Reissner's Membrane Research Articles

Expression profiles of the connexin genes, Gjb1 and Gjb3, in the developing mouse cochlea

Several connexin genes ( GJB1, GJB2, GJB3, GJB6 and GJA1) have been found mutated in patients with non-syndromic and/or syndromic deafness indicating an important role of these proteins in the auditory system. In order to better understand the function of the connexins in the inner ear we have analyzed the gene expression profiles of two connexin genes, Gjb1 (connexin 32) and Gjb3 (connexin 31), by in situ hybridization during the mouse cochlea organogenesis, from early otocyst up to the mature organ in adult. In the developing otocyst epithelium, some restricted domains expressed Gjb3 and Gjb1 whilst high levels of both transcripts were present in the surrounding mesenchymal tissue. As development proceeds, expression of these two genes was found in various subtypes of fibrocytes, either within the spiral limbus or along the spiral ligament, as well as in the basilar membrane cells, in the Reissner's membrane cells, and in subsets of the cellular elements of the cochlear ganglion. Gjb3 and Gjb1 expression was spatiotemporally modulated within the sensory hair cells and the various supporting cells that compose the developing organ of Corti. A transitory expression of Gjb1 was found in the basal and intermediate cells of the stria vascularis. In the adult cochlea Gjb1 transcripts disappeared while Gjb3 expression remained present in fibrocytes with specific expression patterns.

High-resolution magnetic resonance imaging of human cochlea.

High-resolution MRI (MRI) of human inner ear structures provides several advantages over other imaging modalities. High-resolution visualization of inner ear ultrastructure in a noninvasive manner may provide important information about inner ear disease that is not obtainable in other ways. The study was performed to demonstrate the capabilities of MRI at high resolution on the human cochlea, vestibular structures, and facial nerve. Comparative analyses of MRI anatomy with that seen on histological dissection were made. The aim of the study was to define the anatomy of human cadaveric cochlea using a 9.4-Tesla magnetic resonance scanner, currently the most powerful magnetic resonance magnet available. Experimental pilot study of cadaveric human cochleae. Serial scanning using a 9.4-Tesla magnetic resonance imager on normal preserved and fresh cadaveric inner ears was performed in different planes. The images revealed detailed anatomy of the modiolus, utricle, saccule, semicircular canals, and facial nerve. Specifically, identifiable structures within the cochlea included the osseous spiral lamina, Reissner's membrane, membranous spiral lamina, spiral ligament, and others. Data established through the acquisition of images from cadaver cochlea, facial nerve, and vestibular complex provide a foundation for developing steps for testing temporal bones and, eventually, patients with Meniere's disease and other inner ear disease. The present ongoing project will provide information on baseline images of the inner ear using high-resolution MRI.

Cochlear ultrastructure in two-phase endolymphatic hydrops in the guinea-pig.

Endolymphatic hydrops continues to be considered as a pathological factor in the etiology of Meniere's disease. We have developed the two-phase endolymphatic hydrops model, which seems to represent a functional model combining multiple etiologies, and which may resemble the fluctuating characteristics of Meniere's disease. A transmission electron microscopic study was performed on the endolymphatic sacs of four groups of guinea-pig cochleas: (1) controls, (2) non-operated, aldosterone-treated cochleas, (3) operated (dissection of the endolymphatic sac) cochleas, (4) operated and aldosterone-treated cochleas. Light and electron microscopy showed a normal morphology in the controls. Aldosterone as a single treatment resulted in an increased activity of the marginal cells in the stria vascularis. Dissection induced a gradient of degenerative effects and cell loss in the intracellular and extracellular structures of the sensory cells, the stria vascularis and Reissner's membrane, which may be reversible. Subsequent administration of aldosterone induced severe damage and increased cell loss, which may be irreversible. Our findings demonstrate changes that may be reversible due to the compromising effect of a single treatment, and irreversible changes due to interaction of both compromising factors. These findings support our two-phase concept.

