Musashi-1 is the candidate of the regulator of hair cell progenitors during inner ear regeneration.

BACKGROUND: Hair cell loss in the cochlea is caused by ototoxic drugs, aging, and environmental stresses and could potentially lead to devastating pathophysiological effects. In adult mammals, hair cell loss is irreversible and may result in hearing and balance deficits. In contrast, nonmammalian vertebrates, including birds, can regenerate hair cells through differentiation of supporting cells and restore inner ear function, suggesting that hair cell progenitors are present in the population of supporting cells. RESULTS: In the present study, we aimed to identify novel genes related to regeneration in the chicken utricle by gene expression profiling of supporting cell and hair cell populations obtained by laser capture microdissection. The volcano plot identified 408 differentially expressed genes (twofold change, p = 0.05, Benjamini-Hochberg multiple testing correction), 175 of which were well annotated. Among these genes, we focused on Musashi-1 (MSI1), a marker of neural stem cells involved in Notch signaling, and the downstream genes in the Notch pathway. Higher expression of these genes in supporting cells compared with that in hair cells was confirmed by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry analysis demonstrated that MSI1 was mainly localized at the basal side of the supporting cell layer in normal chick utricles. During the regeneration period following aminoglycoside antibiotic-induced damage of chicken utricles, the expression levels of MSI1, hairy and enhancer of split-5, and cyclin D1 were increased, and BrdU labeling indicated that cell proliferation was enhanced. CONCLUSIONS: The findings of this study suggested that MSI1 played an important role in the proliferation of supporting cells in the inner ear during normal and damaged conditions and could be a potential therapeutic target in the treatment of vestibular defects.

No evidence for an association between persistent measles virus infection and otosclerosis among patients with otosclerosis in Japan.

Otosclerosis, which is characterized by disordered bone remodeling, occurs exclusively in the human temporal bone. The etiology of the disease is unknown, but a popular hypothesis is that it is caused by persistent measles virus (MV) infection. Paramyxovirus-like filamentous structures were found in otosclerotic lesions of stapes footplates from patients with otosclerosis. Although MV RNAs have been detected in otosclerotic samples by using reverse transcription-PCR, no complete MV mRNA sequence has been reported, nor has infectious virus been isolated from clinical samples. Furthermore, one study failed to obtain evidence of MV infection in otosclerotic bone samples. In this study, we tested, by three different protocols, for the presence of MV in clinical samples from patients with otosclerosis in Japan. We used a highly sensitive reverse transcription-quantitative PCR method which is able to detect viral mRNA in cells infected with MV at around one infectious unit per well. We obtained no evidence of MV infection in bone samples, primary cell cultures derived from stapes bones, or MV-susceptible cell lines (Vero/hSLAM and II-18 cells) cocultured with bone samples or primary cell cultures derived from them. Thus, our results do not support the hypothesis that persistent MV infection is involved in the pathoetiology of otosclerosis.

Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti.

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.

The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high- and low-frequency cells and evidence for a connection to the lateral membrane.

The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, beta-actin, gamma-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and beta- and gamma-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall.

Dihydrostreptomycin goes through the mechano-electric transduction channel in chick cochlear hair cells.

OBJECTIVE: This study evaluated the ability of dihydrostreptomycin (DHSM) to go through the mechano-electric transduction (MET) channels in hair cells under physiological conditions. MATERIALS AND METHODS: Tall hair cells were isolated from the chick basilar membrane (cochlea). Mechanical stimulation was applied by a glass rod attached to a piezoelectric bimorph, and MET currents were recorded with a whole-cell patch technique. The voltage-dependent block of DHSM to MET channel was estimated by calculating the relative conductances (the ratio of MET current in DHSM saline to DHSM-free saline) at various membrane potentials. RESULTS AND CONCLUSION: At membrane potentials between -100 and +50 mV, DHSM behaves as a voltage-dependent blocker according to a partial block model. At membrane potentials more negative than -100 mV, however, DHSM blocking decreased. This finding differed from the partial block model, but indicated that DHSM escaped through the channel pore into the cytoplasm by acting as a permeant channel blocker due to the large electrical driving force.

