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.

Cholesterol influences potassium currents in inner hair cells isolated from guinea pig cochlea.

OBJECTIVE: There is a correlation between serum hyperlipidemia and hearing loss. Cholesterol is an integral component of the cell membrane and regulates the activity of ion channels in the lipid bilayer. The aim of this study was to investigate the effects of cholesterol on the potassium currents in IHCs by using the cholesterol-depleting drug, MßCD, and water-soluble cholesterol. METHODS: IHCs were acutely isolated from a mature guinea-pig cochlea and potassium currents were recorded. MßCD and water-soluble cholesterol were applied to IHCs under pressure puff pipettes. RESULTS: IHCs showed outwardly rectifying currents (IK,f and IK,s) in response to depolarizing voltage pulses, with only a slight inward current (IK,n) when hyperpolarized. In 10mM MßCD solutions, the amplitude of outward K currents reversely decreased; however, fast activation kinetics was preserved. In contrast, in solution of 1mM water-soluble cholesterol, the amplitude of outward K currents reversely increased. At the membrane potential of +110mV, relative conductances were 0.87±0.07 and 1.18±0.11 in MßCD solutions and cholesterol solutions, respectively. CONCLUSION: The amplitude of K currents in isolated IHCs was reversely changed by cholesterol-depleting drug and water-soluble cholesterol. These results demonstrated the possibility of the involvement of IHC function in hyperlipidemia-induced inner ear disorders.

Nitric oxide influences potassium currents in inner hair cells isolated from guinea-pig cochlea.

OBJECTIVE: Nitric oxide (NO) is a diffusible second messenger, which regulates neurotransmission, serving as the principal endothelium-derived relaxing factor. NO also acts as an ion channel modulator. Nitric oxide synthase (NOS) has been identified in the inner ear, although its physiological role remains unclear. In the present study, the effects of NO onto K currents in inner hair cells (IHCs) were investigated. METHODS: IHCs were acutely isolated and K currents were recorded by conventional whole-cell voltage-clamp recordings. NO donors sodium nitroprusside (SNP) were applied directly to the cells. RESULTS: In 1mM SNP solutions, the amplitude of outward K currents (IK,f and IK,s) reversely decreased; however, fast activation kinetics was preserved. In the current-voltage relationship curves, the maximal slope conductances were 53.2nS and 44.2nS in control solutions and 1mM SNP solutions, respectively. At the membrane potential of +110mV, the amplitudes of outward currents were 9.2±2.9nA in control solutions and 7.3±2.7nA in 1mM SNP solutions, showing a significant difference. CONCLUSION: NO acts as a K channel modulator in IHCs. A fast K current suppression may account for the high-frequency hearing impairment by the prevention of fast repolarization.

Massively parallel DNA sequencing facilitates diagnosis of patients with Usher syndrome type 1.

Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1%) who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%), which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance. This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance.

Membranous band between the tympanic membrane and the external auditory canal.

Two cases of a membranous band between the tympanic membrane and the external auditory canal are presented. These characteristic structures are rare, but observed in several first branchial cleft anomalies. Neither patient presented with an infection. In Case 1, an eleven-year-old girl has this structure in her right ear. In Case 2, a thirteen-year-old boy has this structure in his left ear. Both patients demonstrated slight air-bone gaps in the affected ear with a pure-tone audiometric test, due to limited vibrations of the tympanic membrane.

Temperature enhances activation and inactivation kinetics of potassium currents in inner hair cells isolated from Guinea-pig cochlea.

OBJECTIVES: Until recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs. METHODS: IHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25℃) and physiological temperatures (35℃-37℃). RESULTS: IHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36℃ than at room temperature. Half-time for activation was shorter at 36℃ than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q10 for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36℃ than at room temperature between the membrane potentials of -20 and +60 mV. Q10 for the inactivation time constant was 3.19. CONCLUSION: The results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.

The effect of pressure on potassium currents in the inner hair cells isolated from guinea-pig cochlea.

An increase in hydrostatic pressure in the endolymphatic system causes hydrops-related inner ear diseases such as Meniere's disease or low tone sensorineural hearing loss. In the present study, we investigated the effects of pressure exerted on potassium currents in acutely isolated inner hair cells of the guinea-pig cochlea using whole-cell voltage-clamp techniques. By applying negative or positive pressure via the patch pipette using a syringe, intracellular hydropressure was changed between -40 cm H2O to +20 cm H2O. Negative pressure potentiated the amplitude of potassium currents, whereas positive pressure suppressed the amplitude of potassium currents. Gadolinium, a blocker of stretch-activated cation channels, did not influence pressure-dependent changes in potassium currents; however, cinnarizine blocked pressure-dependent changes in potassium currents. The current changes were not dependent on the sign of the pressure change, that is, similar increases in negative pressures (between -10 cm H2O and -40 cm H2O) and similar decreases in positive pressures (between +10 cm H2O and +20 cm H2O) were observed.

[Problem and assignment for distinguishing the Usher syndrome type].

