The periductal channels of the endolymphatic duct, hydrodynamic implications.

OBJECTIVE: To describe the anatomy of a small network of channels surrounding the human endolymphatic duct. STUDY DESIGN: Archival temporal bone sections and a surgical specimen were studied using a variety of techniques. SETTING: Temporal bone laboratory of the House Research Institute. SUBJECTS AND METHODS: Archival temporal bone sections were examined by light microscopy, 3D reconstruction, and immunohistochemical labeling. A surgical specimen was examined using electron microscopy. Sections from temporal bones with blocked endolymphatic ducts or amputated sacs were examined for the manifestations of endolymphatic hydrops. RESULTS: Peri-endolymphatic duct channels were found to extend from the proximal cisternal part of the endolymphatic sac to the supporting tissue of the saccule and utricle. Tissue in the channels, as seen by conventional and electron microscopy, is continuous with and identical with the tissue surrounding the endolymphatic duct. Tissue in the channels labels with the S100 antibody similar to the spiral ligament and supporting tissue of the vestibular end organs and suggests a neural crest origin, as did the presence of melanocytes. Obstruction of the endolymphatic duct resulted in endolymphatic hydrops whereas amputation of the sac did not. CONCLUSION: Endolymph is probably absorbed in the endolymphatic duct. The peri-endolymphatic duct channels that extend from the proximal sac to the supporting tissue of the saccule label with the S100 antibody and contain melanocytes suggest a neural crest origin and involvement in fluid and potassium hydrodynamics similar to those described for the similarly staining spiral ligament of the cochlea.

An interstitial network of podoplanin-expressing cells in the human endolymphatic duct.

The human endolymphatic duct (ED) with encompassing interstitial connective tissue (CT) is believed to be important for endolymph resorption and fluid pressure regulation of the inner ear. The periductal CT cells are interconnected via numerous cellular extensions, but do not form vessel structures. Here we report that the periductal CT is populated by two distinct cell phenotypes; one expressing podoplanin, a protein otherwise found on lymph endothelia and on epithelia involved in fluid fluxes, and a second expressing a fibroblast marker. A majority of the interstitial cells expressed podoplanin but not the lymphatic endothelial cell markers hyaluronan receptor (LYVE-1) or vascular endothelial growth factor receptor-3 (VEGFR-3). The fibroblast marker positive cells were found close to the ED epithelium. In the mid- and distal parts of the ED, these cells were enriched under folded epithelia. Furthermore, subepithelial CT cells were found to express activated platelet derived growth factor (PDGF)-beta receptors. Cultured CT cells from human inner ear periductal and perisaccular explant tissues were identified as fibroblasts. These cells compacted a three-dimensional collagen lattice by a process that could be promoted by PDGF-BB, a factor involved in interstitial fluid pressure regulation. Our results are compatible with the notion that the periductal CT cells are involved in the regulation of inner ear fluid pressure. By active compaction of the periductal CT and by the formation of villous structures, the CT cells could modulate fluid fluxes over the ED epithelium as well as the longitudinal flow of endolymph in the ED.

Evidence of progression and fluctuation of hearing impairment in branchio-oto-renal syndrome.

We retrospectively analysed long-term serial audiometry data from patients with branchio-oto-renal (BOR) syndrome to show the features of progression and fluctuation in hearing impairment and relate the findings to age and magnetic resonance imaging (MRI) findings in the petrosal bones. Thirty-two clinically affected BOR patients from six Dutch families (A-F) were included. Audiograms were available in 24 cases, covering followup intervals of between 3 and 30 years, and suitable for individual statistical analysis in 16 cases: 14 cases also had MRI findings. Significant progression in hearing impairment was found in 10 cases, while findings of significant fluctuation were made in seven cases. These findings did not clearly correlate with MRI findings. Substantial fluctuation occurred only in cases followed at a relatively young age. Patients with an enlarged endolymphatic duct and/or sac showed significantly higher sensorineural hearing thresholds than those with either normal MRI findings or cochlear/labyrinthine hypoplasia with or without enlarged duct or sac. We conclude that progressive, fluctuant hearing loss occurred in some BOR patients; however, only young patients showed substantial threshold fluctuation. BOR patients with an enlarged endolymphatic duct and/or sac on MRI seemed to be predisposed to developing more severe hearing impairment.

Network organization of interstitial connective tissue cells in the human endolymphatic duct.