The early postnatal development of the hamster cochlea

The Early Postnatal Development of the Hamster Cochlea. Inborn deafness can be diagnosed very early. Deafness in early childhood can be caused by: genetic defects (30 % - 40 %), embryopathies, embryonic noxae (60 %) and pre-, peri- and postnatal noxae. Which structures of the cochlea are disturbed, is unknown in most cases. Systematic studies are necessary with directed experimental impairments of the cochlea in the early development to elucidate these mechanisms. An animal model has to be established in which directed impairments can be carried out in single individuals. The cochlea of the hamster is at birth in a very early state of development. In the apical turn the epithelial layer of the cochlear duct is undifferentiated, whereas in the basal turn the papilla basilaris (Kölliker's organ), the lateral wall and Reissner's membrane are visible. Between the base of the cochlea (1st coil lateral) and the apical part (3rd coil lateral) the difference in the development of the hamster cochlea is exactly four days. This is evident for the 1st and the 5th day after birth. The cochlea development of the hamster is very rapid. The development during one day represents one week of development in the human cochlea. The difference in development in one hamster cochlea from the apex to the base at a certain day representing four days of development is comparable with the development time of four weeks in human. The different developmental stages which are present in the hamster cochlea of a certain day represent the development of the human cochlea of a whole month. The hamster cochlea at the 1st day after birth covers the 2nd embryonic month of the human (4th to 8th week) and at the 5th day the 3rd embryonic month (8th to 12th week). The hamster cochlea at the 1st and the 5th day after birth is especially suitable to study experimental disturbances in the early stages of the development of the cochlear duct when the tectorial membrane, the spiral limbus, the stria vascularis, Reissners membrane and the external spiral Sulcus with the root cells are differentiating. The biggest advantage is that the noxae (hypoxia, teratogenic substances, ototoxic antibiotics and intense noise) can be applied to single individuals which offers better control than the treatment of the mother.

Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the hydropic vestibule after injection of keyhole limpet hemocyanin into the endolymphatic sac of guinea pigs

This study was undertaken to examine the expression of inducible nitric oxide synthase (iNOS / NOS II) in the hydropic vestibule of guinea pigs. Animals were systemically sensitized with 500 microg of keyhole limpet hemocyanin. Two weeks after the first injection, keyhole limpet hemocyanin (100 microg/5 microl) was injected into the endolymphatic sac following the intradural approach, and the next day temporal bones were removed for the immunohistochemical examination. Endolymphatic hydrops was evidenced by the expansion of the Reissner's membrane in the cochlea after direct injection of keyhole limpet hemocyanin into the endolymphatic sac. Inducible nitric oxide synthase expression was increased in the sensory cells, supporting cells and vestibular ganglion cells, while temporal bones, where only phosphate buffered saline was injected, did not show any inducible nitric oxide synthase immunoreactivity. High levels of inducible nitric oxide synthase-catalyzed nitric oxide were detected prior to the development of the inner ear dysfunction. Our results suggest that the occurrence of inducible nitric oxide synthase immunoreactivity parallels the inner ear disturbance as seen in endolymphatic hydrops.

A computational algorithm for computing nonlinear auditory frequency selectivity.

Computational algorithms that mimic the response of the basilar membrane must be capable of reproducing a range of complex features that are characteristic of the animal observations. These include complex input output functions that are nonlinear near the site's best frequency, but linear elsewhere. This nonlinearity is critical when using the output of the algorithm as the input to models of inner hair cell function and subsequent auditory-nerve models of low- and high-spontaneous rate fibers. We present an algorithm that uses two processing units operating in parallel: one linear and the other compressively nonlinear. The output from the algorithm is the sum of the outputs of the linear and nonlinear processing units. Input to the algorithm is stapes motion and output represents basilar membrane motion. The algorithm is evaluated against published chinchilla and guinea pig observations of basilar membrane and Reissner's membrane motion made using laser velocimetry. The algorithm simulates both quantitatively and qualitatively, differences in input/output functions among three different sites along the cochlear partition. It also simulates quantitatively and qualitatively a range of phenomena including isovelocity functions, phase response, two-tone suppression, impulse response, and distortion products. The algorithm is potentially suitable for development as a bank of filters, for use in more comprehensive models of the peripheral auditory system.

