thiazide-sensitive Na-Cl transporter, and SLC34a2 sodium-phosphate transporter. CONCLUSIONS: Several important ion transporters of the SLC family are expressed in the human endolymphatic sac, including Pendrin, the thiazide-sensitive Na-Cl transporter, and the Na-phosphate transporter SLC34a2. The data provide a new knowledge base considering the ion-dependent metabolic mechanisms maintaining inner ear homeostasis. More specifically, the results indicate a strong similarity with the ion transportation occurring in the kidney collecting ducts. In addition, the findings prompt a revision of the theories behind contemporary pharmacological treatment of Ménière's disease and may broaden the understanding of the pathogenesis of BPPV.
Assuntos
Líquidos Corporais/metabolismo , Saco Endolinfático/metabolismo , Expressão Gênica , Homeostase/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Adulto , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
OBJECTIVE: Na(+) and Cl(-) are dominant ions in the endolymphatic fluid in the endolymphatic sac and are important for volume regulation in the endolymphatic sac. An epithelial sodium channel (ENaC) and other Na(+) transporters have been identified in the endolymphatic sac epithelia, and they are involved in the regulation of endolymph. Although the presence of Cl(-) channels in the endolymphatic sac epithelia has been speculated, no Cl(-) channels have been identified. In this study, we confirmed the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the endolymphatic sac by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemical staining. METHODS: Pure mRNA from endolymphatic sac epithelia was prepared using laser capture microdissection (LCM) and examined using RT-PCR. Localization of CFTR and ENaC in the endolymphatic sac was examined using immunohistochemistry. RESULTS: mRNA of the CFTR was expressed in the endolymphatic sac. Immunohistochemical analysis showed the expression of the CFTR on apical side of the endolymphatic sac epithelia and co-localization with the ENaC. CONCLUSION: RT-PCR and immunohistochemistry were used to identify the expression of CFTR in the endolymphatic sac epithelia, which gives us a clue for understanding Cl(-) transport in the endolymphatic sac. These results suggest a pathway for Cl(-), possibly through interaction with the ENaC, which may regulate the endolymph in the endolymphatic sac.