The human endolymphatic duct (ED) and sac of the inner ear have been suggested to control endolymph volume and pressure. However, the physiological mechanisms for these processes remain obscure. We investigated the organization of the periductal interstitial connective tissue cells and extracellular matrix (ECM) in four freshly fixed human EDs by transmission electron microscopy and by immunohistochemistry. The unique surgical material allowed a greatly improved structural and epitopic preservation of tissue. Periductal connective tissue cells formed frequent intercellular contacts and focally occurring electron-dense contacts to ECM structures, creating a complex tissue network. The connective tissue cells also formed contacts with the basal lamina of the ED epithelium and the bone matrix, connecting the ED with the surrounding bone of the vestibular aqueduct. The interstitial connective tissue cells were non-endothelial and non-smooth muscle fibroblastoid cells. We suggest that the ED tissue network forms a functional mechanical entity that takes part in the control of inner ear fluid pressure and endolymph resorption.

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.

The suspensory ligament of the endolymphatic duct in the rat. An ultrastructural study.

Following optimized fixation and specimen handling, the endolymphatic duct was investigated by transmission and scanning electron microscopy. Tubular microfibrils, 10-12 nm thick and of indefinite length, occupied the subepithelial compartment abundantly and seemed to insert into the basal lamina of the epithelial cells of the duct and to merge with the bony wall of vestibular aqueduct thus forming a suspensory ligament of the endolymphatic duct. This ligament is suggested to protect the endolymphtaic duct from collapsing and to play a role in the mechanical protection of the vestibular apparatus from sudden increases in intracranial pressure. The capillaries and the aqueductal vein were located in intimate relationship to the epithelial cells. Both types of vessels were provided with fenestrations of the endothelial cells, supporting the proposal that the endolymphatic duct may be involved in the resorption of endolymph.

Surface morphology of the endolymphatic duct in the rat. A scanning electron microscopy study.

Following intracardiac vascular perfusion fixation of 8 rats with glutaraldehyde in a buffered and oxygenated blood substitute, the vestibular aqueduct and endolymphatic duct were opened by microsurgery of the resulting 16 temporal bones. Optimum preservation of the epithelium for scanning electron microscopy was attained by coating of the specimens with osmium tetroxide and thiocarbohydrazide followed by a continuous dehydration procedure. This technique permitted, for the first time, an investigation of the surface morphology of the epithelial cells in the endolymphatic duct. Three types of cells were identified with the scanning electron microscope. A polygonal and oblong epithelial cell was observed in the largest number throughout the duct, and in the juxtasaccular half of the duct, two additional types of epithelial cells were observed. The scanning electron microscopic observations are compared and discussed with reference to previous transmission electron microscopic studies of the endolymphatic duct.

Ultrastructure of the epithelial cells of the endolymphatic duct in the rat.

The ultrastructure of the epithelial cells of the endolymphatic duct in the rat is described, following vascular perfusion-fixation of live, anaesthetised and artificially respirated animals. The animals were fixed by means of a pressure feed-back controlled peristaltic pump and an isotonic perfusate-fixative containing glutaraldehyde and Dextran. The endolymphatic duct was isolated by microdissection after the perfusion-fixation, to omit the step of a demineralization procedure. The proximal, intermediate and juxta-saccular parts of the endolymphatic duct were embedded, sectioned and studied separately in the electron microscope. Postfixation in a solution containing OsO4 and potassium ferricyanide revealed a well-developed tubulo-cisternal endoplasmic reticulum (TER), not previously described. Serial sectioning and computerized three-dimensional reconstruction demonstrated a continuity of the TER through the cell from subsurface cisterns abutting on the apical cell membrane to subsurface cisterns abutting on the basolateral cell membrane. The TER resembles that found in solute transporting epithelia, e.g., renal proximal tubule, gall bladder, small intestine and choroid plexus. A fluid resorptive capacity of the epithelial cells of the endolymphatic duct is compatible with the fine structure revealed in the present study. Epithelial cells in the juxta-saccular part of the duct display morphological indications of a secretory activity; furthermore, multivesicular bodies were observed in the epithelial cells throughout the endolymphatic duct.

Ultrastructure of the endolymphatic duct in the rat. Fixation and preservation.

Ten rats were vascular-perfused at subphysiologic as well as physiologic pressures, 80 mmHg and 120 mmHg, respectively, employing a pressure feed-back controlled peristaltic pump and an isotonic perfusate/fixative with colloids (2% Dextran) and a hypertonic perfusate/fixative without colloids, 300 and 530 mOsm, respectively. In both experiments the endolymphatic duct and sac were isolated by microdissection after primary fixation. When comparing micrographs from the two experiments we observed that rats perfused at low pressure in isotonic fixative with colloids added had non-dilated lateral intercellular spaces and a subepithelial ground substance loaded with uniformly arranged microfibrils, not previously discovered. In rats perfused at high pressure in hypertonic fixative with no colloids added, we observed an edematous state in the subepithelial space between the solid bony aqueduct and the endolymphatic duct, with a concomitant derangement of previously well organized microfibrils. Furthermore, the epithelium was quite clearly displaced from the capillaries, the intercellular spaces were widely dilated and the endolymphatic duct was compressed into its lumen. Methods of fixation and current theories of endolymph resorption by the endolymphatic duct are discussed.

