Endolymphatic sac tumor misdiagnosed as metastatic renal cell carcinoma: Pitfalls in morphology and immunohistochemistry.

Endolymphatic sac tumor (ELST) is a rare disease that originates from the endolymphatic sac system of the inner ear. Being a low-grade malignant tumor, ELST has a mild morphology and is characterized by a slow but aggressive growth. Most clinicians and pathologists are unfamiliar with this disease. ELST can be misdiagnosed as metastatic renal cancer because of the similarity in morphology and expression of nephrogenic markers such as PAX8. The presented case of a 27-year-old man revealed that observing the characteristic location and confirming the absence of renal neoplasm to rule out the possibility of metastasis are critical for obtaining an accurate final diagnosis.

Innate immune defense in the inner ear - mucines are expressed by the human endolymphatic sac.

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.

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.

Expression of thiazide-sensitive Na+-Cl- cotransporter in the rat endolymphatic sac.

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.

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.

Molecular cloning of otoconin-22 complementary deoxyribonucleic acid in the bullfrog endolymphatic sac: effect of calcitonin on otoconin-22 messenger ribonucleic acid levels.

Anuran amphibians have a special organ called the endolymphatic sac (ELS), containing many calcium carbonate crystals, which is believed to have a calcium storage function. The major protein of aragonitic otoconia, otoconin-22, which is considered to be involved in the formation of calcium carbonate crystals, has been purified from the saccule of the Xenopus inner ear. In this study, we cloned a cDNA encoding otoconin-22 from the cDNA library constructed for the paravertebral lime sac (PVLS) of the bullfrog, Rana catesbeiana, and sequenced it. The bullfrog otoconin-22 encoded a protein consisting of 147 amino acids, including a signal peptide of 20 amino acids. The protein had cysteine residues identical in a number and position to those conserved among the secretory phospholipase A(2) family. The mRNA of bullfrog otoconin-22 was expressed in the ELS, including the PVLS and inner ear. This study also revealed the presence of calcitonin receptor-like protein in the ELS, with the putative seven-transmembrane domains of the G protein-coupled receptors. The ultimobranchialectomy induced a prominent decrease in the otoconin-22 mRNA levels of the bullfrog PVLS. Supplementation of the ultimobranchialectomized bullfrogs with synthetic salmon calcitonin elicited a significant increase in the mRNA levels of the sac. These findings suggest that calcitonin secreted from the ultimobranchial gland, regulates expression of bullfrog otoconin-22 mRNA via calcitonin receptor-like protein on the ELS, thereby stimulating the formation of calcium carbonate crystals in the lumen of the ELS.

Tissue specific levels of glucocorticoid receptor within the rat inner ear.

Individual, rat inner ear tissues were isolated and processed for determination of levels of glucocorticoid (GR) receptor by an Enzyme Linked Immuno-Sorbant Assay (ELISA). Differing levels of GR receptor between seven sampled inner ear regions were measured. Levels of GR receptors in the spiral ligament tissues were found to be significantly higher compared to all other tissue samples. GR levels in the tissues of stria vascularis and organ of Corti were different from one another but both were statistically higher than those detected in the vestibular tissue samples (dark cell regions, cristae ampullares and maculae utriculi), which had the lowest GR receptor levels measured. Intermediate levels of GR receptor were found in the endolymphatic sac region. It is suggested that the varying levels of inner ear GR receptors may be indicative of differing biological responses among the given tissues, as well as differences in the magnitudes of such responses to circulating glucocorticoids.

Morphological changes of the endolymphatic sac induced by microinjection of artificial endolymph into the cochlea.

Morphological changes of the endolymphatic sac were analyzed in guinea pigs following microinjection of artificial endolymph into the cochlea or withdrawal of a quantity of native endolymph. Injections were performed into the second turn of scala media with a micro-pump at a rate of 60-100 nl/min, lasting for a period of 4, 7. 5, 15 or 18 min. In withdrawal experiments, endolymph was aspirated from the second cochlear turn over a period of 8 min. For each procedure the contralateral (non-treated) ear served as a histological control. Following artificial endolymph injections of 7. 5 min or more there was an almost total absence of the normal intraluminal homogeneous substance (HS) on the injected side. Our observations suggest that the disappearance of the HS occurs by both enzymatic and macrophagic activity. After endolymphatic withdrawals the ES was found to contain increased amounts of HS. The results could suggest that the volume of fluid in the ES, and hence the volume of the entire membranous labyrinth, may be regulated by a dynamic relationship between active secretion and enzymatic degradation of a lumen-expanding substance that is intimately related to the intraluminal macrophages. The exact mechanism governing these regulatory systems, and their relationship to ion and water movements across the epithelium of the sac, remain to be elucidated.

Molecular mechanisms underlying ectopic otoconia-like particles in the endolymphatic sac of embryonic mice.

