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Decreased presence or activity of human SLC26A4 at the plasma membrane is a common cause of hearing loss. SLC26A4 (Pendrin) is necessary for normal reabsorption of endolymph, the fluid bathing the inner ear. We identified the μ2 subunit of adaptor protein 2 (AP-2) complex required for clathrin-mediated endocytosis as a protein-partner of SLC26A4 involved in regulating its plasma membrane abundance. We showed that, in the endolymphatic sac, where fluid reabsorption occurs, SLC26A4 is localized along the apical microvilli of mitochondria-rich cells, in contact with the endolymph, and associated with clathrin-coated pits where μ2 and AP-2 are present. Based on SLC26A4 structure, the elements involved in SLC26A4-μ2 interaction were identified and validated experimentally, allowing modeling of this interaction at the atomic level. Pharmacological inhibition of clathrin-mediated endocytosis led to an increased plasma membrane abundance of hemagglutinin-tagged SLC26A4 virally or endogenously expressed in mitochondria-rich cells. These results indicate that the SLC26A4-μ2 interaction regulates SLC26A4 abundance at the apical surface of mitochondria-rich cells.

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 Ca 2+ in the ears with chronic endolymphatic hydrops is likely to be associated with the reduction of these two chelators of Ca 2+ 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., (© 2023. The Author(s) under exclusive licence to Association for Research in Otolaryngology.)

The inner ear, like all organs, interacts with the systemic immune system via lymphatic drainage and vascular circulation to protect itself from infections and stress such as acoustic trauma. The adult mammalian inner ear including the endolymphatic sac is populated with bone-marrow derived resident macrophages. Circulating macrophages continually renew the resident macrophage population. Cells within the endolymphatic sac participate in and affect inner ear immune responses, but specific mechanisms for the interactions are unknown. Resident macrophages are present within the cochlear modiolus, spiral ligament, stria vascularis, on the scala tympani surface of the basilar membrane and in the vestibular ganglia and connective tissue of the vestibular sensory epithelia. In general, the mammalian organ of Corti, on the other hand, does not contain resident macrophages. Although repair of the epithelium following hair cell death is performed by adjacent supporting cells, macrophages in the osseous spiral lamina have been seen to extend processes into the organ of Corti below the inner hair cells where they may assist in reducing synaptopathy. Systemic and middle ear bacterial infections, experimentally simulated by lipopolysaccharide (LPS) injections, cause circulating inflammatory cells to enter the inner ear from venules in the spiral ligament and modiolus. Presumably, this is a surveillance mechanism, and in the absence of cochlear infection, no action is taken, but if noise trauma or ototoxic drug exposure occurs simultaneously, a more aggressive immune response is mounted. Acoustic trauma alone induces influx of circulating immune cells. Vigorous immune responses to pathogens within the cochlea result in fibrotic tissue and osteoid formation within the fluid-filled inner ear spaces. Many of the signals for recruiting and activating immune cells have been identified, but little is known about exactly what the activated cells do, how they interact with resident macrophages and what signals terminate their activity., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

The endolymphatic sac is a small sac-shaped organ at the end of the membranous labyrinth of the inner ear. The endolymphatic sac absorbs the endolymph, in which the ion balance is crucial for inner ear homeostasis. Of the three sections of the endolymphatic sac, the intermediate portion is the center of endolymph absorption, particularly sodium transport, and is thought to be regulated by aldosterone. Disorders of the endolymphatic sac may cause an excess of endolymph (endolymphatic hydrops), a histological observation in Meniere's disease. A low-salt diet is an effective treatment for Meniere's disease, and is based on the assumption that the absorption of endolymph in the endolymphatic sac abates endolymphatic hydrops through a physiological increase in aldosterone level. However, the molecular basis of endolymph absorption in each portion of the endolymphatic sac is largely unknown because of difficulties in gene expression analysis, resulting from its small size and intricate structure. The present study combined reverse transcription-quantitative polymerase chain reaction and laser capture microdissection techniques to analyze the difference of gene expression of the aldosterone-controlled epithelial Na + channel, thiazide-sensitive Na + -Cl - cotransporter, and Na + , K + -ATPase genes in the three individual portions of the endolymphatic sac in a rat model. A low-salt diet increased the expression of aldosterone-controlled ion transporters, particularly in the intermediate portion of the endolymphatic sac. Our findings will contribute to the understanding of the physiological function of the endolymphatic sac and the pathophysiology of Meniere's disease., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2021 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

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.