Human Reissner's membrane in patients with age-related normal hearing and with sensorineural hearing loss.

Morphological features of Reissner's membrane were investigated in 6 patients with age-related normal hearing (ARNH) and in 4 with sensorineural hearing loss (SNHL) stemming from various causes. The membrane consisted of an epithelium, a basement membrane and a mesothelium with melanocytes. There were two major forms of epithelial cells: flat and rounded. In all specimens, the rounded cells formed whorls, clusters, strands and bands. The bands were wider and the whorls larger in the basal turn and decreased gradually in size toward the apex. The number of melanocytes was 2-4 times higher in both the upper half of the basal turn and the lower half of the middle turn than in the rest of the turns. In specimens from patients with SNHL, whorls and clusters were both more numerous and larger, and the number of melanocytes was higher in the upper half of the middle turn and in the apical turn than that in the ARNH group. Ultrastructural examination of epithelial and mesothelial cells as well as of melanocytes showed more pronounced cellular degeneration in patients with SNHL than in those with ARNH. A possible correlation between structural alterations of Reissner's membrane and sensorineural degeneration is discussed.

Ultrastructure and barrier properties of the endolymphatic duct in the guinea pig.

The ultrastructure and barrier properties of the endolymphatic duct (ED) were examined by light and electron microscopy. ED epithelial cells were classified into two types: type I and type II duct cells. The type I duct cells were cuboidal or low columnar and were characterized by a convex apical surface and a few basal processes. The type II duct cells were squamous and were characterized by a flat apical and basal membrane, many small vesicles and a number of small pits along the basal membranes. After electrophoretic horseradish peroxidase (HRP) injection into the ED lumen, no HRP uptake into ED epithelial cells of either type was observed. There was no reaction product either in the lateral intercellular spaces beyond the tight junctions between ED epithelial cells or in ED subepithelial tissues. The ED epithelial cells were considered to play no active role in apical macromolecular absorption and to be impermeable to intraluminal macromolecules.

Differential diagnosis of virus-like particles in the human inner ear.

The entire endolymphatic duct and sac as well as the vestibular epithelia were obtained from four patients with Meniere's disease during translabyrinthine (TL) eighth nerve section and from 12 patients undergoing TL resection of acoustic schwannomas. After these specimens were processed for routine transmission electron microscopy (TEM), they were studied for morphologic evidence of viral infection. Although no virus particles were identified, numerous regularly occurring cell components and artifacts were found to morphologically mimic viruses. An atlas of these structures is presented.

Microorganism transport in the human endolymphatic duct.

There are indications that endolymphatic sac (ES) may be an immunologically active part of the inner ear. So far, no microorganisms or foreign substances have been localized in this area under 'normal' conditions. Only a limited number of human specimens, including the entire endolymphatic duct (ED) and ES, have been collected and analyzed from cadavers or surgical biopsy specimens. In this study, 6 human ED and ES collected from cadavers and at surgery were analyzed by light and electron microscopy. This was done in order to investigate if microorganisms may normally be drained at this route into the ES. Some microorganisms (Mycoplasma pneumoniae) were found in the lumen and subepithelial tissue of 1 human ED. These observations suggest that microorganisms may also be locally processed and disposed at the level of the ED. These results add further evidence as to the immunodefensive role of the human ES.

The effect of endolymphatic sac obliteration on vestibular sensory hair bundles. A high-resolution scanning electron microscopic investigation.

A tannic acid-osmium staining technique and high-resolution scanning electron microscopy were used to demonstrate changes in the glycocalyx and ciliary interconnections of the vestibular sensory cells of guinea pigs after extradural obliteration of the endolymphatic sac and duct. Three months after the obliteration, it was possible to observe degeneration in the glycocalyx and the ciliary interconnections as well as the tip links. These findings suggest that the endolymphatic hydrops causes an endolymphatic ionic imbalance which affects the glycocalyx and ciliary interconnections resulting in further morphological changes of the cilia. The tip links, which are believed to be involved in sensory cell transduction, also seem to be affected.

Technique for transmission and immunoelectron microscopy of the human endolymphatic sac and vestibular epithelia.