Mutations in Cdh23 Cause Nonsyndromic Hearing Loss in waltzer Mice

Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. Here, we use a positional cloning approach to show that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila Fat protein. A single nucleotide deletion in the vJ allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the urticulo-saccular foramen,the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss.

The origin of eponyms used in cochlear anatomy.

The anatomy of the cochlea, one of the most complicated parts of the inner ear, is associated with numerous eponyms, mostly originating from German anatomists of the 19th century. In addition to the organ of Corti and Reissner's membrane, which are the best known, nine other eponymic anatomic terms are associated with the cochlea: Deiters' cells, Hensen's cells, Hensen's strip, Claudius' cells, Boettscher's cells, Rosenthal's canal, Hardesty's membrane, Huschke's teeth, and Nuel's space. All of these anatomic eponyms are described, with their original references.

メニエール病の成因と病態生理

In our previous reports we hypothesized that both vertigo and deafness in Meniere's disease could be explained by high potassium levels in the scala tympani, not in the scala vestibuli. High potassium levels in the scala tympani may be caused mainly by the increased diffusion of potassium through the distended Reissner's membrane. A relative decrease in the perilymphatic space due to endolymphatic hydrops might also play an important role in the elevation of potassium levels in the scala tympani. Thus, the aggravation of vertigo and deafness is closely linked to the degree of hydrops.In Meniere's disease, vertigo and deafness is often induced by stress. Stress is well known to promote the release of vasopressin, resulting in an increase in plasma vasopressin. We previously reported that an increase in plasma vasopressin was observed in Meniere's disease. Although histological and surgical findings support the hypothesis that the dysfunction of the endolymphatic sac is one of the important factors in Meniere's disease, we speculated that a high concentration of plasma vasopressin may be the other causative factor in the formation of endolymphatic hydrops.

Endolymphatic Hydrops by Administration of Vasopressin in the Rat.

The anti-diuretic hormone, vasopressin, has been shown to be important in regulating inner ear fluid. We report on the involvement between Meniere's disease and vasopressin. We designed an experiment to determine whether vasopressin directly affects the fluid level. We chronically infused this hormone in rats and examined them for morphological signs of endolymphatic hydrops. Vasopressin infusion significantly increased the scala media area by 5.9±7.1% (P<0.01), and the length of Reissner's membrane by 5.9±3.2% (P<0.001). The results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps due to down-regulating the number of vasopressin receptors.

A technique for slicing the rat cochlea around the onset of hearing

The cochlea presents a considerable challenge to the study of sound transduction and auditory neurotransmission. This arises from the location of the sensory, supporting and secretory epithelia, and primary auditory neurons within a complex ossified spiral structure comprised of three separate fluid-filled chambers. We have developed a novel cochlear slice preparation, which provides access to the highly differentiated tissues while retaining structural integrity and cell viability. Our technique for slicing the cochlea and imaging tissue structure facilitates the study of peripheral auditory signaling in situ. The preparation was developed in the neonatal rat (postnatal days 4–14) and is based on the use of vibrating blade microtome slicing after perfusing the perilymphatic compartments with chilled Pluronic F127 NF, a block copolymer gel. This material is liquid when cold, and sets when warmed to room temperature, stabilizing the cochlear fluid-filled compartments and thereby supporting the cochlear partition during slicing. Slices (150–300 μm) of neonatal rat cochlea, imaged using infrared videomicroscopy, allow tight-seal voltage clamp recordings from a variety of cell types. Recordings obtained from primary auditory neurons, hair cells, supporting cells, and Reissner's membrane epithelial cells verify the viability of the tissues in the preparation. Data includes novel evidence for glutamatergic and purinergic co-transmission by primary auditory neurons. The preparation has considerable potential in a range of molecular physiological applications requiring cell-specific targeting with retention of cell connectivity.

