Programmed cell senescence during mammalian embryonic development.

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-ß/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.

Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice.

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.

Presence of adrenergic receptors in rat endolymphatic sac epithelial cells.

Intravenous application of catecholamines produces a depression in the endolymphatic sac direct current potential (ESP) and increases endolymphatic pressure via the ß-adrenergic receptor (AR) in guinea pigs, suggesting that catecholamines play a role in the endolymphatic system. However, the localization of ARs in the endolymphatic sac (ES) is still undetermined. The presence of ARs in the rat ES was investigated by reverse transcriptase-polymerase chain reaction using laser capture microdissection (LCM) and immunohistochemical analysis. Expression of α(1A)-, α(1B)-, α(2A)-, α(2B)-, ß(1)-, ß(2)- and ß(3)-ARs was observed in LCM samples of ES epithelia. Immunohistochemical analysis using specific antibodies showed immunofluorescence of ß(2)- and ß(3)-ARs in epithelial cells of the ES intermediate portion, and no specific staining results were obtained for α(1)-, α(2A)-, α(2B)- and ß(1)-ARs. The presence of ß(2)-AR with no clear immunostaining of ß(1)-AR in ES epithelial cells is in accordance with previous electrophysiological and pharmacological results, which suggests that ß(2)-AR mediates the action of catecholamines on the ESP. The presence of ß(3)-AR in the ES epithelial cells and its absence in the stria vascularis implies that ß(3)-AR plays a specific role in the ES.

Establishment of cell lines from the human middle and inner ear epithelial cells.

The middle ear infection is the most common childhood infection. In order to elucidate the cell and molecular mechanisms involved in bacterial recognition and innate immune response, we have established a stable human middle ear cell line, which has contributed to the current knowledge concerning the molecular pathogenesis of the middle ear infection. The inner ear, a sensory organ responsible for hearing and balance, is filled with inner ear fluid, and disturbance of the fluid homeostasis results in dizziness and hearing impairment. It has been suggested that the endolymphatic sac (ES) may play a critical role in the fluid homeostasis of the inner ear. We have established a stable human ES cell line and are undertaking cell and molecular characterization of this cell line.

Interferon-gamma suppresses Na+ -H+ exchanger in cultured human endolymphatic sac epithelial cells.

Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na(+)-H(+) exchanger (NHE) is a major determinant of intracellular pH (pH(i)), and facilitates Na(+) and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN-gamma on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, -2, and -3 isoforms was determined by real-time RT-PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH-sensitive fluorescent dye, 2',7'-bis(carbonylethyl)-5(6)-carboxyfluorescein (BCECF), and a NHE-inhibitor, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, -2, and -3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and -2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na(+)-dependent pH(i) recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN-gamma (50 nM for 24 h) suppressed mRNA expression of NHE1 and -2. IFN-gamma also suppressed functional activity of both NHE1 and -2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN-gamma suppresses the expression and functional activity of NHE1 and -2.

A new approach for selective rat endolymphatic sac epithelium collection to obtain pure specific RNA.

The endolymphatic sac (ES) is an organ that is located in the temporal bone. Its anatomical location makes ES tissue collection without any contamination very difficult, and sometimes accurate molecular analyses of the ES are prevented due to this matter. In the present study, a new selective ES epithelial tissue collection method was attempted using laser capture microdissection to obtain pure ES RNA without any contamination. The validity of this method was demonstrated by RT-PCR with three specific primer pairs against osteocalcin, calponin H1, and NKCC2, which are specific proteins in bone, smooth muscle, and kidney/ES cells, respectively. From the RT-PCR results, the high specificity and sufficient sensitivity of the new method was indicated. It is considered that the new method is optimal for ES collection without contamination and it will be able to contribute to future analyses of the ES.

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells.

CONCLUSION: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. OBJECTIVES: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. MATERIALS AND METHODS: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. RESULTS: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

Effect of inner ear blood flow changes on the endolymphatic sac.

CONCLUSIONS: That the endolymphatic sac (ES) reacts to changes in inner ear blood flow may be important for homeostasis of the inner ear fluid volume and pressure. OBJECTIVES: To elucidate the effect of changes in inner ear blood flow on the ES and to learn more about the volume and pressure regulatory function of the ES. MATERIALS AND METHODS: Epinephrine or sodium nitroprusside (SNP) was injected into the middle ear cavity of adult CBA/J mice. The ES were analyzed morphologically by light microscopy. RESULTS: Epinephrine reduced the luminal size of the ES leading to an accumulation of intraluminal homogeneous substance. Injection of SNP increased the size of the ES lumen, accompanied by a collapse of the lateral intercellular space (LIS) and dense perisaccular tissue. These changes were almost reversed 4 h after injection.