Usher syndrome is an autosomal-recessive disorder that causes bilateral sensorineural hearing loss, retinitis pigmentosa (RP), and occasionally vestibular dysfunction. Usher syndrome types 1, 2, and 3 can be distinguished by differences in audiovestibular features. The objectives of this retrospective study were to evaluate 26 patients with Usher syndrome clinically. The 26 patients (male: 12 cases, female: 14 cases) with Usher syndrome, with a clinical diagnosis based on symptoms of bilateral sensorineural hearing loss and RP, had been registered from 13 hospitals as a multicenter study. We assessed the clinical history and performed audiovestibular and ophthalmologic examinations, and genetic testing. Eleven of the patients were classified as having Usher type 1 (38.5%), 6 with Usher type 2 (23.1%), and 9 with Usher type 3 (38.5%). However, many patients with atypical Usher type 1 (70%) and type 2 (83.3%) were found compared with Usher type 3 (10%). The conductive rate of vestibular examinations including the caloric test (50%) was low. There were many variations in the clinical symptoms in Usher syndrome patients, therefore the classification of Usher types 1, 2, and 3 has been complicated. We have proposed a flowchart for the diagnosis of Usher types 1, 2, and 3.

[Efficacy of levofloxacin 500 mg for middle ear diseases].

We examined the cases that prescribed levofloxacin (LVFX) 500mg for middle ear diseases in our hospital. LVFX 500mg was prescribed in 18 cases (7 male, 11 female, 24-81 years old). LVFX was mainly used for the following clinical conditions, 1) otitis media with granulation proliferation: 3 cases, 2) infection to an artificial materials : 5 cases, 3) treatment for or prevention against the inflammation of the inner ear: 4 cases. Three of eighteen cases were ineffective by LVFX treatment. Bacteriological examination indicated LVFX-resistant MRSA or Pseudomonas aeruginosa were isolated in these three cases.

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.

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.

Partial requirement of endothelin receptor B in spiral ganglion neurons for postnatal development of hearing.

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine ß-hydroxylase promoter (Ednrb(-/-);DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-);DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.

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.

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.

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.

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.

Nystagmus using video-oculography in psychiatric patients.

To evaluate whether nystagmus has clinical significance in psychiatric patients who have functional and/or organic brain dysfunction. We performed gaze, positional and positioning nystagmus tests on 227 patients with psychiatric diseases (144 men, 83 women, with an average age +/- SD of 62.5 +/- 14.0 years) in order to evaluate the frequency and characteristics of nystagmus. Patients were classified according to the underlying disease. Normal control subjects were 107 subjects (26 men, 81 women, with an average age +/- SD of 35.6 +/- 10.0 years). Nystagmus was observed in 56 (24.7%) of 227 cases. Nystagmus was seen in 16 (59.3%) of 27 cases of alcoholism, 14 (22.2%) of 63 cases of organic psychiatric disorders, 25 (20.2%) of 124 cases of schizophrenia, 1 (20.0%) of 5 cases of excited mental retardation, 0 (0.0%) of 7 cases of mood disorders, 0 (0.0%) of 1 case of anxiety disorders and 1 (0.9%) of 107 subjects of normal control. There was a significant difference between psychiatric diseases and normal control. These results indicate that nystagmus may also be a very important clinical finding not only in patients with neurological and neuro-otological diseases, but also in patients with psychiatric diseases.

Salicylate-induced morphological changes of isolated inner hair cells and outer hair cells from guinea-pig cochlea.

OBJECTIVES: The aim of this study is to investigate the salicylate-induced morphological changes of cochlear inner hair cells (IHCs) and outer hair cells (OHCs). METHODS: IHCs and OHCs were acutely isolated from the guinea-pig cochlea. Cells were observed under the inverted microscope and 10mmol/L sodium salicylate solutions or 0.01mmol/L dexamethasone-plus-salicylate solutions were applied. The cell length or the ratio between the length and width was the indices of the morphological changes in cells. RESULTS: Isolated IHCs did not demonstrate any significant changes in sodium salicylate solutions in 20min and in 40min, whereas OHCs were shortened by the 10mmol/L sodium salicylate to 83% in 20min and 75% in 40min. There were no significant differences between in the dexamethasone-plus-salicylate solutions and in the control solutions after 20min and 40min both in IHCs and OHCs. CONCLUSIONS: Although salicylate affected the isolated OHCs from guinea-pig cochlea, IHCs were not changed morphologically by sodium salicylate applications. Dexamethasone inhibited the salicylate-induced morphological changes of OHCs.

Inactivating potassium currents in apical and basal turn inner hair cells from guinea-pig cochlea.

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. IHCs of guinea-pigs were separately isolated from the apical and basal turns and the tonotopic gradient of inactivation kinetics was investigated. TEA-sensitive potassium currents showed voltage-dependent time constant of the inactivation phase both in apical and basal IHCs, however, the degree of inactivation (compared to the ratio between the steady-state current and initial peak current) was voltage-independent. Inactivation time constant was faster in basal IHCs than in apical IHCs and the degree of inactivation was greater in basal IHCs than in apical IHCs, suggesting that inactivation was more predominant in basal IHCs than in apical IHCs.

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.