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.

An exceptionally low calcium (Ca 2+ ) concentration in the inner ear endolymph ([Ca 2+ ] endolymph ) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca 2+ ] endolymph . Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca 2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6-8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca 2+ -ATPases SERCA1 and SERCA2, Na + /Ca 2+ exchanger NCX2, and plasma membrane Ca 2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca 2+ ] endolymph . Structural loss of the distal ES, which is consistently observed in Meniere's disease, may therefore critically disturb [Ca 2+ ] endolymph and contribute to the pathogenesis of Meniere's disease.

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 ( Slc26a4 tm1Dontuh/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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Meniere's disease (MD), a syndromal inner ear disease, is commonly associated with a pathological accumulation of endolymphatic fluid in the inner ear, termed "idiopathic" endolymphatic hydrops (iEH). Although numerous precipitating/exacerbating factors have been proposed for MD, its etiology remains elusive. Here, using immunohistochemistry and in situ protein-protein interaction detection assays, we demonstrate mineralocorticoid-controlled sodium transport mechanisms in the epithelium of the extraosseous portion of the endolymphatic sac (eES) in the murine and human inner ears. Histological analysis of the eES in an extensive series of human temporal bones consistently revealed pathological changes in the eES in cases with iEH and a clinical history of MD, but no such changes were found in cases with "secondary" EH due to other otological diseases or in healthy controls. Notably, two etiologically different pathologies-degeneration and developmental hypoplasia-that selectively affect the eES in MD were distinguished. Clinical records from MD cases with degenerative and hypoplastic eES pathology revealed distinct intergroup differences in clinical disease presentation. Overall, we have identified for the first time two inner ear pathologies that are consistently present in MD and can be directly linked to the pathogenesis of EH, and which potentially affect the phenotypical presentation of MD.

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.

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.

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

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.

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.

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 - -HCO 3 - 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.

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 β 1 - and β 2 -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 β 1 - and β 2 -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., Competing Interests: The authors declare no competing financial interests.

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., (© 2016 Anatomical Society.)

SLC26A4 mutations cause fluctuating and progressive hearing loss associated with enlargement of the vestibular aqueduct (EVA). SLC26A4 encodes a transmembrane anion exchanger called pendrin expressed in nonsensory epithelial cells of the lateral wall of cochlea, vestibular organs and endolymphatic sac. We previously described a transgenic mouse model of EVA with doxycycline (dox)-inducible expression of Slc26a4 in which administration of dox from conception to embryonic day 17.5 (DE17.5) resulted in hearing fluctuation between 1 and 3months of age. In the present study, we hypothesized that Slc26a4 is required to stabilize hearing in DE17.5 ears between 1 and 3months of age. We tested our hypothesis by evaluating the effect of postnatal re-induction of Slc26a4 expression on hearing. Readministration of dox to DE17.5 mice at postnatal day 6 (P6), but not at 1month of age, resulted in reduced click-evoked auditory brainstem response (ABR) thresholds, less fluctuation of hearing and a higher surface density of pendrin expression in spindle-shaped cells of the stria vascularis. Pendrin expression in spindle-shaped cells was inversely correlated with ABR thresholds. These findings suggest that stabilization of hearing by readministration of dox at P6 is mediated by pendrin expression in spindle-shaped cells. We conclude that early re-induction of Slc26a4 expression can prevent fluctuation of hearing in our Slc26a4-insufficient mouse model. Restoration of SLC26A4 expression and function could reduce or prevent fluctuation of hearing in EVA patients., (Published by Elsevier Ltd.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