Hydrops in the cochlea can be induced by sound as well as by static pressure

The Reissner’s membrane (RM) was visualised by confocal microscopy in the isolated temporal bone of the guinea pig. The function of the organ was followed by measuring its physiological response. Static pressure applied in the basal coil caused a distention of the RM in the apical coil into the scala vestibuli. The sensitivity to a test tone was reduced. When the pressure was relieved, the RM returned to its original position and the response recovered. If the increased pressure was maintained, the RM would bulge further. The RM could then be reversibly stretched and return gradually, with a delay, to its original position. Alternatively, it could be over-stretched and return with an over-shoot past its original position toward the organ of Corti. In response to repetitive tone pulses of above 80 dB, hydrops of the RM also developed. This was accompanied by a reduced sensitivity. A slow recovery to the original position, or over-shoot, and return of responsiveness could be seen. Above 106 dB sustained loss was generally seen. It is concluded that the RM can accommodate increased scala media pressure by distention. This will relieve the organ of Corti from part of the pressure and may protect the organ from trauma.

Human Nonsyndromic Hereditary Deafness DFNA17 Is Due to a Mutation in Nonmuscle Myosin MYH9

The authors had previously mapped a new locus-DFNA17, for nonsyndromic hereditary hearing impairment-to chromosome 22q12.2-q13. 3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle-myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase-PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G-->A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated.

Rupture pressures of membranes in the ear.

The rupture pressures of the tympanic membrane, Reissner's membrane, the round window membrane, and the annular ligament have all been measured in cadaver ears from Norwegian cattle. For the tympanic membrane, a static overpressure was applied to the ear canal; for Reissner's membrane, to the endolymph; and for the round window membrane, to the perilymph. The rupture pressure of the annular ligament equals the rupture force to the footplate divided by the area of the oval window. The mean rupture pressures are 0.39 atm for the tympanic membrane, 0.047 atm for Reissner's membrane, greater than 2 atm for the round window membrane, and 29.4 atm for the annular ligament. This last pressure corresponds to 0.68 kilogram force applied to the footplate. The ruptures of the tympanic membrane appeared without exception as small tears in the pars flaccida. The rupture pressure of the tympanic membrane was also measured in a few ears from foxes.

Detection of single-stranded DNA in the hydropic vestibule after the direct injection of antigen into the endolymphatic sac of guinea pigs

Immunohistochemical study for single-stranded DNA (ssDNA) with vestibule of guinea pigs was performed after the injection of keyhole limpet hemocyanin (KLH) into the right endolymphatic sac. Endolymphatic hydrops became evident by expansion of the Reissner's membrane in the cochlea of all animals 1 day after the injection of KLH. Increased ssDNA expression was detected in the sensory epithelium and transitional area, while temporal bones in the control group did not show any ssDNA immunoreactivities. ssDNA is accompanied with the apoptotic change in the vestibule. Our results suggest that apoptotic changes could be involved in the hydropic vestibule and these phenomena lead inner ear disturbance as seen in endolymphatic hydrops.

Vibrations of the guinea pig organ of Corti in the apical turn

Vibrations of the organ of Corti were measured in response to sound applied to the ear in the apical turn of a living guinea pig. Measurements were made at 29 points on the Reissner’s membrane (RM) at 10 μ spacing along a radial track. Measurements also included 22 points on the reticular lamina (RL), Claudius’ cells and osseous spiral lamina. Our goal was to characterize the vibration of the RM and the RL with high spatial resolution along a radial axis. The tuning and spatial patterns of the RM are compared in the radial direction with those for the RL at the fundamental frequency and at the second harmonic. The shape of the RM tuning curve changes with radial position, and differ significantly from those observed at the RL. These results support our earlier findings (Hao and Khanna, Hear. Res. 99 (1996) 176–189).

