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By using the whole-cell patch-clamp technique, an amiloride-sensitive Na+-selective conductance was found in epithelial cells from the endolymphatic sac (ES) epithelia of guinea-pigs. In the current-clamp configuration, the average resting membrane potential was -41.7+/-8.4 mV (n = 22). Application of amiloride at a concentration of 20 microM elicited a decrease in cation conductance that was responsible for a membrane hyperpolarization by 17.9+/-6.0 mV (n = 22). Substitution of N-methyl d-glucamine chloride (NMDG-Cl) for external NaCl led to a more significant membrane hyperpolarization by 28.4+/-8.3 mV (n = 22). At holding potential of -70 mV, amiloride and ethylisopropylamiloride (EIPA) blocked the inward current in a concentration-dependent manner over the range of concentrations of between 0.1 microM and 50 microM, with an inhibitory constant (Ki) of 1.3+/-0.4 microM (n = 7) and 1.5+/-0.3 microM (n = 5), respectively. In the voltage-clamp configuration, substitution of NMDG-Cl for external NaCl significantly reduced the inward current (n = 9), indicating that the whole-cell conductance has a high permeability for Na+. Superfusion with 20 microM amiloride induced a significant reduction of the inward current, shifted the reversal potential from -39.4+/-8.8 mV to -60.4+/-10.5 mV (n = 12), and decreased the inward conductance from 5.0+/-1.3 nS to 3.7+/-1.5 nS (n = 12). The permeability ratio of Na+ over K+, calculated from the difference in reversal potential between the currents before and after application of amiloride, was approximately 5:1. Additionally, the conductance was not activated by application of forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo-cAMP (8-Br-cAMP). These findings suggest that a low-amiloride-affinity Na+ channel localized in the ES epithelial cells may be involved in uptake of Na+ in the ES.

The present study examined the effect of glycerol administration on 3-fucosyl-N-acetyl-lactosamine (CD15) epitope expression in the endolymphatic sac (ES) of the guinea pig's inner ear. Adult guinea pigs were injected intravenously with glycerol (2 g/kg body wt.). CD15 expression was studied at 80 min up to 5 h after treatment. In untreated animals single cells and cell groups in the ES expressing CD15 epitope intra- and intercellularly were identified by immunohistochemistry to be mainly in the epithelial layer of the rugosal and distal part of the sac. Glycerol administration modulated the expression of CD15 epitope. In the epithelial layer, expression decreased and was nearly depleted after 3 h. After 4 h of glycerol administration, CD15 expression reappeared and reached the comparable level of controls. The numbers of CD15-positive cells in the lumen of the ES increased steadily and arrived at their the highest levels in 2-h specimens. The localization of CD15-epitope expression and its modulation after glycerol administration within the ES implies that this molecule may play a role in re-establishing the sac's normal function. In addition, we speculate that CD15 may be associated with processes of an immune response in the inner ear.

Aquaporin CHIP (AQP-CHIP) is a water channel protein previously identified in red blood cells and water transporting epithelia. The inner ear is an organ of hearing and balance whose normal function depends critically on maintenance of fluid homeostasis. In this study, AQP-CHIP, or a close homologue, was found in specific cells of the inner ear, as assessed by immunocytochemistry with the use of affinity-purified polyclonal antibodies against AQP-CHIP.AQP-CHIP was predominantly found in fibrocytes in close association with bone, including most of the cells lining the bony labyrinth and in fibrocytes lining the endolymphatic duct and sac. AQP-CHIP-positive cells not directly apposing bone include cells under the basilar membrane, some type III fibrocytes of the spiral ligament, fibrocytes of the spiral limbus, and the trabecular perilymphatic tissue extending from the membranous to the bony labyrinth. AQP-CHIP was also found in the periosteum of the middle ear and cranial bones, as well as in chondrocytes of the oval window and stapes. The distribution of AQP-CHIP in the inner ear suggests that AQP-CHIP may have special significance for maintenance of bone and the basilar membrane, and for function of the spiral ligament.

The diffusion pattern of horseradish peroxidase (HRP) injected into the scala tympani of the cochlear basal turn of guinea pigs was studied to test whether antigen presented in this manner can gain access to the endolymphatic sac. By two hours, HRP reaction product was found throughout the cochlea, with the greatest amounts in the spiral ligament, spiral limbus, basilar membrane, and organ of Corti. In several cochleas, very weak labeling was seen in the stria vascularis. HRP reaction product was maximal in the basal turn. By two hours, HRP reaction product was also observed in the endolymphatic sac lumen, epithelial cells, subepithelial tissue, and perisaccular connective tissue. It was more common in the proximal portion. At this time, macrophages within the lumen already appeared to have phagocytosed the HRP. By 72 hours after injection, the inner ear was cleared of HRP. The results of this study support the hypothesis that antigen in the scala tympani gains access to the endolymphatic sac lumen, where it may be presented by macrophages to the systemic immune system. Antigen most likely does not gain access to the endolymphatic space in the cochlea, but it gets to the endolymphatic sac through the perilymph and the perisaccular tissue.

The presence of separate bundles of nerve fibres in the gerbilline endolymphatic sac (ES) is described, paying particular attention to their ultrastructure and localization. One bundle, localized in the area of the subepithelium which separates the sigmoid sinus from the ES, is composed only of myelinated fascicles which, moreover, seem to have an isolated contact with the ES area. Other two single nerve fibres, much smaller in caliber, are localized in the ES subepithelium and laterally to the ES area, still close to the sigmoid sinus. These fibres, composed of myelinated and unmyelinated fascicles, seem to have a rather longitudinal orientation and, moreover, contract close relationships with the rich vascular network of the ES subepithelial tissue. As far as the course is concerned, the serial sectioning technique would suggest that the nerve fibres get very close to the ES epithelial cell layer, going proximal to distal. Speculations on the origin of this nerve contingent in the ES are proposed and discussed in view of possible new theories for pathogenesis and therapy of some inner ear diseases.