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., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Control over ionic composition and volume of the inner ear luminal fluid endolymph is essential for normal hearing and balance. Mice deficient in either the EphB2 receptor tyrosine kinase or the cognate transmembrane ligand ephrin-B2 (Efnb2) exhibit background strain-specific vestibular-behavioral dysfunction and signs of abnormal endolymph homeostasis. Using various loss-of-function mouse models, we found that Efnb2 is required for growth and morphogenesis of the embryonic endolymphatic epithelium, a precursor of the endolymphatic sac (ES) and duct (ED), which mediate endolymph homeostasis. Conditional inactivation of Efnb2 in early-stage embryonic ear tissues disrupted cell proliferation, cell survival, and epithelial folding at the origin of the endolymphatic epithelium. This correlated with apparent absence of an ED, mis-localization of ES ion transport cells relative to inner ear sensory organs, dysplasia of the endolymph fluid space, and abnormally formed otoconia (extracellular calcite-protein composites) at later stages of embryonic development. A comparison of Efnb2 and Notch signaling-deficient mutant phenotypes indicated that these two signaling systems have distinct and non-overlapping roles in ES/ED development. Homozygous deletion of the Efnb2 C-terminus caused abnormalities similar to those found in the conditional Efnb2 null homozygote. Analyses of fetal Efnb2 C-terminus deletion heterozygotes found mis-localized ES ion transport cells only in the genetic background exhibiting vestibular dysfunction. We propose that developmental dysplasias described here are a gene dose-sensitive cause of the vestibular dysfunction observed in EphB-Efnb2 signaling-deficient mice., (Published by Elsevier Inc.)

Conclusion: We found that aquaporins (AQPs) in the fluid transporting cells, such as vestibular dark cells and endolymphatic sac epithelial cells, seem to be of importance in fluid transport in the inner ear, while those in the sensory and ganglion cells may play a functional role in sensory cell transduction., Objective: Expression of AQPs (0-12) was analyzed in normal mouse vestibular end organs., Methods: CBA/J mice were used in this study. Localization of AQPs 0-12 in the vestibular end organs and endolymphatic sac was investigated by immunohistochemistry., Results: The AQPs were found abundantly distributed in many structures in the vestibular end organs, i.e. vestibular sensory and supporting cells, vestibular dark cells, vestibular ganglion cells, and the endolymphatic sac.

Slc26a4 (Δ/Δ) mice are deaf, develop an enlarged membranous labyrinth, and thereby largely resemble the human phenotype where mutations of SLC26A4 cause an enlarged vestibular aqueduct and sensorineural hearing loss. The enlargement is likely caused by abnormal ion and fluid transport during the time of embryonic development, however, neither the mechanisms of ion transport nor the ionic composition of the luminal fluid during this time of development are known. Here we determine the ionic composition of inner ear fluids at the time at which the enlargement develops and the onset of expression of selected ion transporters. Concentrations of Na(+) and K(+) were measured with double-barreled ion-selective electrodes in the cochlea and the endolymphatic sac of Slc26a4 (Δ/+), which develop normal hearing, and of Slc26a4 (Δ/Δ) mice, which fail to develop hearing. The expression of specific ion transporters was examined by quantitative RT-PCR and immunohistochemistry. High Na(+) (∼141 mM) and low K(+) concentrations (∼11 mM) were found at embryonic day (E) 16.5 in cochlear endolymph of Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. Shortly before birth the K(+) concentration began to rise. Immediately after birth (postnatal day 0), the Na(+) and K(+) concentrations in cochlear endolymph were each ∼80 mM. In Slc26a4 (Δ/Δ) mice, the rise in the K(+) concentration occurred with a ∼3 day delay. K(+) concentrations were also found to be low (∼15 mM) in the embryonic endolymphatic sac. The onset of expression of the K(+) channel KCNQ1 and the Na(+)/2Cl(-)/K(+) cotransporter SLC12A2 occurred in the cochlea at E19.5 in Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. These data demonstrate that endolymph, at the time at which the enlargement develops, is a Na(+)-rich fluid, which transitions into a K(+)-rich fluid before birth. The data suggest that the endolymphatic enlargement caused by a loss of Slc26a4 is a consequence of disrupted Na(+) transport.

Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 (Δ/Δ) mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 (Δ/Δ) line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4., Competing Interests: The authors have declared that no competing interests exist.