Three-dimensional regular arrangement of the annular ligament of the rat stapediovestibular joint.

The stapes footplate articulates with the vestibular window through the annular ligament. This articulation is known as the stapediovestibular joint (SVJ). We investigated the ultrastructure of adult rat SVJ and report here on the characteristic ultrastructure of the corresponding annular ligament. Transmission electron microscopy showed that this annular ligament comprises thick ligament fibers consisting of a peripheral mantle of microfibrils and an electron-lucent central amorphous substance that is regularly arranged in a linear fashion, forming laminated structures parallel to the horizontal plane of the SVJ. Scanning electron microscopy revealed that transverse microfibrils cross the thick ligament fibers, showing a lattice-like structure. The annular ligament was vividly stained with elastica van Gieson's stain and the Verhoeff's iron hematoxylin method. Staining of the electron-lucent central amorphous substance of the thick ligament fibers by the tannate-metal salt method revealed an intense electron density. These results indicate that the annular ligament of the SVJ is mainly composed of mature elastic fibers.

Dissociation enzyme effects on the potassium currents of inner hair cells isolated from guinea-pig cochlea.

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. Some enzymes using for IHCs dissociation affect these inactivation kinetics. IHCs were dissociated from guinea-pig cochlea by 1 mg/ml trypsin or 0.25 mg/ml protease VIII, and the properties of the K+ currents were compared using conventional whole-cell voltage-clamp recordings. TEA-sensitive potassium currents showed fast inactivation kinetics in both trypsin-dissociated cells and protease VIII-dissociated cells. The time constant of the inactivation phase in trypsin-treated cells was similar to that in protease VIII-treated cells. However, the rate of inactivation (compared by the ratio between the steady-state current and initial peak current) in protease VIII-treated cells was larger than that in trypsin-treated cells. In protease VIII-dissociated cells, the time constant of recovery from inactivation elucidated by paired-pulse protocol was 3.5 ms. Papain is another enzyme that is sometimes used for dissociating IHCs, so effects of papain were observed. Extracellular papain application (8 unit/ml) demonstrated a slight increase of the outward potassium currents.

Glutamate induced currents in isolated inner hair cells from guinea-pig cochlea.

We investigated the direct action of glutamate (Glu) on the membrane current of isolated inner hair cells of guinea-pig cochlea. Glu elicited inward currents at a holding potential of -70 mV. Eight of 13 cells showed a steady inward current, while five of 13 cells showed a fast and rapidly desensitized current. I-V relationships demonstrated that the reversal potential of Glu-induced current was near 0 mV. Glu-induced currents were dose-dependent, where the half maximum concentration (K(d)) was 41 microM and Hill coefficient (n) was 1.75.

Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea.

Cyclopiazonic acid (CPA) is a reticulum-like intracellular Ca(2+) store depletory, which raises intracellular Ca(2+) concentration. The effect of CPA on membrane currents in isolated inner hair cells (IHCs) from guinea-pig cochlea was investigated by the patch-clamp technique in the whole-cell configuration. Four out of eight IHCs showed an augmentation of the currents and the other four cells showed an inhibition of the currents by extracellular CPA application. The activation kinetics of outward currents were not changed by CPA. Three out of four IHCs obtained from the basal part of the cochlea demonstrated augmentation, whereas three out of four IHCs from the apical part demonstrated inhibition of the currents. This result suggests that Ca(2+)-activated currents were dominant in the basal IHCs of the cochlea.

Expression of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocators in human adenoid tissue.