The fate of cationized ferritin (CF) injected into the endolymphatic space of the endolymphatic sac was observed by transmission electron microscopy. At 10 min after the injection, CF particles bound to the apical plasma membrane of epithelial cells of the sac and were then endocytosed with coated pits. However, they never passed through the junctional complexes between the epithelial cells. At 30 min after the injection, the CF particles were transferred to endosomes and lysosomes by small vesicles of 100-150 nm in diameter. CF particles were also found in small vesicles close to Golgi cisternae and in multivesicular bodies. Acid phosphatase positive lysosomes were found close to endosomes containing CF particles. In addition, a small fraction of the small vesicles containing CF particles became inserted into the basolateral plasma membrane. At 60 min after the injection, many CF particles were found in acid phosphatase positive secondary lysosomes. These observations suggest that endocytosis of endolymph is actively performed by the epithelial cells of the sac, and transepithelial vesicular transport across the epithelial cells occurs.

The expression of the CD 15 (3-fucosyl-N-acetyl-lactosamine) epitope was immunohistochemically studied on paraffin sections of adult guinea pig inner ears. Two regions of the inner ear expressed the epitope for CD 15: the tectorial membrane of the cochlea and the endolymphatic sac. The upper part of the main body of the tectorial membrane was deeply stained. In the rugosal and distal part of the endolymphatic sac several unevenly distributed cells showed strong intra- and extracellular localization of the CD15 epitope. The CD15 epitope is associated with a transduction structure (tectorial membrane) and with a "volume regulating" compartment (endolymphatic sac) and may be involved in the maintenance of the structural integrity of both.

The etiopathogenesis of Menière's disease has remained controversial since the early 1900s. Many investigators have studied the histopathology of the inner ear in patients with this disorder. Three basic pathologic mechanisms have emerged: fibrosis of the endolymphatic sac and vestibular epithelia, altered glycoprotein metabolism, and inner ear viral infection. This article reviews the current understanding of these three basic pathologic processes.

This study was designed to investigate the expression of intercellular adhesion molecule-1 (ICAM-1) on the spiral modiolar vein (SMV) with its collecting venules (CVs) and the venules of the endolymphatic sac during inner ear inflammation. These data will further elucidate the role of adhesion molecules in extravasation of inflammatory cells from blood vessels during an inner ear immune response. Labyrinthitis was induced in rats by inoculation of keyhole limpet hemocyanin into the scala tympani of animals who had been systemically sensitized to it. Expression of ICAM-1 was examined with a mouse monoclonal antibody to rat ICAM-1 by immunohistochemistry. ICAM-1 was found weakly on the epithelium of SMVs and CVs as early as 6 hours postchallenge, reaching a maximum by day 2 and then fading away gradually. The maximum influx of immunocompetent cells into the cochlea was seen between days 3 and 7. Staining for ICAM-1 was observed on the epithelium of the endolymphatic sac and perisaccular region at 12 and 24 hours, respectively, and this was associated with infiltration of cells into these areas 3 days postchallenge. By day 28, the inner ear had developed endolymphatic hydrops, but at this time it showed almost no significant staining with anti-ICAM-1. The molecule was also expressed in the mesothelium of perilymph, the perineurium of cochlear nerves, the spiral ligament, and the basal cells of the stria vascularis following immunization. Our data provide evidence that endothelial cells of the SMV and its CVs, as well as other inner sites, have the potential to express ICAM-1. This expression precedes the influx of immune cells; therefore, it is possible that this ligand plays a pivotal role in the onset of inflammation in the inner ear. This study also confirmed that the immune response results in endolymphatic hydrops as a long-term consequence.

The distribution of mRNAs coding for three different isoforms of the alpha and two of the beta subunit of Na,K-ATPase was studied in the rat vestibular system using in situ mRNA hybridization. The dark cells of the utricular macula and of the ampullae of the semicircular canals expressed high levels of mRNA encoding the alpha 1 and beta 2 isoforms of the Na,K-ATPase, a composition that in the cochlea has been uniquely found in the stria vascularis. However, in the dark cells it was coupled with a weak expression of beta 1. The sensory epithelia of the vestibular system showed alpha 1 and beta 1 expression at much higher levels than in the cochlear sensory epithelium. Weak expression limited to the alpha 1, beta 1, and beta 2 isoforms was observed in the endolymphatic sac, contrasting previous cytochemical results which suggested extensive Na,K-ATPase activity to the sac. The results support the widely held hypothesis that the vestibular dark cells play a role similar to that of the stria vascularis in endolymph production. They indicate that the ion transport requirements of the vestibular sensory epithelia may be different than those in the cochlea. They also suggest that the endolymphatic sac may not be a major site of inner ear ion exchange.

Adrenergic innervation of the human endolymphatic sac (ES) has not been verified previously. To investigate this question a sensitive histofluorescence method for visualization of catecholamines and serotonin, using a solution composed of sucrose-potassium phosphate-glyoxylic acid (SPG) in cryostat sections, was employed. Three human ES specimens were obtained during surgery for acoustic neuroma. Distinct fluorescence in the subepithelial tissue, indicating the presence of monoaminergic neurones and their axonal varicosities, was observed. SPG-positive terminal nerve fibres around small ES capillaries and subendothelially were also seen. Like the effects of sympathetic stimulation elsewhere in the human body, the ES might respond to such stimulation with, for example, vasoconstriction and increased transepithelial water transport. Since the ES is thought to be responsible for maintaining inner ear fluid homeostasis, adrenergic influence could be important for it to function properly.

