Neuronal Fibers and Neurotransmitter Receptor Expression in the Human Endolymphatic Sac.

INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression, using adjacent dura mater as control. The expression of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.

[Investigation of the innervation of endolymphatic sac and kidney in rats].

OBJECTIVE: To confirm the existence of the nerve fibers on the endolymphatic sac (ES) and compare the innervation of endolymphatic sac with that of kidney to reveal their relationship. METHODS: Fifteen healthy Swards white rats were processed by heart pouring. The temporal bones and kidneys were taken out and were processed and sectioned by paraffin-embedded technique. The sections of ES and kidney were both stained by the antibody that was not labeled by biotin, including antibodies for neurone specific enolase (NSE), glial fibrillary acidic protein (GFAP) and neurofilament (NF). The slides were observed by light-microscope and analyzed by image-analyzer system and the subsequent data was treated statistically by SPSS 11.0. RESULTS: On light-microscope, the brown positive staining was observed both in the epithelia of ES and the principal cell of renal collecting duct. However, there was not any positive staining on the negative control slides. Through image analysis to the grey and density, there was no difference between the expression of ES and that of the principal cell of renal collecting duct (P > 0.05). CONCLUSIONS: Nerve fibres exactly exist in the epithelia of ES. There is similar density innervation between the ES and the principal cell of renal collecting duct.

The endolymphatic duct and sac of the rat: a histological, ultrastructural, and immunocytochemical investigation.

A study of the ultrastructure, vascularization, and innervation of the endolymphatic duct and sac of the rat has been performed by means of light- and electron-microscopic and immunocytochemical methods. Two different types of epithelial cells have been identified: the ribosome-rich cell and the mitochondria-rich cell. These two cell types make up the epithelium of the complete endolymphatic duct and sac, although differences in their quantitative distribution exist. The morphology of the ribosome-rich cells varies between the different parts of the endolymphatic duct and sac; the morphology of the mitochondria-rich cells remains constant. According to the epithelial composition, vascularization, and structural organization of the lamina propria, both duct and sac are subdivided into three different parts. A graphic reconstruction of the vascular network supplying the endolymphatic duct and sac shows that the vascular pattern varies among the different parts. In addition, the capillaries of the duct are of the continuous types, whereas those supplying the sac are of the fenestrated type. Nerve fibers do not occur within the epithelium of the endolymphatic duct and sac. A few nerve fibers regularly occur in the subepithelial compartment close to the blood vessels; these fibers have been demonstrated in whole-mount preparations by the application of the neuronal marker protein gene product 9.5. Single beaded fibers immunoreactive to substance P and calcitonin-gene related peptide are observed within the same compartment. Dopamine-beta-hydroxylase-immunoreactive axons are restricted to the walls of arterioles. Morphological differences between the different portions of the endolymphatic duct and sac are discussed with regard to possible roles in fluid absorption and immunocompetence.

Nerve fibres of the endolymphatic sac: electronmicroscopic findings in the Mongolian gerbil.

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.

Adrenergic innervation of the human endolymphatic sac.

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.

Sympathetic and CGRP-positive nerve supply to the endolymphatic sac of guinea pig.

The distribution of sympathetic fibers and calcitonin gene related peptide (CGRP)-positive fibers was examined in the endolymphatic sac of 10 guinea pigs by using immunohistochemical techniques to demonstrate tyrosine hydroxylase (TH) and CGRP. A meshwork of TH-positive fibers, found around the sigmoid sinus, sent branches to the distal and the intermediate parts of the endolymphatic sac. In these areas. TH-positive fibers traveled freely from blood vessels and formed a loose plexus beneath the lining epithelium. Such fibers were rare in proximal portion of the endolymphatic sac and in the endolymphatic duct. Seven days after elimination of the ipsilateral superior cervical ganglion, some of the TH-positive fibers were gone, however, there were still a few fibers in the endolymphatic sac as well as around the sigmoid sinus, thus their origin remains obscure. CGRP-positive fibers also branched from the fibers around the sigmoid sinus, and were distributed throughout the endolymphatic sac, some occasionally extending to the endolymphatic duct. They not only formed a dense plexus in the sublining space, but also spread through the lining cell layer. Elimination of the ipsilateral superior cervical ganglion did not affect the distribution of CGRP-positive fibers, indicating that they are probably not sympathetic fibers.