The rat cochlea in the absence of circulating adrenal hormones: an electrophysiological and morphological study

Circulating adrenal hormones affect strial function. Removal of endogenous levels of adrenal steroids by bilateral adrenalectomy (ADX) in rats causes a decrease of Na +/K +-ATPase activity in the cochlear lateral wall [Rarey et al., 1989. Arch. Otolaryngol. Head Neck Surg. 115, 817–821] and a decrease of the volume of the marginal cells in the stria vascularis [Lohuis et al., 1990. Acta Otolaryngol. (Stockh.) 110, 348–356]. To study further the effect of absence of circulating adrenocorticosteroids on cochlear function, 18 male Long Evans rats underwent either an ADX or a SHAM operation. Electrocochleography was performed 1 week after surgery for tone bursts in a frequency range of 1–16 kHz. Thereafter, the cochleas were harvested and examined histologically. No significant changes in the amplitude growth curves of the summating potential (SP), the compound action potential (CAP) and the cochlear microphonics (CM) were detected after ADX. However, visually, there appeared to be a decrease of endolymphatic volume (tentatively called imdrops). Reissner’s membrane (RM) extended less into scala vestibuli in ADX animals than in SHAM-operated animals. The ratio between the length of RM and the straight distance between the medial and lateral attachment points of RM were used as an objective measure to quantify this effect in each sub-apical half turn of the cochlea. The decrease in length of RM was statistically significant. Thus, circulating adrenal hormones appear to be necessary for normal cochlear fluid homeostasis. Absence of one or more of these hormones leads to shrinkage of the scala media (imdrops). However, the absence of adrenal hormones does not affect the gross cochlear potentials. Apparently, the cochlea is capable of compensating for the absence of circulating adrenal hormones to sustain the conditions necessary for proper cochlear transduction.

Effect of cisplatin on the expression of glutathione-S-transferase in the cochlea of the rat.

Glutathione-S-transferase (GST) has been found to conjugate glutathione (GSH) to diverse electrophiles and play a major role in the detoxification of alkylating and platinating agents. However, there is little information regarding the pattern of GST expression in the cochlea. Although cisplatin is ototoxic, its effect on GST in the cochlea is unknown. In the present study we investigated the pattern of GST expression in control and cisplatin-treated cochleas by using the laboratory rat as animal model. Sixteen mature rats were randomly assigned to control or cisplatin groups. After treatment, cochleas were procured and tissues stained by immunohistochemical methods to detect the presence of GST. Optical density measurements were determined to gauge intensity of GST immunostaining. Strong positive GST immunostaining was demonstrated in all cochleas, with the most intense staining found in the spiral ligament and the least in Reissner's membrane. Mean optic density scores were lower for cisplatin-treated cochleas than for control cochleas in all areas analyzed. These findings showed that GST is expressed throughout the rat cochlea, with cisplatin treatment causing its decreased expression.

Expression of inducible nitric oxide-synthase in the vestibular system of hydropic guinea pigs

Immunohistochemical investigations of the guinea pig vestibular system, using a specific antibody to the inducible isoform of NO-synthase (iNOS/NOS II), have been performed 3 weeks after surgical closure of the right endolymphatic duct ( n=7). Endolymphatic hydrops (ELH) of the right temporal bone became evident by excavation of the Reissner's membrane in all seven animals. Those animals revealed iNOS-expression in ganglion cells, in the wall of blood vessels and in nerve fibers of the right vestibular system, while the corresponding left temporal bones and temporal bones of non-operated controls ( n=6) as well as of sham-operated animals ( n=3) did not show any iNOS-positive structures. iNOS-generated NO could be involved in the pathophysiology of vestibular dysfunction in Meniere's disease.