This study was undertaken with the aim of localizing hyaluronan (hyaluronic acid, HYA) in tissue sections of the human endolymphatic sac by use of a hyaluronan-binding affinity protein and the avidin-biotin/peroxidase staining procedure. Five human endolymphatic sacs were removed during surgery for acoustic neuroma. After microwave-aided fixation and decalcification, paraffin-embedded sections were prepared by routine histological methods. HYA was detected in some of the intraluminal substance as well as in parts of the epithelial lining, mainly in the rugose portions of the endolymphatic sac. HYA was observed intracellularly in epithelial cells. It was also found in the subepithelial tissue near the epithelia and close to the bony aqueduct. The distribution of HYA was uneven at all locations. The finding of HYA within the human endolymphatic sac may imply that this substance has important functions in the control of inner ear fluid homeostasis.

The distribution of Na+, K(+)-ATPase, Ca(++)-ATPase, carbonic anhydrase, and calcium-binding proteins were investigated immunohistochemically in paraffin sections of guinea pig inner ears. Marginal cells of the stria vascularis, type II fibrocytes of the spiral ligament, and cells in supralimbal and suprastrial regions, were positive for Na+, K(+)-ATPase. Type I fibrocytes of the spiral ligament were positive for Ca(++)-ATPase, carbonic anhydrase, calmodulin and osteopontin. In the vestibular system, dark cells were positive for Na+, K(+)-ATPase. However, these cells and subepithelial fibrocytes were negative for Ca(++)-ATPase, carbonic anhydrase, and the calcium-binding proteins. In the endolymphatic sac, epithelial cells in intermediate and distal portions were positive for Na+, K(+)-ATPase, but the reaction was less than that in the stria. The same endolymphatic sac cells that were positive for Na+, K(+)-ATPase were also positive for Ca(++)-ATPase and calcium-binding proteins, but negative for carbonic anhydrase. The presence of Ca(++)-ATPase and calcium-binding proteins in the type I fibrocytes of the spiral ligament suggests that these cells are involved in mediating Ca++ regulation. Lower levels of Na+, K(+)-ATPase and the co-existence of Ca(++)-ATPase and calcium-binding proteins in the epithelial cells of the endolymphatic sac indicate that these cells have a distinctive role in ion transport that is different from that of the cells of the stria vascularis and vestibular dark cells.

A constant volume of horseradish peroxidase (HRP) was injected directly into the endolymphatic sac (ES) lumen of the guinea pig to investigate the detailed absorption activity and the barrier properties of the ES. The reaction products were analyzed using an ultrastructural and morphometric method 1 to 10 h after the injection of this tracer. The epithelial cells in the proximal portion did not absorb the intraluminal HRP at any intervals after the tracer injection. The epithelial cells of the intermediate portion were classified clearly into two types according to their absorption activity: active-absorptive, and non-active cells. Uptake in the active-absorptive cells reached its maximal rate 8 h after the injection and then decreased. The active-absorptive cells in teh intermediate portion are considered to play a major role in the macromolecular absorption in the ES epithelium. The non-active cells scarcely absorbed the intraluminal HRP, suggesting that these cells are not involved in the macromolecular absorption. The absorption activity in the distal portion was lower than that in the active-absorptive cells in the intermediate portion 1 to 8 h after the HRP injection, while higher 10 h after the injection. Not only the intermediate portion but also the distal portion may play an active role in the macromolecular absorption. In no portion of the ES did intraluminal HRP penetrate beyond the junctional complexes between epithelial cells or through the cytoplasm. It is conceivable that there is a tight barrier to the intraluminal macromolecules in the epithelial linings of the ES.

We investigated electron microscopically the changes of anionic sites of a charge barrier in the capillary basement membrane of the stria vascularis and endolymphatic sac following inner ear immune reactions. Hartley guinea pigs were immunized with bovine type II collagen, keyhole limpet hemocyanin, or horseradish peroxidase, with boosted and challenged antigens through the stylomastoid foramen. Animals were killed painlessly from several days up to 56 days after the antigen challenge. Polyethylenimine was used as a cationic tracer in order to observe the localization of anionic sites of the charge. In the animals immunized with bovine type II collagen or horseradish peroxidase, a significant decrease of anionic charge in the stria and the sac was found in the early stage of immunization. However, the keyhold limpet hemocyanin immunization group did not show any remarkable changes in the charge because of its lesser transfer into the inner ear due to of its high molecular weight and negative surface charge. A decrease of the charge under immunologic conditions may induce a hyperpermeability of vessels and a malabsorption of endolymph, and thus may cause endolymphatic hydrops.

Using a newly developed injection technique, the absorption of horseradish peroxidase (HRP) in the endolymphatic sac (ES) of the guinea pig was examined by light and electron microscopy. HRP (molecular weight: 40,000; molecular diameter: about 5-nm) was directly injected into the lumen of the ES by electrophoresis after the recording of a direct current potential in the ES lumen. Both the macrophages floating in the ES lumen and the epithelial cells in the intermediate portion of the ES absorbed intraluminal HRP. The macrophages internalized the intraluminal HRP at a higher rate than the epithelial cells, suggesting that macrophages play a major role in macromolecular absorption in the ES. It was considered that the macrophages took up intraluminal HRP by phagocytosis, while the epithelial cells of the intermediate portion took it up by pinocytosis. In contrast, the epithelial cells in the proximal portion of the ES absorbed little HRP. No penetration through the junctional complexes between epithelial cells was observed in either the intermediate or the proximal portion at any interval after the injection of HRP. This finding indicates that these junctional complexes are impermeable to intraluminal HRP.