Tricellulin Expression and its Deletion Effects in the Endolymphatic Sac.

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.

Toll-like receptor ligands induce cytokine and chemokine production in human inner ear endolymphatic sac fibroblasts.

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.

Co-expression of pendrin, vacuolar H+-ATPase alpha4-subunit and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac.

The endolymph in the endolymphatic sac (ES) is acidic (pH 6.6-7). Maintaining this acidic lumen is believed to be important for the normal function of the ES. The acid-base regulation mechanisms of the ES are unknown. Here we investigated the expression patterns of acid-base regulators, including vacuolar (v)H+-ATPase (proton pump), carbonic anhydrase (CA) II, and pendrin in the murine ES epithelium by immunohistochemistry (IHC) and compared their expression patterns by double immunostaining. We found that pendrin and vH+-ATPase were co-localized in the apical membrane of a specific type of ES epithelial cell. Pendrin- and vH+-ATPase-positive cells also expressed cytoplasmic CA II. Co-expression of pendrin, vH+-ATPase, and CA II in the same subgroup of ES cells suggests that this specific type of ES cell is responsible for the acid-base balance processes in the ES and pendrin, vH+-ATPase, and CA II are involved in these processes.

The cell coat of the sensory and supporting cells of the rainbow trout saccular macula as demonstrated by reaction with ruthenium red and tannic acid.

The surface of most cells is covered by glycoconjugates. The composition and thickness of the surface coat varies among different cell types. The purpose of the present study was to demonstrate the presence of and to characterize the cell coat surrounding the cells in the saccular macula of the rainbow trout. Tissues were fixed in Karnovsky's fixative containing either ruthenium red (0.5, 1, or 2%) or tannic acid (1, 2, or 4%). The apical surface of the sensory and supporting cells reacted with both agents. Varying the concentration of the compounds within a certain range did not significantly affect the degree of tissue staining. Whereas ruthenium red staining was distributed evenly along the luminal surface of the epithelium and along the length of the stereocilia, tannic acid formed electron-dense clumps on the luminal surface of sensory and non-sensory cells and in the basal region of the macular epithelium. The stereocilia of the sensory cells also exhibited tannic acid-positive, electrondense precipitate, particularly near the distal ends of these processes, while uniform staining of the plasma membrane was seen along their lengths. The results of this study suggest that the trout saccular macula is provided with extracellular microenvironments which may be necessary for functional integrity.

Changes in ultrastructural characteristics of endolymphatic sac ribosome-rich cells of the rat during development.

It has recently been demonstrated that endolymphatic sac (ES) ribosome-rich (dark) cells respond to induced endolymph changes and are thus likely to be involved in endolymph homeostasis. Therefore, we studied the ultrastructural characteristics of rat ES ribosome-rich cells during development in order to determine the cellular distribution of organelles involved in protein metabolism, secretion and absorption, indicative for their contribution to endolymph homeostasis. During embryonal stages ribosome-rich cells contain a limited number and variety of organelles and are predominantly involved in the production of components for cell growth and differentiation. In the young adult stage (P60) three different states of ribosome-rich cells may be distinguished. State A resembles a cell with only limited metabolic activities whereas state B is characterized by numerous different intracellular organelles and is considered to be involved in production and secretion as well as absorption and degradation of complex proteins. A third cellular state, state C, is filled with phagolysosomes and contains very few other organelles. This is considered to be a final (pre)apoptotic state. Autoradiography data suggest that ES ribosome-rich cells are capable of synthesis and secretion of tyrosine-containing proteins and may thus be involved in regulation of the osmolarity of endolymph based on the capacity to bind cations as well as water molecules. In addition, ES ribosome-rich cells appear to synthesize and secrete fucosylated glycoproteins into the endolymph. In conclusion, the present data suggest that ES ribosome-rich cells are actively involved in endolymph homeostasis through secretion and absorption of complex proteins and it is hypothesized that they are able to adapt their function or activities in response to changes in endolymph composition.