OBJECTIVE: The aim of this study was to determine whether aryl hydrocarbon receptor (AhR) and AhR nuclear translocators (ARNTs) are expressed in human adenoid tissue. METHODS: Paraffin-embedded human adenoid tissue specimens were obtained from eight children with adenoid hypertrophy. Sections were stained immunohistochemically with specific polyclonal antibodies to evaluate the expression pattern of AhR, ARNT1 and ARNT2 were evaluated. RESULTS: AhR-immunoreactivity was ubiquitously seen in human adenoid tissue such as the epithelium, subepithelial layer, germinal center, mantle zone and interfollicular area. ARNT1 was also widely expressed in the same regions as AhR. Although ARNT2 was expressed in the human adenoid, the expression level was significantly lower than that of ARNT1. CONCLUSION: These data suggest that the relationship between AhR and ARNTs may play an important role in the local immune response in adenoid and that ARNT1 may be more important than ARNT2 especially in adenoid tissue.

Effect of salicylate on potassium currents in inner hair cells isolated from guinea-pig cochlea.

Although salicylate is one of the most widely used nonsteroidal anti-inflammatory drugs, it causes moderate hearing loss and tinnitus at high-dose levels. In the present study, salicylate effects on the K currents in inner hair cells were examined. Salicylate reversibly reduced the outward K currents (I(K,f)), but did not affect the inward current (I(K,n)). Salicylate blocked the outward K currents in a concentration-dependent manner according to Hill equation with a half-blocking concentration of 1.66mM, and the Hill coefficient of 1.86.

Property of I(K,)(n) in inner hair cells isolated from guinea-pig cochlea.

One of the potassium currents, I(K,)(n), is already activated at the resting potential of the cell and thus determines the membrane potential. KCNQ4 channel has been identified as the molecular correlate of I(K,)(n). In the present study, we measured I(K,)(n) in acutely isolated IHCs of guinea-pig cochlea using the whole-cell voltage-clamp techniques, and investigated the properties of the currents. I(K,)(n) was 70% activated around the resting potential of -60 mV and deactivated on hyperpolarization. I(K,)(n) was blocked by the KCNQ-channel blockers, linopirdine (100 microM) and XE991 (10 microM), but was insensitive to both I(K,f) blocker, tetraethylammonium (TEA), and I(K,s) blocker, 4-aminopyridine (4-AP). There was no significant difference in the size of I(K,)(n) between the apical and basal turn IHCs.

Effect of hydrogen peroxide on potassium currents in inner hair cells isolated from guinea pig cochlea.

Hydrogen peroxide (H2O2) is a ubiquitous reactive oxygen species that can induce several inner ear disorders. In this study, we recorded the potassium (K) currents in acutely isolated inner hair cells of guinea pig cochlea, and investigated the effects of H2O2. We also observed the morphological changes in inner hair cells induced by H2O2. In the H2O2 solutions, the amplitude of outward K currents (I(K,f) and I(K,s)) clearly decreased after perfusion for approximately 15 min. Despite the decrease in outward currents, small inward currents (I(K,n)) did not show any reduction. H2O2 induced morphological changes in the inner hair cells. All the inner hair cells in the H2O2 solutions showed shrinkage and granularity of the cell body and led to loss of viability. These results showed the vulnerability of inner hair cells to reactive oxygen species-induced inner ear disorders.

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid.

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Ultrastructural transformation of gastric parietal cells reverting from the active to the resting state of acid secretion revealed in isolated rat gastric mucosa model processed by high-pressure freezing.

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H(+)/K(+)-ATPase demonstrated a progressive translocation of H(+)/K(+)-ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H(+)/K(+)-ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.

Inactivating and non-inactivating potassium currents in isolated inner hair cells from guinea pig cochlea.

Using conventional whole-cell voltage-clamp recordings, we examined the 4-aminopyridine (4-AP)- and tetraethylammonium (TEA)-sensitive K(+) currents in the cochlear inner hair cells (IHCs) of guinea pigs. 4-AP-sensitive currents were activated slowly and sustained the same current level, whereas TEA-sensitive currents were activated rapidly, followed by inactivation. The inactivation time course of TEA-sensitive currents was voltage-dependent, becoming faster at more depolarized levels. The inactivation of TEA-sensitive currents almost recovered within 5 ms. 4-AP- and TEA-sensitive K(+) currents coexisted in the same IHC.