The endolymphatic sac (ELS) has been implicated in the maintenance of endolymph volume and pressure in the membranous labyrinth through fluid resorption and secretion of osmotically active substances, known as glycosaminoglycans (GAGs). To assess whether the ELS cells synthesize the GAG, hyaluronan (HA), and to further elucidate the secretory function of the ELS, a series of experiments were carried out on in vitro tissue-cultured, fetal murine ELSs. In phase 1 of the investigation, the ELSs that were attached to a small portion of the posterior labyrinth, were resected from whole otocyst specimens and studied in tissue culture. This model was chosen to determine whether a change in endolymph homeostasis affects ELS activity. Radiolabeled 14C glucose incorporation was used to evaluate HA synthesis by ELS cells when cultured in vitro. Approximately 43 percent of the incorporated 14C glucose radiolabel was digested by Streptomyces hyaluronidase (an HA-specific hyaluronidase), suggesting HA synthesis by sac cells. In phase 2 of our experiments, the ELSs were not resected from the otocysts. Instead, they were left attached to intact membranous labyrinths, and whole otocysts were cultured. Studies analogous to those of phase 1, assessing the ability of the ELS cells to incorporate 14C glucose into HA, were performed on these specimens. Streptomyces hyaluronidase treatment of these ELS specimens resulted in a reduction in the removal of radiolabel. Therefore, the ELS cells of intact otocysts incorporated less 14C glucose into HA when compared to the ELS cells of the resected specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

The endolymphatic sac (ELS) has been the subject of investigation for many years and yet its overall function remains unclear. It is believed mainly to be involved in the regulation of endolymph through fluid resorption and secretion of osmotically active substances. The present study was performed using in vitro cultured, fetal ELSs of 18 to 19 day gestational mice, to assess whether the ELS cells can synthesize the osmotically active polysaccharide, hyaluronan (HA). The ELS and portions of the membranous labyrinth were dissected from whole otocyst specimens and placed in 14C glucose-enhanced tissue culture media. A light microscopic (LM), autoradiographic study was performed to assess whether 14C glucose could be incorporated by the tissue into HA. Both the ELS cells and the adjacent cartilage demonstrated radiolabel incorporation within 4 hours of incubation in tissue culture medium, with increased radiolabel density in ELS cells after 24 hours of incubation. HA-specific hyaluronidase (HAase) resulted in removal of HAase-sensitive compounds in the ELS in both 4-hour and 24-hour cultured specimens when compared to adjacent cartilage cells (p = 0.001). Approximately 43 percent of the radiolabel was incorporated into HA in ELS specimens, as compared to a 22 percent HA synthesis in the adjacent cartilage tissue, suggesting preferential synthesis by ELS cells. The dissected murine otocysts demonstrate viability in vitro as measured by their ability to incorporate 14C glucose from tissue culture medium. Under these conditions the cultured ELS demonstrates an ability to synthesize HA. A theory of ELS function is proposed.

The endolymphatic sac is believed to play a major role in membranous labyrinth homeostasis by controlling the volume of endolymph, removing debris, and participating in the immune response of the inner ear. The endolymphatic sac is postulated to absorb endolymph and to synthesize and secrete high-molecular-weight and osmotically active glycosaminoglycans (GAGs). The present study examines the ability of in vitro adult guinea pig endolymphatic sac cells to synthesize complex proteins and polysaccharides. The intent is to characterize the nature of these compounds by studying carbon-14 (14C) glucose incorporation in tissue cultured endolymphatic sac specimens using autoradiographic and specific enzymatic digestion techniques. Our results suggest that sac cells can synthesize GAGs and proteins in vitro in proportionately larger amounts than surrounding connective tissue and dura. The principal GAG synthesized by the endolymphatic sac appears to be hyaluronan.

This study aimed to investigate the origin of the free cells in the lumen of the endolymphatic sac (ES). Activation of the cells was accomplished through osmotic induction using glycerol. The ES and the perisaccular tissue were analyzed with special reference to the activity of periaqueductal bone marrow cells after different time intervals following the injection of hyperosmotic agents. The results show that the perisaccular or periaqueductal bone marrow space may constitute a source of some of the free cells occurring in the ES. Osmotic challenging of the inner ear may cause activation of the periaqueductal bone marrow, initiating the locomotion and migration of cells (mostly monocytes, neutrophils and eosinophilic leukocytes) along bone marrow sinusoids that frequently anastomose with the ES vessels. The free cells show signs of transepithelial diapedesis and, in the lumen of the ES, cells may develop into phagocytes which initiate the ingestion and degradation of secreted macromolecular aggregates. It is thought that osmotic alterations in the inner ear may give rise to local changes in or around the ES, leading to the chemotactic attraction of bone marrow cells. The results verify the existence of a complex sugar/protein aggregate metabolism over the wall of the ES, which is linked to the turnover of free cells. The findings may indicate that ES macrophages are important in the regulation of inner ear fluid homeostasis.

Modern immunohistochemical methods allow a functional characterization of the human endolymphatic sac (ES) and its associated cell populations. The currently available immunohistochemical data on the extraosseous part of the human ES support the assumption that the epithelium is metabolically active and capable of both secretion and absorption. The reactivity of some epithelial cells with antibodies against neuroendocrine antigens implies a paracrine activity of the human ES. Further results provide evidence for a possible role of the human ES in inner ear immune defense and indicate a putative functional relationship of the human ES to the common mucosa-associated immune system.