Hypothesis: Pendrin acts as a Cl-/HCO3- exchanger and is responsible for endolymphatic fluid volume and pH homeostasis in human endolymphatic sac epithelial cells., Background: The endolymphatic sac (ES) is part of the membranous labyrinth in the inner ear that plays an important role in maintaining homeostasis of the endolymphatic fluid system. However, the exact mechanism of fluid volume and pH regulation is not fully understood yet. We aimed to demonstrate the expression of various anion exchangers (AEs), including pendrin, in cultured human endolymphatic sac epithelial (HESE) cells., Methods: Endolymphatic sac specimens were harvested during acoustic neuroma surgery (n = 24) using the translabyrinthine approach and then subcultured with high epidermal growth factor (EGF) (25 ng/ml) media and differentiated using low-EGF (0.5 ng/ml) media. The cultured cells were classified according to the morphology on TEM. The Cl-/HCO3- exchanger activity was assessed by pHi measurement using pH sensitive dye 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM). We performed reverse transcriptase-polymerase chain reaction and immunohistochemical staining for AEs., Results: We determined that 7.3 ± 6.7% of cells differentiated into mitochodria-rich cells and 50.2 ± 15.1 of cells differentiated into ribosome-rich cells. bAE3, AE4, SLC26A4, SLC26A6, and SLC26A11 were also expressed in cultured HESE cells. The cultured cells had Cl-/HCO3- and Cl-/formate exchange activity on the luminal membrane, which is sensitive to anion channel inhibitors (DIDS 500 μM). Furthermore, we showed that pendrin (SLC26A4) was expressed in cultured HESE cell membranes., Conclusion: Our results suggest that AEs, including pendrin, are expressed in epithelia of ES and may have role in maintaining ionic homeostasis, and the HESE culture system are useful for uncovering the functional role of ES epithelial cells.

Conclusion: It is suggested that aquaporins (AQPs) 1, 2, and 3, and vasopressin type 2 receptors (V2Rs) in the fluid transporting cells, such as stria vascularis, vestibular dark and transitional cells, and endolymphatic sac epithelial cells, have an important role in fluid transport in the inner ear, while those in the sensory and ganglion cells may play a functional role in the sensory cell transduction system., Objective: To analyze expression of AQP1, AQP2, and AQP3 as well as V2Rs in the normal mouse inner ear., Methods: CBA/J mice were used in this study. Localization of AQP1, AQP2, AQP3, and V2Rs in the inner ear, i.e. cochlea, vestibular end organs, and endolymphatic sac, was investigated by immunohistochemistry., Results: The results show that AQP1, AQP2, AQP3, and V2Rs are abundantly distributed in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, vestibular dark and transitional cells, and the endolymphatic sac.

Objective: Claudins are a family of membrane proteins which localize to tight junctions (TJs). Recent studies have shown that claudins can form pores for ions in the TJs and regulate the permeability of epithelial paracellular ion transport. The endolymphatic sac (ES) is a part of the inner ear, absorbing the endolymphatic fluid. ES dysfunction may result in endolymphatic hydrops. In this study, we focused on the paracellular transport and examined claudin mRNA expression in the ES epithelia., Materials and Methods: Total RNA was isolated from whole ES epithelia of rats by laser capture microdissection. RT-PCR was used to evaluate the expression of claudins. The expression of each claudin mRNA in the epithelial cells of rat ES was confirmed by in situ hybridization., Results: RT-PCR indicated the expression of cldn2, cldn4, cldn6, cldn7, cldn9, cldn11, cldn12, and cldn14. The expression of these claudin mRNAs in the epithelial cells of rat ES was confirmed by in situ hybridization., Conclusion: We demonstrated mRNA expression of multiple claudins in the rat ES epithelia. These results in the ES epithelia were consistent with a role of claudins in paracellular ion transport., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The endolymphatic sac tumor (ELST) is a low-grade carcinoma originating in the ear. These extremely rare tumors are capable of invading the cerebellopontine angle and might be mistaken for choroid plexus tumors (CPTs) in this region. Currently, these tumors are distinguished by conventional morphologic and immunohistochemical studies for S-100, cytokeratin, and GFAP expression, but all markers are variably expressed by both tumors. Therefore, we examined new promising markers such as EAAT-1 and Kir7.1 in 4 ELSTs and 35 CPTs located in the fourth ventricle or at the cerebellopontine angle in adults. Immunohistochemical expression of Kir7.1 was found in 30 (100%) of 30 and EAAT-1 in 32 (91%) of 35 CPTs tested and was absent in all ELSTs. Expression of GFAP was found in 16 (55%) and S-100 in 29 (100%) of 29 CPTs tested, but both markers were also expressed in 2 of 4 ELSTs examined. Specificity and sensitivity of Kir7.1 (both 100%) and EAAT-1 (100% and 91%, respectively) were superior to the values for S-100 (50% and 100%, respectively) and GFAP (50% and 55%, respectively) for distinguishing CPT from ELST.