A surgical technique is presented to obtain the entire vestibular aqueduct, containing the endolymphatic duct and sac as well as the vestibular epithelia, from the maculae and cristae during labyrinthectomy. The inner ear tissue was fixed in 3% glutaraldehyde, decalcified in 0.1 mol/L Na-EDTA, and routinely processed for transmission electron microscopy, including post-fixation with osmium tetroxide. Postembedding protein A-colloidal gold immunoelectron microscopy was performed after exposure of cellular antigenic sites by sodium metaperiodate. To validate this technique, thin sections from the crista ampullaris and the endolymphatic sac were incubated with antibodies raised against S-100, a protein present in specific types of neural tissue. Specific immunoreactivity was observed in the crista ampullaris, but not in the endolymphatic sac. The surgical biopsy technique described provides a means for the neuro-otologist to collect inner ear tissue from surgical patients that can be used for pathological studies using immunohistochemistry and electron microscopy.

Ultrastructure and organization of circumventricular organs and endolymphatic tubules in the treefrog, Hyla versicolor.

The morphology and anatomical relationship of the paraphysis, anterior choroid plexus, and endolymphatic tubules of Hyla versicolor suggest that these structures may act as a functional unit to regulate composition of the cerebrospinal fluid (CSF). Ependymal cells of both the paraphysis and the choroid plexus exhibit ultrastructural features (an extensive microvillar border, large intercellular spaces containing an amorphous material, and apically located mitochondria) which indicate active exchange between the blood and CSF. Mast cells (which may play a role in regulating vascular permeability) also occur on the brush border. The tubules of the endolymphatic sacs were found to lie in close relation with the dorsal portion of the paraphysis. The endolymphatic organ is known to play a role in systemic buffering during respiratory acidosis and may influence Na/K fluxes via calcium release. The association of the paraphysis with the endolymphatic tubules may therefore represent an important mechanism for ion regulation in the CSF of amphibians. The ultrastructure of the pineal organ agrees with descriptions for other species. No pineal stalk was present. Photoreceptor inner and outer segments project into a central lumen continuous with the third ventricle. Outer segments show signs of disorganization. Photoreceptor/neural dendrite contacts involve synaptic ribbons and conventional synapses.

Ultrastructure of the organ of Corti in experimental hydrops.

The early ultrastructural changes of the organ of Corti in the guinea pig after obliteration of the endolymphatic sac and duct were examined in a time-sequence study. Initial loss of outer hair cells was followed by inner hair cell degeneration, both starting at the apical part of the cochlea. Morphological changes of the sensory cells were found to start at the endolymphatic surface and were characterized by a variety of sterociliary pathologies, distortion of the cuticular plate and a less prominent contrast-staining of glycocalyx. Further degeneration was marked by pathological changes of intracellular organelles and the afferent and efferent nerve endings. Details of the ultrastructural changes in the organ of Corti are discussed with regard to the pathophysiology of experimental hydrops.

The pre- and postnatal maturation of the epithelium in the endolymphatic sac. An electron microscopic survey.

The cellular development of the endolymphatic sac was studied in the CBA/CBA mouse, starting from day 10 of gestation following the different stages of maturation up to an adult age of one month. The first immature cylindrical cells lining the future sac in several cell layers are seen at day 12 of gestation. At day 18 of gestation, a true sac appears and a floccular precipitate is frequently found in its lumen together with signs of increased activity in the still immature epithelial cells. Approximately one day before birth the first signs of the future light and dark cells can be distinguished. At day 4 post partum the cells are more differentiated with some showing signs of secretory activity indicating that these cells start to function at this stage. Eight days after birth differentiation into distinguishable almost mature light and dark cells is seen. Two days later these epithelial cells have obtained a fully mature appearance. At 14 days after birth widened lateral intercellular spaces separating the epithelial cells can be visualized and a few free floating cells are found in the sac lumen. The sac epithelium is thus considered to have completed its maturation process at this stage.

Imbalanced calcium homeostasis and endolymphatic hydrops.

In this paper the current state of knowledge of the development in experimental endolymphatic hydrops (EEH) is summarized, with particular emphasis on calcium. An imbalanced Ca2+ homeostasis in the inner ear is demonstrated using EEH as an animal model for Meniere's disease. The possibility of a receptor-mediated Ca2+ transport across the epithelial layer, especially the light cells, and of 'chemical signal' as an initiating modulating factor in the disturbance of Ca2+ homeostasis was suggested. It is pointed out that melanin is capable of binding calcium and may act as a buffering system. Finally, the possible malfunction of the Ca-overloaded melanocytes on the inner ear function is discussed.