Otoconin-90, the principal otoconial matrix protein, provided a tool to investigate the molecular mechanism of otoconial morphogenesis. The endolymphatic sac of the embryonic chick and guinea pig contain otoconia. Here, we show that the embryonic mouse transiently expresses ectopic otoconia in the endolymphatic sac. Massive precipitate of otoconin-90-positive material is detectable in the lumen of the endolymphatic sac between embryonic day 14.5 and 17.5 with frequent accretion into more heavily staining otoconia-like particles. Otoconin-90 was also localized at the surface and the interior of epithelial cells lining the endolymphatic sac as well as incorporated into free floating cells. In contrast, in situ hybridization failed to detect mRNA in the endolymphatic duct and sac, even though the adjacent nonsensory vestibular structures are heavily stained. Because of ample expression of otoconin-90 protein in the absence of the corresponding mRNA, we conclude that the luminal otoconin-90 is imported via longitudinal flow from the vestibular compartments, where both mRNA and protein are strongly expressed. Because of absence of mRNA, the expression of the corresponding protein by the epithelia lining the endolymphatic sac can only be explained by a resorptive process, as previously proposed on the basis of the movement of luminal macromolecules. The data do not support the previous hypothesis that the transient expression of otoconia-like particles of the endolymphatic sac represents a vestigial phenomenon from the amphibian stage, since amphibia express ample mRNA encoding otoconin-22 in the endolymphatic sac system.

Insulin receptors on the endolymphatic sac: an autoradiographic study.

Light microscopic autoradiographs were prepared of isolated guinea pig endolymphatic sac (ES) tissue incubated with iodine (I)-125-labeled insulin (insulin I 125). These demonstrated specific binding of the insulin I 125 to receptors in the ES. Receptor-mediated endocytosis of the insulin-receptor complex is postulated to occur, and the relevance of insulin receptors in the ES is discussed, with particular emphasis on the previously observed synthesis of hyaluronan from glucose by the ES.

Immunohistochemical localization of collagen types I, III, and IV, laminin, fibronectin, and keratin in the endolymphatic sac.

We have employed immunohistochemistry to obtain baseline information on the molecular constituents of the extracellular matrix (ECM) of the endolymphatic duct (ED) and endolymphatic sac (ES) of the chinchilla. The results demonstrated that collagen types I and III were distributed in the subepithelial layer in the ED and ES, type IV collagen and laminin in the basement membranes, and fibronectin in the subepithelial layer and partly in the conglomerated cells in the ES. Collagen type III was diffusely distributed in the whole subepithelial layer of the ES, whereas collagen type I was concentrated densely in the deep layer of the interstitium, although gradually, the cuboidal epithelium in the ES was transformed into a flatter type in the ED. The epithelial cells of the ED and ES were clearly positive for keratin. This study deals, in particular, with the normal distribution of ECM components of the ED and ES of the chinchilla.

Localization of glucocorticoid receptors in the murine inner ear.

We performed an immunohistochemical investigation of the distribution of glucocorticoid receptors (GRs) in the murine inner ear and found that GRs were expressed extensively, but with various degrees of immunoreactivity in different regions. We observed the strongest GR expression in the type III fibrocytes of the spiral ligament. Although the immunoreactivity of the cochlear hair cells and of the vestibular sensory epithelia was weak, the neighboring cochlear supporting cells and the subepithelial regions of the vestibular sensory epithelia were immunostained. Staining for GRs was also positive in the spiral ganglia and vestibular ganglia, as well as in the endolymphatic sac. The role of GRs in the inner ear is discussed.

Constituents of the endolymphatic tubules as demonstrated by three-dimensional morphometry.

The various constituents of the tubules of the human adult intraosseous endolymphatic sac were stained with lectins and Millon reaction to reveal some of the carbohydrate and protein-containing substances in the endolymph. The amount of each substance was determined by digitizing sequential temporal bone sections. Differing amounts of the various identified substances were found in sacs from patients with Meniere's disease, patients with labyrinthine fibrosis, and controls.

Glycolipidic component of the epithelial cell coat of the endolymphatic sac.

The endolymphatic sac (ES) is thought to synthesize and secrete glycoconjugates such as sulfated glycoproteins into the endolymphatic lumen. Ganglioside Gm1 is a specialized glycolipid containing one sialic acid molecule, which is generally found in the outer leaflet of the cell membrane. This glycolipid, which is known to be a specific receptor for cholera toxin (CT), acts as a membrane transducer and is involved in the modulation of cell metabolism, growth and regeneration. In the present study we identified Gm1 by studying the distribution of the FITC-labeled CT-subunit B in the ES epithelium of adult guinea pigs. Our findings indicate the presence of this ganglioside in the ES, with a predominant localization in the basolateral aspect of the epithelial cell layer. No detectable differences between ES cell types could be identified, whilst the ES distal and intermediate portions showed more reactivity than the proximal portion. This study, which represents the first description of a lipidic glycoconjugate component in the ES, provides evidence in support of the role of the ES in the turnover and regulation of inner ear fluids.

Distribution of endothelin-1 like activity in the endolymphatic sac of normal guinea pigs.

Endothelin-1 (ET-1), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, has been reported to act as a local hormonal regulator of pressure, fluid, ions as well as neuropeptides. The present study investigated the distribution of ET-1 in the endolymphatic sac and duct of normal guinea pigs, by immunohistochemistry. ET-1 like activity was identified in the epithelial cells and subepithelial connective tissue of the endolymphatic sac and duct. No significant difference was seen in the intermediate and proximal portions. These findings suggest that ET-1 may play an important role in the regulation of inner ear pressure, fluid volume, and ion balance.