We present a case of a papillary tumour at the cerebellopontine angle in a 41-year-old man. He presented with left-sided facial and ear pain associated with dizziness, nystagmus and hearing loss. CT scan of the temporal bone showed a destructive tumour at the left cerebellopontine angle. Surgical excision was performed and the diagnosis of the endolymphatic sac tumour was made. Endolymphatic tumour is a low grade adenocarcinoma that originates from the endolymphatic sac. The definitive diagnosis requires a combination of clinical features, radiological finding and pathological correlation.

A homeostasis of the electrochemical properties and volume of the endolymph in the inner ear is essential for hearing and equilibrium sensing and is maintained by ion-transport across an epithelial tissue, the endolymphatic sac. One of the key proteins in the maintenance is Na(+), K(+)-ATPase. Although we previously found that the Na(+), K(+)-ATPase in the sac plays a pivotal role in the control of the endolymphatic volume, the mechanism remains unclear. Therefore, in this study, we examined the expression of FXYD6, a functional modulator of the Na(+), K(+)-ATPase, in the epithelial cells of the endolymphatic sac using various approaches. Laser capture microdissection RT-PCR was used to identify FXYD6 mRNA in the endolymphatic sac. Immunolabeling with the specific antibody showed that FXYD6 was predominantly expressed in the intermediate portion of the endolymphatic sac, and it was colocalized with the Na(+), K(+)-ATPase. Because the Na(+), K(+)-ATPase in this region is known to exhibit a high level of activity, an interaction of FXYD6 with this transporter may be critically involved in the regulation of the characteristics of the endolymph., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Conclusion: The endolymphatic sac is part of the membranous inner ear and is thought to play a role in the fluid homeostasis and immune defense of the inner ear; however, the exact function of the endolymphatic sac is not fully known. Many of the detected mRNAs in this study suggest that the endolymphatic sac has multiple and diverse functions in the inner ear., Objectives: The objective of this study was to provide a comprehensive review of the genes expressed in the endolymphatic sac in the rat and perform a functional characterization based on measured mRNA abundance., Methods: Microarray technology was used to investigate the gene expression of the endolymphatic sac with the surrounding dura. Characteristic and novel endolymphatic sac genes were determined by comparing with expressions in pure dura., Results: In all, 463 genes were identified specific for the endolymphatic sac. Functional annotation clustering revealed 29 functional clusters.

Laser-capture microdissection (LCM) is an excellent tool to selectively obtain target tissue or cells. The endolymphatic sac (ES) is part of the inner ear, and a large part of the ES is located in the temporal bone. The rat ES is conventionally harvested using stereomicroscopy. In this method, contamination is unavoidable because of its size and location; therefore, additional checks, such as in situ hybridization, are necessary to confirm the cellular localization, and quantitative analysis is difficult in the ES. We have shown a selective epithelial tissue method using LCM to obtain RNA without contamination from ES epithelial tissue.

The endolymphatic sac (ES) is an inner ear organ that is connected to the cochleo-vestibular system through the endolymphatic duct. The luminal fluid of the ES contains a much higher concentration of proteins than any other compartment of the inner ear. This high protein concentration likely contributes to inner ear fluid volume regulation by creating an osmotic gradient between the ES lumen and the interstitial fluid. We characterized the protein profile of the ES luminal fluid of patients (n = 11) with enlarged vestibular aqueducts (EVA) by proteomics. In addition, we investigated differences in the protein profiles between patients with recent hearing deterioration and patients without hearing deterioration. The mean total protein concentration of the luminal fluid was 554.7±94.6 mg/dl. A total of 58 out of 517 spots detected by 2-DE were analyzed by MALDI-TOF MS. The protein profile of the luminal fluid was different from the profile of plasma. Proteins identified from 29 of the spots were also present in the MARC-filtered human plasma; however, the proteins identified from the other 25 spots were not detected in the MARC-filtered human plasma. The most abundant protein in the luminal fluid was albumin-like proteins, but most of them were not detected in MARC-filtered human plasma. The concentration of albumin-like proteins was higher in samples from patients without recent hearing deterioration than in patients with recent hearing deterioration. Consequently, the protein of ES luminal fluid is likely to be originated from both the plasma and the inner ear and considering that inner ear fluid volumes increase abnormally in patients with EVA following recent hearing deterioration, it is tempting to speculate that albumin-like proteins may be involved in the regulation of inner ear fluid volume through creation of an osmotic gradient during pathological conditions such as endolymphatic hydrops.

Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4(-/-), results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4(+/-) and Slc26a4(-/-) mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4(-/-) compared to Slc26a4(+/-) mice. In Slc26a4(+/-) and Slc26a4(-/-) mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4(+/-) mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4(-/-) mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.

Meniere's disease, characterised by episodic vertigo, fluctuating hearing loss and tinnitus, can occur under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although its pathogenesis has been proposed to be a disorder of water transport in the inner ear, subsequently, it remains unsolved, until now. A recent study revealed that both plasma stress hormone, vasopressin (pAVP) and its receptor, V2 (V2R) expression in the inner ear endolymphatic sac were significantly higher in Meniere's patients. In the present study, to link V2R-related molecules and inner ear hydrops, we examined V2R-linked water channel molecule, aquaporin-2 (AQP2) expression and translocation in human endolymphatic sac. AQP2 mRNA expression in the endolymphatic sac was significantly higher in Meniere's patients by using real-time polymerase chain reaction, as further confirmed by western blotting. AQP2-like immunoreactivity (-LIR) was translocated from luminal to basolateral side with endosomal trapping in the endolymphatic sac at the time of AVP exposure in human endolymphatic sac tissue culture. The similar AQP2-LIR translocation was also demonstrated by forskolin and blocked by vasopressin/V2R specific antagonist, OPC31260 and protein kinase A (PKA) specific antagonists, H-89 and KT-5720. We concluded that in the pathogenesis of inner ear hydrops resulting in Meniere's attacks, pAVP elevation as a result of stress and subsequent V2R-cAMP-PKA-AQP2 activation and endosomal trapping of AQP2 in the endolymphatic sac, might be important as a basis of this disease. Further experimental and clinical studies are needed to better clarify the neuroscientific relationship between stress and Meniere's disease., (© 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.)

Conclusion: Bone marrow-derived cells (BMDCs) are constitutively present in the vestibular end organs and in the endolymphatic sac, and may play a role in the maintenance of inner ear homeostasis., Objectives: The aim was to examine the distribution and characteristics of BMDCs in the vestibular end organs and in the endolymphatic sac., Methods: Bone marrow-chimeric mice were generated by bone marrow transplantation from mice genetically labeled with enhanced green fluorescent protein to C57 Bl/6 mice to visualize BMDCs. Three months after bone marrow transplantation, inner ear specimens were processed for histochemical analyses., Results: BMDCs were widely distributed in the vestibular end organs and the endolymphatic sac, whereas there were differences in the phenotype and the distribution between the vestibular end organs and the endolymphatic sac. A subpopulation of BMDCs in the vestibular end organs expressed antigen-presenting protein MHC class II. Moreover, the density of BMDCs in the vestibular end organs increased in response to local mechanical stress.

Conclusion: Intake of antisecretory factor (AF)-inducing SPC-flakes significantly reduced vertigo in patients suffering from Ménière's disease (MD). The positive effect may be due to a modulation of the transport of water and ions in the endolymphatic space., Objective: To evaluate the effects of a 3-month treatment period with SPC-flakes in patients suffering from MD., Patients and Methods: A prospective, double-blind, placebo-controlled study was performed. A total of 51 adult patients with MD were included in the study: 27 subjects treated with SPC-flakes and 24 subjects with control cereals. The patients received SPC-flakes or control cereals (1 g per kg body weight per 24 h in two servings) for 3 months. Otoneurological examinations were carried out before and after this period., Results: The severity of MD was classified according to the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) grading system. Fourteen of the 27 patients randomized to intake of the AF-inducing SPC-flakes reported decreased vertigo, compared with 2 of 24 in the control group (p < 0.001). No consistent change in the otoneurological examinations could be demonstrated in any of the groups of patients.