A time sequence study was performed on experimental animals to investigate long-term effects of intravenously administered glycerol on the epithelial cell activity in the endolymphatic sac (ES) and on the ES volume. Fifteen to 60 min after systemic glycerol administration, the ES volume decreased. During this time, the ES lumen was often obliterated. Subsequently, the lumen dilated. Meanwhile, many light epithelial cells showed granules with floccular and/or lamellar contents. Concomitant deposition of floccular material into the luminal space suggested secretion of macromolecular substances, presumably from these transformed light cells. The number of granule-containing cells was significantly increased 2 h (p < 0.01) and 4 h (p < 0.01) after glycerol administration. The ES was significantly dilated after 4 h (p < 0.01) and 6 h (p < 0.05). Thus systemic alterations in osmotic pressure led to a reversible change in ES volume, with initial collapse followed by dilation and normalization after 8 h. The secretory response of the ES preceded the volume increase. A great variability in ES volume indicated high compliance of this organ system. A secretion/degradation system or turnover of osmotically active macromolecular complexes in the epithelial lining and ES lumen seems to be linked to the ability of the ES to hold fluid volumes within a wide range. This may serve as a micromechanical pressure-volume-regulating device for monitoring endolymph fluid homeostasis.

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.

The results of a light and transmission electron microscopic analysis of an endolymphatic sac (ES) from a patient suffering from episodic vertigo, tinnitus, and hearing loss are presented. A biopsy of the intraosseous portion of the ES was obtained during a translabyrinthine approach to section the vestibular nerve in the internal acoustic meatus. The material consisted mainly of tubular epithelial structures filled with heavily stained material. Pathologically dilated and degranulated rough endoplasmic reticuli and disaggregation of polyribosomes with accumulation of solitary ribosomes in the cytosol and endoplasmic reticulum suggested a disturbed epithelial cell protein synthesis. Ultrastructural evidence of an increased merocrine secretion of glycoprotein conjugates into the ES was noted. This made it possible to analyze the presumed intracellular secretory pathways. An increased number of intraepithelial lymphocytes and monocytes was observed. Since the inner ear had been subjected to surgical intervention before the vestibular nerve section, no conclusions can be drawn as to whether the patient's symptoms were related to the disturbed protein metabolism and hypersecretion of glycoprotein conjugates into the ES. The findings support earlier experimental results that indicate that the ES has not only a resorptive function but also a secretory one.

The capillary permeability and the basolateral endocytic pathway of the mouse endolymphatic sac (ES) epithelium were examined in vivo using intravenous injection of horseradish peroxidase (HRP). The capillaries of the ES were classified as either fenestrated or non-fenestrated. Because dense reaction products were observed soon after injection of HRP in macrophages near both types of capillaries, they were both considered to be permeable to macromolecules. In non-fenestrated capillaries, the basement membrane and small vesicles of the endothelium of the ES were stained with reaction product. These non-fenestrated capillaries were considered to be of muscle type. After 15 min, ES epithelial cells absorbed HRP basolaterally, and the multivesicular bodies and lysosomes of epithelial cells were stained with reaction product. The process of basolateral absorption in the ES epithelium was similar to that in the intestinal epithelium. Our results provide further evidence that the ES is a metabolically active organ which plays an important role in fluid transport in the inner ear.

Unilateral endolymphatic hydrops was produced in guinea pigs by cauterization of the endolymphatic sac. Measurements of compound action potential (CAP), cochlear microphonics (CM) and negative summating potential (-SP) confirmed endolymphatic hydrops three months after surgery. In both control and hydropic ears, reaction product of HRP was observed only on the perilymphatic surface of the epithelial cells of Reissner's membrane after 10 min perfusion, while it was observed on both the endolymphatic and perilymphatic surfaces after 30 min perfusion. Epithelial tight junctions were not stained and labelled pinocytotic vesicles were observed in the epithelial cells. These findings suggest that the transport of HRP through Reissner's membrane is unchanged in endolymphatic hydrops and that the epithelial junctions are tight regardless of the distension of Reissner's membrane.

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.

The growth rate of calcium carbonate (CaCO3) crystals in the endolymphatic sac was modulated, and morphological changes in this organ were observed by light and electron microscopy. When calcium chloride (CaCl2) was given to the three frog for a short period (3 days to 2 weeks), CaCO3 crystal production was accelerated. Epithelial cells enlarged, their rough endoplasmic reticulum (rER) and Golgi apparatus developed, and dense material increased around CaCO3 crystals and/or in the endolymphatic lumen. In addition, multiluminal endolymphatic chambers appeared in some frogs. On the other hand, as the CaCl2 loading period lengthened and CaCO3 crystal formation decreased or stopped, the epithelial cells became flat and extended with scanty cytoplasm, and the rER and Golgi apparatus decreased in number and size. Furthermore, the amount of dense material around CaCO3 crystals and in the lumen decreased. These findings suggest that the rER, Golgi apparatus and dense material have key roles in the production of CaCO3 crystals.