Morphological and functional characteristics of cells cultured from the endolymphatic sac.

The endolymphatic sac is a part of the homeostasis-regulating system of the membranous labyrinth of the inner ear. Disturbances in the function of the endolymphatic sac are believed to be involved in the genesis of different inner ear disorders, such as endolymphatic hydrops and Ménière's disease. To make studies of the ion- and fluid-regulating mechanisms of the sac possible, a method to culture the tissue in vitro was developed. Epithelial cells and fibroblasts were morphologically characterised in the cell cultures with light and electron microscopy as well as immunohistochemically using antibodies against cytokeratin and vimentin. Since mesenchymal cells have been shown to express vimentin and epithelial cells cytokeratin, the antibodies against these two intermediate filament proteins were used to further confirm the morphological identification. In addition, some functional characteristics of the cultured cells from the endolymphatic sac were studied. ATP and K(+) were added to the cell cultures and changes in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) were determined with the fura-2 method. A rapid and transient increase in [Ca(2+)](i) could be seen in both epithelial cells and fibroblasts after applying ATP (200 microM) extracellularly. However, when K(+) was added in concentrations of 50 mM and 100 mM, no changes in [Ca(2+)](i) could be seen in either the epithelial cells or the fibroblasts. The results show that the cultured endolymphatic sac cells preserve their morphological characteristics and maintain a high viability. Accordingly, this method provides a tool for further studies of ion transport mechanisms and fluid homeostasis in the endolymphatic sac.

Atrial natriuretic peptide-like immunoreactive cells in the guinea pig inner ear.

Using specific antibodies against cardiodilatin/atrial natriuretic peptide (CDD/ANP) in a conventional immuno-histochemical method (PAP) we located ANP/CDD-like immuno-reactive cells related to the secretory area, to the sensory and to the neuronal area in the compartments of the inner ear (cochlea, utricle/ampulla, and endolymphatic sac). Immunoreactive cells were unevenly distributed in the different compartments as well as within the cochlear space. Our findings suggest that ANP/CDD may play a role in the local control of fluid and electrolyte homeostasis of the inner ear. ANP/CDD-binding sites and ANP/CDD-like immunoreactivity in the inner ear may also indicate that the peptide has an additional paracrine and/or autocrine function in the organ.

Localization of pH regulating proteins H+ATPase and Cl-/HCO3- exchanger in the guinea pig inner ear.

Mechanisms that regulate endolymphatic pH are unknown. It has long been recognized that, because of the large positive endolymphatic potential in the cochlea, a passive movement of protons would be directed out of endolymph leading to endolymphatic alkalization. However, endolymphatic pH is close to that of blood, suggesting that H+ is being secreted into endolymph. Since the kidney and the inner ear are both actively engaged in fluid and electrolyte regulation, we attempted to determine whether proteins responsible for acid secretion in the kidney also exist in the guinea pig inner ear. To that end, a monoclonal antibody against a 31 kDa subunit of a vacuolar vH+ATPase and a polyclonal, affinity purified antibody against the AE2 Cl-/HCO3- exchanger (which can also recognize AE1 under some conditions) were used. In the cochlea, the strongest immunoreactivity for the vH+ATPase was found in apical plasma membranes and apical cytoplasm of strial marginal cells. These cells were negative for the Cl-/HCO3- exchanger. Certain cells of the inner ear demonstrated both apical staining for vH+ATPase and basolateral staining for the Cl-/HCO3- exchanger; these included interdental cells and epithelial cells of the endolymphatic sac. Cochlear cell types with diffuse cytoplasmic staining for vH+ATPase and a basolaterally localized Cl-/HCO3- exchanger included inner hair cells, root cells and a subset of supporting cells in the organ of Corti. Hair cells of the utricle, saccule and cristae ampullaris also expressed both vH+ATPase and the Cl-/HCO3- exchanger, but immunostaining for the vH+ATPase was less intense and less polarized than in the cochlea. These immunocytochemical results support a role for the vH+ATPase and Cl-/HCO3- exchanger in the regulation of endolymphatic pH and suggest that certain cells (including strial marginal cells and epithelial cells of the endolymphatic sac) may be specialized for this regulation.

Postnasal maturation of the human endolymphatic sac.

