Robert S. Kimura

Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis

Gap junctions in the rat cochlea were investigated using immunostaining for connexin26 and transmission electron microscopy. Electron microscopy of normal and pre-embedded immunostained material showed that there were gap junctions between and among all cells that light microscopy showed to have immunostained appositions. Light microscopy showed immunostaining between and among all cell types that electron microscopy showed to be joined by gap junctions. Immunostaining for connexin26 was therefore taken as providing a reasonable approximation of the locations of gap junctions throughout the cochlea and was used to provide an overview of the extent of those locations. Cells interconnected via gap junctions fell into one of two groups. The first group consists of nonsensory epithelial cells and includes interdental cells of the spiral limbus, inner sulcus cells, organ of Corti supporting cells, outer sulcus cells, and cells within the root processes of the spiral ligament. The second group consists of connective tissue cells and includes various fibrocyte types of the spiral limbus and spiral ligament, basal and intermediate cells of the stria vascularis, and mesenchymal cells which line the scala vestibuli. The present work represents a first attempt towards a description of how serial gap junctions among cochlear cells reflect a level of organization of the tissue. The organization described here, together with a great deal of information from previous investigators, suggest that serially arranged gap junctions of both epithelial and connective tissue cells serve as the strucural basis for recycling endolymphatic potassium ions that pass through the sensory cells during the transduction process.

Experimental blockage of the endolymphatic duct and sac and its effect on the inner ear of the guinea pig. A study on endolymphatic hydrops.

In 1965, Kimura and Schuknecht! reported the consistent development of endolymphatic hydrops after obliteration of the endolymphatic duct and sac in the guinea pig. This preliminary histological report was limited to specimens of short postoperative periods, ranging from 1 to 30 days. The present communication reports the results of a longer term experiment with guinea pigs kept up to 14 months, and includes both light and electron microscopic findings. Other experiments were performed on the endolymphatic duct and sac to provide further information on the physiological processes concerned in the development of endolymphatic hydrops.

Gap junction systems in the mammalian cochlea.

Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of K(+) ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of K(+) and result in hearing loss.

The pathology of sudden deafness.

Pathological studies were performed on eight temporal bones, of which six were from individuals with unilateral sudden deafness and two from one individual with bilateral sequential sudden deafness. The hearing losses were profound in four ears, severe in three ears and moderate in one ear. At the time of onset of the sudden deafness two reported having headcolds, one had acute pharyngitis, two had pneumonia and two complained of headache. Vertigo as an associated symptom was severe in one case and mild in two cases. The principal pathological changes consisted of atrophy, in varying combinations and severity, of the organ of Corti, tectorial membrane and stria vascularis. These pathological alterations were judged to be more like those occurring in labyrinthitis of known viral etiology than those following experimentally induced vascular lesions in animals.

Distribution of HRP in the Inner Ear after Injection into the Middle Ear Cavity

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 Reissners 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 Reissners 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.

Atrophy of the stria vascularis, a common cause for hearing loss.

Atrophy of the stria vascularis is a genetically determined deafness of aging characterized by a bilateral symmetrical sensori‐neural hearing loss showing flat audiometric patterns and excellent speech discrimination. The temporal bones of individuals exhibiting this type of hearing loss were studied by serial sections and surface preparations for light microscopy and by electron microscopy. The atrophic changes are most severe in the apical regions of the cochleas and involve the marginal, intermediate and basal cells in that order of severity. It seems probable that atrophy of the stria vascularis causes some deficiency in the quality of endolymph throughout the cochlear duct, regardless of the location of the atrophy. Typically all other structures of the cochlear duct are normal, and the sense organ when stimulated within its sensitivity range is capable of normal stimulus coding, thus accounting for the usually excellent speech discrimination.

Immunolocalization of Na+, K(+)-ATPase, Ca(++)-ATPase, calcium-binding proteins, and carbonic anhydrase in the guinea pig inner ear.

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.

Ultrastructural Study of the Human Spiral Ganglion

Electron microscopic study of the human spiral ganglion was conducted on 17 ears from 12 individuals aged 9 months to 92 years. Two types of neurons, large and small, with distinct cytological characteristics were found. Both types of neurons were myelinated and unmyelinated; however, a majority of the population was unmyelinated (94%). The distribution of myelinated neurons varied greatly among individuals, though a slight increase in their population was noted in aged individuals, the highest count being 28% in a specimen from an individual 75 years old. The small neurons constituted 6% of the population, and their cytoplasms were highly filamentous in both myelinated and unmyelinated types. The findings of this study provide no evidence as to the functional significance of the myelination of spiral ganglion cells.

Animal models of endolymphatic hydrops

Endolymphatic hydrops in the animal can be produced by various methods. The most promising is obliteration of the endolymphatic duct in the guinea pig. The extent of hydrops is similar to that of Ménières disease specimens, but the animals are asymptomatic. A better model, utilizing a less invasive method and having the ability to elicit episodic vestibular symptoms, is needed. There is a reasonable doubt that one of the causes for Ménières disease lies in the abnormal endolymphatic duct and sac. A wide ablation of such pathologic tissue may provide additional pathogenetic information.

Gap Junction Systems in the Rat Vestibular Labyrinth: Immunohistochemical and Ultrastructural Analysis

The distribution of gap junctions within the vestibular labyrinth was investigated using immunohistochemistry and transmission electron microscopy. Connexin26-like immunoreactivity was observed among supporting cells in each vestibular sensory epithelium. Reaction product was also present in the transitional epithelium of each vestibular endorgan and in the planum semilunatum of crista ampullaris. No connexin26-like immunoreactivity was observed among thin wall epithelial cells or among vestibular dark cells. In addition, fibrocytes within vestibular connective tissue were positively immunostained. Reaction product was also detected in the melanocyte area just beneath dark cells. Ultrastructural observations indicated that a gap junction network of vestibular supporting cells extends to the transitional epithelium and planum semilunatum and forms an isolated epithelial cell gap junction system in each vestibular endorgan. In contrast, no gap junctions were found among wall epithelial cells or among dark cells. Fibrocytes and melanocytes were coupled by gap junctions and belong to the connective tissue cell gap junction system, which is continuous throughout the vestibular system and the cochlea. The possible functional significance of these gap junction systems is discussed.