The murine endolymphatic sac (ELS) can survive for several weeks in tissue culture. The epithelial cells mature in vitro and demonstrate functional activity under these artificial conditions. The goal of the present study was to investigate fluid and particle transport between the ELS cells, the ELS lumen, and the surrounding tissue culture medium. A series of tracers, including anti-mouse IgG-labelled colloidal gold, horseradish peroxidase (HRP), and ruthenium red (RR) were used to elucidate different aspects of ELS transport. Results indicated that both light and dark cells of the ELS took up HRP from both the luminal and the basal side of the epithelial cells, forming coated vesicles. Only luminal uptake occurred when the HRP was injected into the lumen, and only basal uptake was observed when the HRP was placed in the culture media. HRP introduced into the tissue culture media never entered the lumen of the ELS. Anti-mouse IgG labelled with gold showed no uptake at all from either side of the epithelial cells. Gold could be found only in membrane-bound vesicles in the cytoplasm of reticuloendothelial (RES) cells in the ELS lumen and lateral intercellular spaces (LIS) of the sac. This suggests that there are no IgG sites in sac cells and that the circulating RES cells seen in the lumen of the ELS come from the surrounding connective tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of endolymphatic sac (ES) dysfunction in the etiopathogenesis of Meniere's disease has remained controversial since the early 1900s. The first reports of the ultrastructural (transmission electron microscopy, TEM) pathology of the human ES in Meniere's disease have been published only in the last decade. These studies have been based on biopsies of the extraosseous (intradural) ES and in no cases has the TEM appearance of the intraosseous ES been described. Likewise the control material used has been from biopsies of extraosseous ES taken from patients with acoustic schwannomas. To date, no reports have compared the ultrastructure of the intrasosseous ES from normal control patients to patients with Meniere's disease. Since the intraosseous ES is believed to be the most active portion of the entire ES, studies were made of the ultrastructure of ten normal interosseous human ESs fixed immediately after death and obtained at autopsy (control material). Fourteen patients undergoing translabyrinthine (TL) neurotologic procedures (10, TL resection of acoustic schwannoma; 4, TL eighth cranial nerve section for Meniere's disease) had the entire vestibular aqueduct, containing the endolymphatic duct and the intraosseous ES, removed and processed for TEM. The roles of the epithelium, subepithelial space, and vasculature were morphologically studied to evaluate possible ES pathology in Meniere's disease and in patients with acoustic schwannoma. Wide anatomic variation in the distribution and density of the subepithelial connective tissue was observed in all groups. There was no difference in the TEM appearance of the intraosseous ES from normal controls and patients with eighth nerve schwannoma, nor was there any difference in the ES collagen deposition in patients with Meniere's disease. The ESs from two patients with Meniere's disease showed evidence of abnormal glycoprotein metabolism; one with possible hypersecretion and one with possible alteration of degradation of resorbed glycoprotein. The results of this preliminary study suggest that "perisaccular fibrosis" of the intraosseous ES was not a pathologic feature in these four cases of Meniere's disease and that alteration of ES glycoprotein secretion/resorption may be of etiopathologic significance.

A recent report from our laboratory describes techniques for growing fetal murine endolymphatic sac (ELS) in vitro in tissue culture. The purpose of the present study is twofold: first, to determine whether the in vitro endolymphatic cells function in their artificial environment; and second, to begin to understand the nature of luminal cell function in vitro when separated from subepithelial connective tissue and blood. The endolymphatic sac was dissected from 18th gestational day fetal mouse otocysts and grown in DME tissue culture media for 7 days. Light microscopic sections of the endolymphatic sac were stained with Periodic Acid Schiff (PAS) reagent, alcian blue, Verhoff's elastin stain, and van Gieson's collagen stain to reveal deposits of glycogen as well as mucopolysaccharides, elastic fibers, and collagen. Controls for glycogen staining were prepared using the amylase enzyme. For electron microscopical evaluation, 'en bloc' staining was used, to confirm the location of cellular glycogen. Results indicate that in vitro luminal cells of the murine ELS are viable and show signs of functional activity with the markers used. The luminal substance and apical cytoplasm shows distinct purple metachromasia with toluidine blue and PAS-positive staining that disappears with amylase digestion. The ELS cells and luminal substance were negative for alcian blue at pH 1.0 and pH 2.5. These findings are similar to those seen in in vivo murine control sac specimens and demonstrate the ability of cultured sac cells to store glycogen and produce complex carbohydrates. The similarity in staining between the luminal substance and the cytoplasm of sac cells in all in vitro specimens suggests some secretory function by sac cells.

A panel of monoclonal and polyclonal antibodies has been used to study the epithelium of the extraosseous part of the human endolymphatic sac (ES) by immunohistochemistry. The ES epithelium reacted with several epithelial markers such as Lu-5, different anticytokeratins, antiepithelial membrane antigen, and anticarcinoembryonic antigen. Unexpectedly, all epithelial cells also revealed a strong positive reaction for the mesenchymal marker vimentin and for S-100 protein. 'Neuroendocrine', a neurosecretory antigen, and neuron-specific enolase reactivity was detected in a few epithelial cells. The results support the assumption that the ES epithelium is metabolically active and capable of secretion and resorption. These findings are in keeping with results of functional experiments in animals. The demonstration of neurosecretory antigen and neuron-specific enolase in some cells indicate that the epithelium may also have paracrine functions.

The distribution patterns of horseradish peroxidase (HRP) reaction products in the inner ears of guinea pigs were studied after injections into the middle ear cavities and perilymphatic and subarachnoid spaces. The normal round window membrane resisted HRP penetration from the middle ear side, but when it became pathological after repeated applications, its permeability increased. HRP deposits were found in the cochlear and vestibular sensory cells and in the lumen of the endolymphatic sac. HRP reaction products were minimal at the cochlear apex even after long survival times, suggesting that perilymph flow, if it exists, is rather weak toward this direction. Whereas the stria vascularis is impermeable to HRP, the vestibular dark cells were accessible; thus, the metabolic activity of the dark cells can be more readily controlled by drug applications through the middle ear cavity. The finding of HRP deposits on the scala vestibuli surface of Reissner's membrane and the absence of HRP in the upper portion of the spiral ligament at the basal turn suggests that the oval window is a secondary route of passage for these particles from the middle ear cavity to the inner ear. In order to determine the route of HRP into the endolymphatic sac from the middle ear cavity or scala tympani, the cochlear and/or vestibular aqueducts were obliterated singly or together. The route of HRP was determined to be the vestibular aqueduct. HRP is believed to enter the sac lumen through Reissner's and saccular membranes and the sac epithelium. Drugs and other large molecular substances instilled in or gaining access to the middle ear cavity may reach the endolymphatic sac causing its functional alteration.

