Masaya Takumida

The endolymphatic sac and inner ear homeostasis. I: Effect of glycerol on the endolymphatic sac with or without colchicine pretreatment

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.

Geometrical Array of the Vestibular Sensory Hair Bundle

The stereometrical arrangement of the sensory hair bundles in vestibular epithelia of the guinea pig was studied with high-resolution scanning electronmicroscopy. Besides the previously known arrangement in tall and short hair bundles, a new morphological criterion dividing the hair bundles into two separate populations is described. Thus the hair bundles can be separated into a loose and a tight type, depending on the interrelationship between the individual stereocilia. The cilia in the loose hair bundle are arranged in longitudinal rows parallel with the cells plane of polarization, so that the stereocilia increasing stepwise in height are compressed tightly together. The stereocilia in the tight hair bundle type are arranged in equally tall transverse rows in which the cilia are situated directly adjacent to each other. While all the stereocilia are connected by thin filaments along the entire course of their shafts, their bevelled tips are connected by a single tip link to the shaft of the adjacent, taller stereocilium. The tip link morphology differs between the two hair bundle types. It is assumed that these two different types of hair bundles have different mechanical properties, which could indicate a higher degree of selectivity on the hair cell level in the vestibular end organs.

The effect of gentamicin on the glycocalyx and the ciliary interconnections in vestibular sensory cells: A high resolution scanning electron microscopic investigation

Changes in glycocalyx structure and ciliary interconnections of the vestibular sensory cells are demonstrated after gentamicin administration. A special high resolution scanning electron microscope and a tannic acid-osmium staining technique giving an almost three dimensional view were used to achieve this purpose. Guinea pigs were injected with a single dose of 5 mg of gentamicin directly into the middle ear. Seven days after the injection, it was possible to observe the degenerative process of the glycocalyx and the ciliary interconnections. The first detectable change was a disarrangement of the cilia with a loosening of the interconnections. The ciliary membrane presented with an irregular appearance. The tip links connecting the tips of the stereocilia to their neighbours were also affected showing elongation or even disappearance. In the later stages of the degeneration process, the sensory hairs presented with different degrees of fusion whereafter they finally disappeared totally. These findings suggest that the glycocalyx acts to maintain a normal stability and shape of the ciliary membrane and to keep the regular distance between cilia in order to maintain the arrangement of the whole ciliary bundle. Gentamicin probably affects the glycocalyx and the ciliary interconnections resulting in a disarrangement, detachment and fusion of cilia. The tip links, which are suggested to be involved in sensory cell transduction, seem to be also affected by gentamicin.

Effect of gentamicin on the carbohydrates of the vestibular end organs:An investigation by the use of FITC lectins

The effect of gentamicin on the glycoconjugates in the vestibular end organs is demonstrated using FITC-lectins. Five milligrams of gentamicin dissolved in 0.1 ml. saline was injected in a single dose into the middle ear of adult guinea pigs. Seven days after the injection, the fluorescent reactivity of Wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Concanavaline A (Con A), and Latyrus odoratus agglutinin (LOA) was decreased in the apical epithelial cell surface as well as in the gelatinous layer of the otolithic membrane of the maculae. In the cupula of the crista ampullaris, the reactivity for WGA and Con A was reduced. The otoconia, however, showed no detectable changes. This indicates that gentamicin may induce an altered carbohydrate metabolism resulting in a decrease of N-acetyl-glucosamine, mannose, galactose and glucose in the glycocalyx lining the epithelial cells as well as in the gelatinous layer of the otolithic membrane or cupula in the vestibular end organs.

Lectin detection of carbohydrates in the endolymphatic sac.

SummaryThe carbohydrate contents of the guinea pig endolymphatic sac were investigated by the use of lectins. The lumen of the endolymphatic sac was filled with stainable precipitate containing N-acetyl glucosamine, mannose, glucose, galactose and fucose. N-Acetyl galactosamine was also detected but in minute amounts. This composition corresponded to other areas in the inner ear, such as the cupula, the otolithic membrane and the tectorial membrane. The function of these carbohydrates may play an important role in preventing the lumen of the endolymphatic sac from collapsing as well as in regulating transepithelial fluid transport.