Identification of substances in the endolymphatic sac.

The composition of the contents of the endolymphatic sac (ES) has yet to be fully defined. Carbohydrates have been found in the ES of human fetuses and animals but not identified in the adult human ES. In the present study, celloidin was removed from previously prepared human temporal bone sections and a histochemical method was used to detect carbohydrates and protein in the ES. Six biotinylated lectins were used to identify specific carbohydrates in 15 ears: beta-D-N-acetylglucosamine, beta-D-galactose, D-galactose, alpha-D-mannose, D-N-acetylgalactosamine and alpha-L-fucose. The intensity of staining was graded qualitatively. A substance in the ES tubules that did not stain with any lectin was identified by the Millon reaction as containing protein. The carbohydrates and protein may exist in the different tubules or in the same tubule without mixing. This finding seems to support the idea that at least some of these substances are produced locally in the ES. Our observations support the hypothesis of the existence of a secretion and degradation system in the endolymphatic sac.

Aquaporin-2 in the human endolymphatic sac.

OBJECTIVE: To detect and localize aquaporin-2 (AQP-2), a water channel regulated by the antidiuretic hormone, in human endolymphatic sac. MATERIAL AND METHODS: Human endolymphatic sacs were sampled during removal of vestibular schwannomas via a translabyrinthine approach. Samples were immediately fixed in 10% formalin (24 h) and embedded in paraffin; in situ hybridization and immunohistochemistry were performed with an AQP-2-specific probe and a polyclonal antibody. RESULTS: Both AQP-2 mRNA and protein were detected in the epithelium of the endolymphatic sac. AQP-2 immunostaining was mainly cytoplasmic, suggesting that most AQP-2 was located in intracellular pools. CONCLUSIONS: In the endolymphatic sac, AQP-2 probably participates in the homeostasis of endolymph; the possibility of reducing the volume of endolymph by inhibiting its expression and membranous insertion using an antidiuretic hormone inhibitor represents a new therapeutic approach for the treatment of Ménière's disease.

Expression of mRNAs encoding hormone receptors in the endolymphatic sac of the rat.

The endolymphatic sac (ES) is believed to absorb the endolymphatic fluid produced by the stria vascularis and vestibular dark cells. Recent studies have implied that the function of the ES may be controlled by circulating hormones, suggesting that hormone receptors should exist there. In the present study, the expression of genes encoding receptors for aldosterone, atrial natriuretic peptide (ANP) and vasopressin in the ES was examined by reverse transcription-polymerase chain reaction (RT-PCR). Next, the cellular localization of the expression of these genes was investigated by in situ hybridization. RT-PCR indicated that aldosterone. ANP-A and vasopressin V1a receptor genes were expressed in the ES. In contrast, neither ANP-B nor vasopressin V2 receptor gene expression was detected. In situ hybridization experiments demonstrated aldosterone receptor gene expression in epithelial cells of the intermediate potion of the ES, while expression of ANP-A or V1a receptor genes was not detected. The present results suggested that aldosterone may play a specific role in the function of the ES. However, we could not conclude that ANP and vasopressin play physiological roles in the ES because receptors for these hormones were detected only by highly sensitive PCR.

Sex hormones in the kidney and endolymphatic sac. An immunohistological study.

Autopsy specimens of kidney and endolymphatic sac were studied in terms of progesterone (P), estradiol (E), testosterone (T), dihydrotestosterone (DHT) and aldosterone (A) using the peroxidase-antiperoxidase method. The results obtained were as follows: The epithelial cells of the renal tubules were positive to P, E, T, DHT and A. The epithelial cells of the endolymphatic sac were also positive to these sex hormones though varying in stainability among cases. In view of the immunohistological findings of sex hormones and aldosterone, the resorptive and regulative function of the epithelium in the kidney and endolymphatic sac is discussed.

Localization of type IV collagen and laminin in the guinea pig inner ear.

The distribution of type IV collagen (C-IV) and laminin, which are important components of the basement membrane (BM), was studied immunohistochemically in the inner ear of healthy Hartley guinea pigs. Antibodies against C-IV and laminin were used in this study. The distribution of C-IV in the inner ear was almost the same as that of laminin, but the extent of staining for laminin was less than that for C-IV in some sites. The sites of the inner ear in which these components were most densely localized were the areas surrounding the spiral ganglion cells and nerve fibers, the capillary vessels in the stria vascularis and the spiral prominence, and an area directly beneath the epithelium of the endolymphatic sac. Type IV collagen and laminin were also localized around the other vascular BM and the epithelial BM in the inner ear, but the tectorial membrane, the cupula of the crista ampulla, and the sensory epithelium did not take up stain. These results suggest that the vascular BM of the stria vascularis and spiral prominence, as well as the epithelial BM of the endolymphatic sac, may play an important role in fluid transport, and that the perineural BM of the inner ear might play an important role in the functional maintenance of the optimal environment of the inner ear nervous system.