Histologic and histochemical studies of the endolymphatic sac of full-term rat fetuses were undertaken to clarify the anatomy of the endolymphatic sac, and the mechanism of endolymph circulation and regulation. Hitherto undescribed structure enveloping the sac with blood vessels extending from the sigmoid sinus resembling the kidney glomerulus was seen. This endolymphatic "glomerulus" is believed to provide a means of active exchange of water and electrolytes between blood vessels and the endolymphatic sac.

After obliteration of endolymphatic sac and duct, no significant alteration in d.c. potential, K+ activity or protein content during the development of endolymphatic hydrops could be observed. The K+ activity of endolymphatic sac was only one-ninth of that in the cochlear part of the endolymph. After injection of Thorotrast into the endolymphatic sac, aggregated particles were found in the cochlear endolymph 2 days later. Ethacrynic acid (60 mg/kg) caused a decrease in K+ activity in the cochlear endolymph, but an increase in the endolymphatic sac. Intercellular edema was observed both in stria vascularis and in endolymphatic sac by light and electronmicroscopy after ethacrynic acid administration. These results suggest the existence of an active transport mechanism in the endolymphatic sac epithelium.

The review of literature about the inner ear fluid circulation theory was presented. The creation and absorption of fluids were discussed as well as the role of endolymphatic sac in fluid pression regulation.

In the present investigation we studied the morphology o the endolymphatic sac in guinea pig fetuses (age 20-, 30-, 45-, 60-days-old and newborns). Twenty-day and 30-days-old guinea pig fetuses often displayed small prismatic or hexagonally shaped granules, presumably representing miniature otoconia. The granules appeared freely in the lumen of the endolymphatic sac as well as incorporated in the cytoplasm of the freely floating cells or macrophages. The origin of these "sac otoconia' as well as the possible role of the endolymphatic sac in statoconis turnover and metabolism is discussed.

Ca++ activity and DC potential were measured in vivo in the endolymphatic sac (ES) of guinea pigs by means of double-barrelled ion-sensitive microelectrodes. We found a positive DC potential of 14 mV and a Ca++ activity of 4.7 X 10(-4) M. Anoxia induced a decrease in the DC potential and an increase in Ca++ activity; however, no negative DC potential was measured during permanent anoxia. The Ca++ activity measured was in agreement with the Ca++ value calculated with the Nernst equation, assuming a positive DC potential. On the basis of these data, it was suggested that the Ca++ in the ES is in electrochemical equilibrium with the surrounding fluid and no active Ca++ transport is necessary in the ES of guinea pigs.

We examined the effects of anoxia and ethacrynic acid on the endolymphatic potential and cation activity in the superior ampulla of the guinea pig, using double-barrelled ion-exchanger microelectrodes. In normal guinea pigs the ampullar endolymphatic potential was + 3.9 +/- 1.2 mV (n = 32), the Cl- activity 130 +/- 4.6 mM (n = 9), and the Na+ activity 18.4 +/- 4.4 mM (n = 20). After anoxia, the ampullar DC potential decreased rapidly and reversed its polarity within 5 min. It then decreased gradually for 60 min and increased afterwards to approximately zero. K+ activity decreased gradually after a latency of 10 min, whereas Na+ activity increased. During the gradual decrease of a negative ampullar endolymphatic potential, an increase in Na+ activity was observed. Thirty minutes after the intravenous injection of ethacrynic acid (100 mg/kg), the potential began to decrease, changed to a negative polarity, and approached a maximum negative level 100 min after the injection. The decrease in K+ activity corresponded to the reduction of potential whereas Na+ activity remained unchanged. The DC potential of the endolymphatic sac in normal guinea pigs was + 14.7 +/- 5.1 mV (n = 17). The Na+ concentration was 103.3 +/- 14.7 mM (n = 14) and the K+ concentration was 11.6 +/- 0.8 mM (n = 4). After anoxia, the DC potential decreased rapidly and approached 0 mV within 8 min. No negative potential could be observed. The Na+ concentration began to increase 2 min after anoxia and reached the extracellular Na+ concentration about 30 min later.(ABSTRACT TRUNCATED AT 250 WORDS)

The lateral intercellular spaces (LIS) in the epithelium of the endolymphatic sac in different mammals were studied using light and transmission electron microscopy. The reason for the study was that widened LIS are known to occur in fluid transporting epithelia, where they may reflect transepithelial flux of water and solutes. Increased knowledge about LIS may lead to further insight into the mechanisms of endolymph resorption in the endolymphatic sac. The effects of various fixatives, fixation methods and osmolality, and also of surgical labyrinthectomy, on the ultrastructure of LIS were investigated. Widened LIS regularly occurred in the mammalian endolymphatic sac and seem to reflect an in vivo condition. It is thought that LIS may form a pathway for transepithelial water flow in the endolymphatic sac. A hypothetical model of the function of LIS during transepithelial fluid movement in the sac is presented.

There have been several reports of perisaccular fibrosis of the endolymphatic sac in some autopsied cases of Meniere's disease. We attempted to initiate perisaccular fibrosis of the sac in guinea pigs by injecting a small amount of 10% silver nitrate solution that cauterizes the tissue readily to produce scar, ie, fibrosis. Ninety-nine guinea pigs out of 108 (92%) that were treated with silver nitrate showed the endolymphatic hydrops in varied extent, from slight to profound. The extent of hydrops might depend on the extent of destruction of the sac following the cauterization. The lumen of the sac was sometimes occupied with dense fibrous connective tissue with deposition of black-silver particles in it. The silver nitrate injection method is an easy way to produce endolymphatic hydrops in guinea pigs.