Conclusion: Estrogen receptor (ER) alpha and beta were expressed in the inner ear, and expression decreased with increasing age. ERalpha may alter cochlear and vestibular sensory transduction, and ERbeta may have a neuroprotective function in the inner ear., Objective: Expression of ERalpha and ERbeta in the mouse inner ear and its alterations with sex and aging were analyzed., Materials and Methods: Male and female CBA/J mice aged 8 weeks and 24 months were used. The localization and the intensity of ERalpha and ERbeta immunoreactivity in the inner ear of young and old mice of both sexes were investigated by immunohistochemistry., Results: ERalpha and ERbeta were co-expressed in the inner ear, i.e. in the nuclei of stria vascularis, outer and inner hair cells, spiral ganglion cells and vestibular ganglion cells, vestibular dark cells and endolymphatic sac. Strial marginal cells, outer hair cells and type II ganglion cells showed less expression of ERalpha. No gender- or age-related difference was noted in the expression pattern of ERalpha or ERbeta, but fluorescence intensity of ERalpha was stronger in young female mice than in young male mice. In contrast, ERbeta revealed no significant difference. In the old mice, fluorescence intensities of both ERalpha and ERbeta were significantly decreased in both sexes.

Objective: There are some kinds of sicknesses provoked by inadequate adaptation to physical and/or psychogenic stress in daily life. Delayed endolymphatic hydrops (DEH) is an inner ear disease like Ménière's disease (MD) characterized by episodic vertigo in the setting of preexisting unilateral deafness that especially occurs in civilized people with a stressful lifestyle. Its otopathologic finding was demonstrated to be inner ear endolymphatic hydrops through a temporal bone study in 1976, as in the case with MD in 1938. To elucidate the relationship between stress and the inner ear, we examined the plasma antidiuretic stress hormone vasopressin (pAVP) and its type 2 receptor (V2R) expression in the endolymphatic sac in patients with DEH., Study Design: A prospective molecular biological study., Methods: Between 1998 and 2007, we enrolled 20 patients with ipsilateral DEH to examine their pAVP during remission from vertigo attacks. Plasma vasopressin was also examined in 87 patients with unilateral MD and 30 control patients with chronic otitis media. Using the real-time polymerase chain reaction method with tissue samples obtained during surgery, we examined V2R mRNA expression in the endolymphatic sac in 6 patients with ipsilateral DEH, 9 patients with unilateral MD, and 6 control patients with acoustic neuroma., Results: Plasma vasopressin (1.5 times versus controls; unpaired t test, p = 0.140) and V2R mRNA expression in the endolymphatic sac (35.8 times versus controls; unpaired t test, p = 0.002) were higher in patients with DEH compared with those with acoustic neuroma. There were no significant differences in pAVP or V2R expression in the endolymphatic sac between DEH and MD. Patients with DEH showed a significantly negative correlation between pAVP and V2R (Pearson test, r = -0.92, p = 0.009) as in those with MD (Pearson test, r = -0.68, p = 0.043)., Conclusion: Civilized people are frequently exposed to stress in their daily life, and pAVP can easily become elevated at any time. Therefore, a negative feedback system between pAVP and V2R in the endolymphatic sac may function for inner ear fluid homeostasis against stress-induced increases in pAVP. For the pathogenesis of endolymphatic hydrops resulting in vertigo attacks in patients with DEH as well as MD, pAVP may represent a matter of consequence, but V2R overexpression in the endolymphatic sac could be much more essential as a basis for these diseases.

Conclusion: Since many water channels and pumps, such as aquaporin (AQP) 1-4, AQP6-9, vasopressin type 2 receptor (V(2)-R), Na(+)-K(+)-Cl(-) cotransporter (NKCC) 1, and NKCC2 were expressed in the rat endolymphatic sac, it should play an important role in the physiological function of acid-based metabolism and water balance in endolymphatic fluid homeostasis., Objective: To evaluate the expression and immunolocalization of AQP1-9, V(2)-R, NKCC1, and NKCC2 in the rat endolymphatic sac., Materials and Methods: Wistar rats were used. Expression of AQP1-9, V(2)-R, NKCC1, and NKCC2 mRNA in the rat endolymphatic sac was investigated using the reverse transcription-polymerase chain reaction (RT-PCR) method, and detailed immunolocalization of AQP1-9, V(2)-R, NKCC1, and NKCC2 was investigated using immunohistochemical methods, including immunofluorescence microscopy., Results: mRNAs and proteins of AQP1-4, AQP6-9, V(2)-R, NKCC1, and NKCC2 were expressed, but AQP5 was not expressed in the rat endolymphatic sac.