Innervation of the endolymphatic sac.

Previous studies suggest that the endolymphatic sac plays an important role in the homeostasis of endolymph. Factors that influence blood flow in the sac may affect its function. This blood flow may be influenced by autonomic innervation; however, no such innervation has been demonstrated. The purpose of this study was to demonstrate catecholaminergic and cholinergic fibers on the endolymphatic sac. Endolymphatic sacs from Hartley guinea pigs were stained either immunocytochemically for tyrosine hydroxylase to reveal catecholaminergic fibers or histochemically for acetylcholinesterase to reveal cholinergic fibers. For tyrosine hydroxylase immunostaining, the endolymphatic sacs were treated with dilute hydrogen peroxide and then incubated in the primary antiserum. The tissue was further processed by the avidin-biotin immunoperoxidase method and reacted with diaminobenzidine. For acetylcholinesterase histochemistry, the tissue was processed by a modification of the direct thiocholine method. Light microscopy of the whole-mounted endolymphatic sacs revealed tyrosine hydroxylase-positive and acetylcholinesterase-positive fibers. Some of the acetylcholinesterase-positive fibers were clearly associated with vessels. This innervation, which has not been described previously, may significantly influence blood flow and function of the endolymphatic sac.

Origin of sympathetic and sensory innervation of the endolymphatic sac. A retrograde axonal tracing study in the guinea pig.

Lectin-conjugated horseradish peroxidase (WGA-HRP) was used as a retrograde axonal tracer to determine endolymphatic sac (ES) innervation and the nature of such a nervous supply. WGA-HRP placed into the ES of the guinea pig resulted in labelling of neurons in the ipsilateral sympathetic superior cervical ganglion and the sensory trigeminal ganglion. The number of labelled neurons in the cervical superior ganglion varied according to the survival time with peak labelling occurring after 48 h. Sparse labelling of the sensory trigeminal ganglion was also present after 48 h. The results indicate that sympathetic neurons from the superior cervical ganglion and to a certain extent trigeminal somatosensory neurons innervate the ES or perisaccular tissue. If these findings reflect the existence of a sympathetic functional reflex unit remains to be elucidated.

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.

Organization of ascending auditory pathways in the pigeon (Columba livia) as determined by autoradiographic methods.

A mixture of tritiated proline and fucose was injected into the labyrinthine endolymphatic space of 5 white king pigeons (Columba livia). Using standard autoradiographic techniques, we observed transsynaptic labeling in ascending auditory pathways to the level of the mesencephalon. Auditory system structures, ipsilateral to the injection site, which labeled heavily were the cochlear nerve, the magnocellular and angular nuclei, and the superior olive. Those ipsilateral structures which were slightly labeled were the lateral lemniscus and the dorsal part of the lateral mesencephalic nucleus. Contralateral structures which labeled were the superior olive, lateral lemniscus, and dorsal part of the lateral mesencephalic nucleus. The results of this study suggest that ascending auditory pathways (to the level of the mesencephalon) in the pigeon are more similar to those described for mammals in general than previously thought.

Comparative scanning electron microscopic investigations of the sensory epithelia in the teleost sacculus and lagena.

Scanning electron microscopic studies were conducted on the sensory epithelia of the auditory portions of the ears in teleost species representing wide taxonomic diversity. A number of the features of the ears investigated resembled features found in other teleost species, although some major exceptions to earlier patterns were found, particularly in the saccular sensory epithelium. The saccular maculae of all but one species contained basically similar ciliary bundles on the sensory hair cells while there was some significant variation on the lagenar maculae. Hair cell orientation patterns on the sacculus contained four orientation groups in all of the species, other than the mormyrid, Gnathonemus, which only had two groups. Lagenar maculae had two orientation groups, and the orientation patterns were similar to one another. The most divergent form of lagenar macula was found in gnathonemus. These data, combined with data from earlier investigations, provide a broad overview of the surface features of the ear in teleost fishes. Most significantly, it now appears that there are at least five different saccular hair cell orientation patterns among teleost fishes, and all of these patterns are found spread through many major teleost taxa. While there is some similarity in ear structures among some groups of closely related species, such as the Elopomorpha and the Gadiformes, it is becoming more apparent that there is extensive convergence in a number of features of he teleost ear that most likely reflect similar selective pressures during the evolution of the ear. The nature of these selective pressures, however, are not well understood.