A time sequence study of the effect of hemilabyrinthectomy on the endolymphatic sac was performed in mice. Light and transmission electron microscopy of the sac showed significant morphological changes of the epithelial lining and adjacent structures. Initially (2 and 4 days post-labyrinthectomy) the sac lumen was collapsed, but later (7 days post-labyrinthectomy) it was dilated or 'ballooned' and filled with a darkly staining homogeneous substance. This substance, which has been identified histochemically as a proteoglycan, appeared to be secreted from the epithelium of the endolymphatic sac. This finding suggests that the endolymphatic sac may be capable both of absorbing and of secreting endolymph.

The combined effects of glycerol and colchicine on the endolymphatic sac were investigated in mice. Glycerol induced signs of secretion from the epithelium with formation of secretory granules in the light epithelial cells. Other characteristics of the epithelial lining were also changed resulting in an increased widening of the lateral intercellular spaces, a partial collapse of the lumen and with a deposition of a stainable substance within the lumen. This reaction lasted from 30 min to 24 h following the injection. Pretreatment with colchicine was found to decrease or inhibit the glycerol-induced secretion of macromolecules into the sac. The lumen collapsed but frequently there was no presence of stainable substance. Animals treated with both glycerol and colchicine showed marked signs of inner ear malfunction which could indicate that the secretory activity in the sac might be closely related to the regulation of inner ear fluid homeostasis and that functional disturbances in this system may lead to disorders of inner ear function.

The protein content and the d.c. potential of the endolymph differs in the various parts of the endolymphatic space (cochlea, utricle, semicircular canals and endolymphatic sac) as also does the ion composition (chloride, potassium, sodium). 12 months after obliteration of the endolymphatic sac and duct in guinea pigs the d.c. potential falls, whereas, the sodium activity increases. The endolymphatic hydrops is not caused by an increased colloid osmotic pressure. The increased water-binding capacity of the cochlear endolymph is correlated with the increased Na+ activity.

Carbohydrate complexes were investigated in the murine endolymphatic sac by means of histochemical techniques in normal untreated animals as well as after ethacrynic acid treatment. The light epithelial cells were classified into three different types: normal, granular and vacuolar. The granular and vacuolar cells were believed to secrete glycoproteins and/or proteoglycans, the presence of which was closely correlated with the component of the precipitate in the lumen of the endolymphatic sac. This finding suggested that the light cells not only absorb endolymph but may also be involved with secretory activity. Such a dual modality in function may have several important implications, since it suggests that the endolymphatic sac has both absorptive and secretory functions.

The accumulation and degradation of a homogeneous precipitate in the lumen of the endolymphatic sac (ES) was studied in mice. Filling of the endolymphatic sac was induced by surgical labyrinthectomy and the sacs were studied 1-8 weeks postoperatively. The initial phase (1-2 weeks postlabyrinthectomy) was characterized by filling of the ES with the homogeneous precipitate. The number of freely floating cells in the lumen was increased after two weeks. Three weeks postoperatively the ES lumen was generally clear, with apparently no stainable material. Ultrastructural analysis of the ES showed that this clearance of the endolymphatic space resulted from degradational activity in the epithelial cells initiated in the proximal portion of the sac. Breakdown of the homogeneous substance seemed to result from cellular ingestion with concomitant lysosomal digestion. Four weeks postoperatively cell clusters were observed subepithelially and were filled with densely staining precipitate, indicating that these cells or macrophages were involved in the turnover of the homogeneous substance in the ES. The functional significance of a degradational system of this substance in the ES is discussed.

The effect of amiloride on the murine endolymphatic sac was investigated. The amiloride caused collapse of the lateral intercellular spaces in the endolymphatic sac epithelium and a subsequent mild endolymphatic hydrops. These changes indicated a decreased absorption of endolymph in the endolymphatic sac. Amiloride is known to inhibit the transcellular fluid transport without inducing any changes in the paracellular fluid transport. It is therefore suggested that amiloride specially inhibits the fluid and ion exchange in the apical portion of the epithelial cells resulting a decrease in transcellular fluid transport across the endolymphatic sac epithelium. The transcellular fluid transport seems to be one of the main mechanisms in the endolymphatic sac fluid exchange system.

Intravenously injected kanamycin sulfate (KM) was located in the endolymphatic sac (ES) of guinea pigs by an immunohistologic method. Kanamycin sulfate had passed through the capillaries in the subepithelial layer and reached the epithelial layer as early as 1 minute after injection. The amount of KM in the epithelial layer gradually increased with time (until 30 minutes after injection). The KM also accumulated in floating cells in the ES. These results tend to indicate that the ES is rather easily permeable to systemically administered drugs, which readily get into the regional endolymph. In conclusion, much more attention should be paid to the ES and drug ototoxicity associated with drug therapy in the management of patients with inner ear disturbances.

Complex macromolecules are suggested to play an important role for the function of the endolymphatic sac (ES). As proposed in previous experimental studies, proteoglycans are supposed to be present in the ES. As they are composed of a proteic core linked to chains of glycosaminoglycans, chemical identification of the glycosaminoglycans is of particular importance, bearing in mind that all but hyaluronic acid contain sulphur. In order to follow the short-term turnover of sulphur in the ES, an autoradiographic study has been carried out in the Mongolian gerbil using 35S as tracer. Radioactive labelling of the gerbilline ES was controlled 15, 20, 30 and 60 min after intraperitoneal injection. While the first signs of the presence of radioactive sulphur were noticed after 20 min in the blood vessels and in the basal aspect of the ES epithelium, after 60 min it was possible to observe the presence of the tracer both within the epithelial cell layer as well as in the lumen of the ES. These findings are consistent with the presence of a fast turnover of sulphur molecules in the gerbilline ES.