OBJECTIVE/HYPOTHESIS: The objective of this study was to trace the postnatal development of the human endolymphatic sac. The endolymphatic sac starts as a pouch-like structure during the fetal period in the human but later develops into a complex network of tubules in its mature form. STUDY DESIGN: Observational study. METHODS: Light microscopic examination of the morphology of the endolymphatic sac in a population of temporal bones. RESULTS: Examination of 28 temporal bones from 19 infants and children reveals that endolymphatic sac morphological maturation is usually completed in the first year of life, although it may occur as late as 4 years of age. There was good correlation of maturation levels between sides when pairs of temporal bones were evaluated. Immature pouch-like endolymphatic sacs may appear as late as 4 years of age. CONCLUSIONS: Endolymphatic sac maturation to its adult tubular form is usually completed by the first year. This finding contrasts with other studies examining the growth of the endolymphatic sac to adult size after birth. Histological evidence suggests that the endolymphatic sac, although not morphologically mature by appearance, may still be participating at some level of functioning.

Effects of endolymphatic duct occlusion on the structure and function of the endolymphatic sac in the adult guinea pig.

The endolymphatic sac (ELS) has been shown to respond rapidly to sudden disruptions in fluid balance such as labyrinthectomy or systemic administration of hyperosmolar agents. The present study was designed to determine the ELS response to slower changes in fluid dynamics by occluding the endolymphatic duct (ELD), thereby interrupting the longitudinal flow of endolymph to the ELS. Morphologic studies and autoradiographic techniques were used to evaluate the effects of ELD obstruction on the structure and function of the ELS after 48 hours. There were no significant changes in cellular morphology and a slight decrease in the incorporation of radiolabeled glucose when compared with normal ELS cells. We conclude that it is rapid change in fluid balance that triggers the ELS response, which is not seen with disruption of longitudinal flow.

Differences in endolymphatic sac mitochondria-rich cells indicate specific functions.

OBJECTIVE/HYPOTHESIS: The purpose of the study was to examine the specific involvement of endolymphatic sac mitochondria-rich cells in endolymph homeostasis. STUDY DESIGN: Transmission electron microscopy and immunohistochemistry were performed on the endolymphatic sac of young adult rats, and two important developmental stages were also investigated. METHODS: Ultrastructural characteristics of endolymphatic sac mitochondria-rich cells were studied more concisely and compared with renal mitochondria-rich cells (i.e., the intercalated cells). In addition, expression of cytokeratins 7 and 19 was determined. RESULTS: Until birth, only one type of mitochondria-rich cell is observed in the rat endolymphatic sac. In young adult animals, distinct differences in mitochondria-rich cell ultrastructure in the endolymphatic sac enables classification into subtypes or configurations. Comparison of endolymphatic sac mitochondria-rich cells with renal intercalated cells reveals striking similarities and provides additional information on their specific function in endolymph homeostasis. Furthermore, differences in cytokeratin expression are determined in endolymphatic sac mitochondria-rich cells. CONCLUSIONS: Differences in morphology of endolymphatic sac mitochondria-rich cells develop after birth and may reflect a distinct functional or physiological state of the cell. In analogy to renal intercalated cells, the distribution patterns of H+-adenosine triphosphatase and Cl-/HCO3- exchanger may differ between subtypes. We propose that subtype A mitochondria-rich cells, from which protruding A mitochondria-rich cells are the activated state, are involved in proton secretion (apical H+-adenosine triphosphatase) and thus are potential candidates for hearing loss accompanying renal tubular acidosis. Subtype B mitochondria-rich cells are the most likely candidates to be affected in Pendred syndrome because of the assumed function of pendrin as apical Cl-/HCO3- exchanger.

Somatostatin (somatostatinlike) immunoreactive cells in the human inner ear.

Certain epithelia of the human inner ear and human endolymphatic sac display somatostatin and/or somatostatin-like immunoreactivity. Histologic sections from 13 human temporal bones and from 15 endolymphatic sacs were studied using the unlabeled antibody peroxidase-antiperoxidase technique. The somatostatin and/or somatostatin-like immunoreactive cells were located exclusively in the covering epithelium of the spiral prominence and in the epithelium of the intermediate and rugosal part of the endolymphatic sac. In the epithelium of the spiral prominence and endolymphatic sac, secretory granules of the same size and appearance as those of intestinal or pancreatic somatostatin-producing cells were demonstrated ultrastructurally. The findings are consistent with a local exocrine, paracrine, and/or endocrine system of the inner ear.