The basement membrane and associated structures in the murine endolymphatic sac

SummaryThe ultrastructure of the basement membrane of the murine endolymphatic sac was studied under various experimental conditions in labyrinthectomized and ethacrynic acid-treated animals and was compared with normal anatomy. The basement membrane was clearly visualized after staining with ruthenium red or dialyzed iron. The basement membrane of the murine sac consists of two different layers: the lamina rara and the lamina densa. It demarcates the border between the epithelial cells and the subepithelial connective tissue. Our findings suggest that the basement membrane acts as a physical support to the epithelium in the endolymphatic sac. The basement membrane also shows a dynamic capacity to form a new basement membrane, with the result that the lateral intercellular space between contiguous epithelial cells may be integrated with the subepithelial space. This system is believed to act as a macromolecular and bulk water transport system. In the subepithelial space, collagen, reticular and elastic fibrils are found having a close relationship to the basement membrane. The elastic fibres are presumed to play a role in the pressure-regulating mechanism in the endolymphatic sac.

The endolymphatic sac and inner ear homeostasis. II: Effect of glycerol on the sensory end organs with or without colchicine pretreatment

The effects of glycerol and colchicine on the sensory end organs of the inner ear were investigated in mice. Glycerol alone induced a widening of the intercellular spaces lining vestibular dark and transitional cells as well as the marginal cells of the stria vascularis. This was noted within 30 min after the injection of glycerol and was normalized again within 4 h after the injection. Colchicine induced some morphological changes in the inner ear sensory cells, such as dissociation of Golgi complexes etc. These isolated glycerol or colchicine injections did not cause any signs of inner ear functional impairment. Treatment with glycerol following pretreatment with colchicine, however, induced marked inner ear dysfunction with impaired sense of balance and audition. The inner ear morphology revealed a combination of changes as compared with what was observed after isolated treatment with glycerol or colchicine i.e. edema of the stria vascularis, and vestibular dark and transitional cells as well as dissociation of Golgi complexes in the sensory cells. The cochlea showed moderate endolymphatic hydrops. These findings indicate that colchicine affects the inner ear fluid regulating mechanisms which may lead to severe functional derangement after additional glycerol treatment. It is conceivable that the present experiment may serve as a useful model for further studies on inner ear changes related to endolymphatic hydrops and Ménières disease.

Initial changes in the sensory hair-cell membrane following aminoglycoside administration in a guinea pig model

SummaryThis study demonstrates the initial changes affecting the sensory hair-cell plasma membranes in the vestibular end organs of gentamicin-treated guinea pigs by using a ruthenium red staining technique. First, 0.1 ml of a solution containing 5 mg gentamicin sulfate was injected into the middle ear. After 7 days, the sensory hair cell cilia were observed to be degenerating. The various stages of this degeneration process were classified into two types: the decrease in glycocalyx was designated type I fusion, while type II fusion was characterized by a bleb formation of the plasma membrane of the sensory hair cells, followed by a decrease in glycocalyx. The latter mechanism allowed plasma membrane contact, with subsequent fusion of the plasma membrane of neighboring sensory hair-cell cilia. The material also illustrates the degeneration of ciliary actin filaments. These findings suggest that the aminoglycoside affects both the glycocalyx and the plasma membrane, and that the decrease in glycocalyx may be the first sign of sensory hair-cell fusion.

Observation of the Glycocalyx of the Organ of Corti: An investigation by electron microscopy in the normal and gentamicin-treated guinea pig

The ultrastructure of the glycocalyx of the organ of Corti in the normal and gentamicin-treated guinea pig was investigated at the electron microscopic level by using the ruthenium red staining technique. The glycocalyx was evident over the whole apical surface of both sensory and supporting cells. The entire length of the stereocilia of both inner and outer hair cells was seen to be covered by a glycocalyx, which interconnected the neighbouring stereocilia. In the gentamicin-treated animals, the fusion process between the stereocilia was revealed and a decrease of the glycocalyx was noticed at the point where fusion started. These findings suggest that the glycocalyx may play an important role in holding the stereocilia together in a bundle, yet prevent their close contact by means of its strong negative charge.

Ultrastructural localization of carbonic anhydrase in the vestibular end organs of the guinea pig

SummaryCarbonic anhydrase activity was demonstrated cytochemically on an ultrastructural level in the vestibular end organs of the guinea pig. Reaction product was found in the dark cells, transitional cells, cells of the planum semilunatum and supporting cells. In the dark cells, reaction product was observed in the cytoplasm as well as in the basal infoldings. Reaction product was also observed in the basal infoldings of the transitional cells and the cells of the planum semilunatum. The globular structures inside the supporting cells, transitional cells and the cells of the planum semilunatum were also surrounded by the reaction product. These findings suggest that carbonic anhydrase may have different functions, such as water and ion transport, respiration, nutrition and calcium carbonate deposition in the vestibular end organs.