Earlier studies have suggested that activation of the purinergic receptor causes Na absorption from the endolymph through nonselective cation channels in endolymphatic sac (ES) epithelia. In this study, the mRNA expression patterns of the P2Y(1, 2, 4, and 6) receptors in the rat ES were examined by conventional reverse-transcription PCR. Their cellular localization was investigated by high-specificity reverse-transcription PCR using laser capture microdissection and in-situ hybridization. Our experiments showed that the mRNA of these receptors is expressed in ES epithelia. These results indicate that extracellular nucleotides may regulate ion transport by several purinergic pathways that operate through these receptors in the ES and that some of these receptors may be responsible for regulating Na+ absorption through the activation of nonselective cation channels.

Objective: To investigate the distribution and role of alpha, beta and gamma subunits of epithelial sodium channel (ENaC) in the cochlea and endolymphatic sac of guinea pig., Methods: The expression of alpha-, beta- and gamma-ENaC subunits proteins was studied by immunohistochemistry with the specific polyclonal rabbit antibodies against the alpha, beta and gamma subunits of rat ENaC. Alpha-ENaC mRNA was detected by in situ hybridization with digoxin labeled cDNA probe., Results: All three subunits of ENaC, alpha-, beta- and gamma-, were widely distributed in the labyrinth. In the cochlea, strong labeling of alpha-ENaC protein was found in the spiral limbus, and to a less extent, in the spiral ligament, organ of Corti and Reissner's membrane. The immunoreactivity of beta-ENaC was observed in the spiral ligament, spiral limbus, spiral ganglion, organ of Corti and Reissner's membrane with a less intensity than that of alpha-ENaC. Gamma-ENaC was presented primarily in the superior part of the spiral ligament, spiral limbus, spiral ganglion, and weakly in the organ of Corti and Reissner's membrane. In the endolymphatic sac, intensive immunoreactivities of all three subunits were seen in the epithelial cells and the subepithelial cells at similar intensity. Alpha-ENaC mRNA was localized in the spiral limbus, the inferior part of spiral ligament, stria vascularis, and epithelial cells and subepithelial cells of endolymphatic sac., Conclusion: Different subunits of the ENaC expressed in various cell regions of the cochlea and endolymphatic sac in distinct patterns may form the functional sodium channel to regulate the endolymph, thus serve to maintain homeostasis in inner ear.

The endolymphatic sac (ES) is a part of the membranous labyrinth and is believed to absorb endolymph. It has been well-established that the endolymph absorption is dependent on several ion transporters in a manner similar to that in the kidney, and the ES is regulated by hormones such as aldosterone and vasopressin that also affect on the kidney. The thiazide-sensitive Na(+), Cl(-) cotransporter (TSC) is an electroneutral cotransporter specific to the kidney that plays an important role in absorption of NaCl in renal tubules. In the inner ear, TSC expression has never been examined. The expression of TSC in the rat ES was examined by RT-PCR, in situ hybridization and immunohistochemistry. These analyses indicated that TSC genes and proteins were expressed in the rat ES. In contrast, it was not observed in the rat cochlea by RT-PCR. This is the first report confirming the expression of TSC in the ES.

Objective: The endolymphatic sac (ES) is part of the membranous labyrinth of the inner ear. Its central role in immunologic activity within the inner ear has been confirmed by numerous studies. The aim of this study was to investigate the expression of histamine receptors (H(1), H(2), H(3)) in the rabbit ES., Methods: A total of 10 healthy male New Zealand white rabbits weighing 2 to 3 kg were used in the experiments. For immunohistochemical studies, immunostaining was performed according to the avidin-biotin-peroxidase complex technique., Results: Serial sections of the ES of rabbits revealed the presence of H(1), H(2), and H(3) receptor immunoreactivity. Immunoreactive cells for all H(1), H(2), and H(3) were found in the epithelial and subepithelial layers of the duct and the proximal ES. In conclusion, this study showed the immunohistochemical localization of H(1), H(2), and H(3) receptors in the ES of rabbits. These receptors may be important in the homeostasis of the inner ear. In addition, they may be target receptors in the medical treatment of inner ear disorders such as endolymphatic hydrops.