Ryuhei Inamoto

Deletion of Tricellulin Causes Progressive Hearing Loss Associated with Degeneration of Cochlear Hair Cells

Tricellulin (also known as MARVELD2) is considered as a central component of tricellular tight junctions and is distributed among various epithelial tissues. Although mutations in the gene encoding tricellulin are known to cause deafness in humans (DFNB49) and mice, the influence of its systemic deletion in vivo remains unknown. When we generated tricellulin-knockout mice (Tric−/−), we found an early-onset rapidly progressive hearing loss associated with the degeneration of hair cells (HCs); however, their body size and overall appearance were normal. Tric−/− mice did not show any morphological change pertaining to other organs such as the gastrointestinal tract, liver, kidney, thyroid gland and heart. The endocochlear potential (EP) was normal in Tric−/− mice, suggesting that the tight junction barrier is maintained in the stria vascularis, where EP is generated. The degeneration of HCs, which occurred after the maturation of EP, was prevented in the culture medium with an ion concentration similar to that of the perilymph. These data demonstrate the specific requirement of tricellulin for maintaining ion homeostasis around cochlear HCs to ensure their survival. The Tric−/− mouse provides a new model for understanding the distinct roles of tricellulin in different epithelial systems as well as in the pathogenesis of DFNB49.

Expression of thiazide-sensitive Na+–Cl− cotransporter in the rat endolymphatic sac

The endolymphatic sac (ES) is a part of the membranous labyrinth and is believed to absorb endolymph. It has been well-established that the endolymph absorption is dependent on several ion transporters in a manner similar to that in the kidney, and the ES is regulated by hormones such as aldosterone and vasopressin that also affect on the kidney. The thiazide-sensitive Na(+), Cl(-) cotransporter (TSC) is an electroneutral cotransporter specific to the kidney that plays an important role in absorption of NaCl in renal tubules. In the inner ear, TSC expression has never been examined. The expression of TSC in the rat ES was examined by RT-PCR, in situ hybridization and immunohistochemistry. These analyses indicated that TSC genes and proteins were expressed in the rat ES. In contrast, it was not observed in the rat cochlea by RT-PCR. This is the first report confirming the expression of TSC in the ES.

Endolymphatic sac is involved in the regulation of hydrostatic pressure of cochlear endolymph

To clarify the role of the endolymphatic sac (ES) in the regulation of endolymphatic pressure, the effects of isoproterenol, a beta-adrenergic receptor agonist, and acetazolamide, a potent carbonic anhydrase inhibitor, both of which decrease ES direct current potential on cochlear hydrostatic pressure, were examined in guinea pigs. When isoproterenol was applied intravenously, hydrostatic pressures of cochlear endolymph and perilymph were significantly increased with no change in endocochlear potential or the hydrostatic pressure of cerebrospinal fluid. Acetazolamide produced no marked change in the hydrostatic pressure of cochlear endolymph. In ears with an obstructed ES, the action of isoproterenol on the hydrostatic pressure of cochlear endolymph and perilymph was suppressed. These results suggest that the ES may regulate the hydrostatic pressure of the endolymphatic system via the action of the agents such as catecholamines on the ES.

Comparison of 18 F-FLT PET and 18 F-FDG PET for detection of cervical lymph node metastases in head and neck cancers

Abstract Conclusion: Despite low uptake of tracer, 3′-deoxy-3′-18F-fluorothymidine (FLT) PET could detect cervical lymph node metastases as well as 2-deoxy-2-18F-fluoro-d-glucose (FDG) PET. Objective: The diagnostic efficacy of FLT PET was compared with that of FDG PET regarding nodal staging of head and neck squamous cell cancers. Methods: Twenty-three patients were examined with FLT PET and FDG PET. PET images were evaluated qualitatively for regions of focally increased metabolism and maximum standardized uptake values (SUV) were calculated for semiquantitative analysis. Results: The mean (± SD) FLT SUV in visualized metastatic lymph nodes was 4.8 ± 2.9 as compared with 6.9 ± 4.9 for FDG SUV (p < 0.001). Significant correlations were found between the area of metastatic lymph nodes and both FLT SUV (r = 0.8; p < 0.0001) and FDG SUV (r = 0.84; p < 0.0001). The false-positive (over-staged) and false-negative (under-staged) rates for lymph node staging by FLT PET were 4% (1/23) and 17% (4/23), respectively. Those for FDG PET were 9% (2/23) and 13% (3/23). All metastatic lymph nodes measuring more than 9 mm in short-axis diameter were correctly detected by FLT PET. However, both FLT and FDG PET had low sensitivity for detecting the lymph node metastases ≤ 9 mm in short-axis diameter and tumor deposits < 5 mm.

A new approach for selective rat endolymphatic sac epithelium collection to obtain pure specific RNA

The endolymphatic sac (ES) is an organ that is located in the temporal bone. Its anatomical location makes ES tissue collection without any contamination very difficult, and sometimes accurate molecular analyses of the ES are prevented due to this matter. In the present study, a new selective ES epithelial tissue collection method was attempted using laser capture microdissection to obtain pure ES RNA without any contamination. The validity of this method was demonstrated by RT-PCR with three specific primer pairs against osteocalcin, calponin H1, and NKCC2, which are specific proteins in bone, smooth muscle, and kidney/ES cells, respectively. From the RT-PCR results, the high specificity and sufficient sensitivity of the new method was indicated. It is considered that the new method is optimal for ES collection without contamination and it will be able to contribute to future analyses of the ES.

Presence of Adrenergic Receptors in Rat Endolymphatic Sac Epithelial Cells

Intravenous application of catecholamines produces a depression in the endolymphatic sac direct current potential (ESP) and increases endolymphatic pressure via the β-adrenergic receptor (AR) in guinea pigs, suggesting that catecholamines play a role in the endolymphatic system. However, the localization of ARs in the endolymphatic sac (ES) is still undetermined. The presence of ARs in the rat ES was investigated by reverse transcriptase-polymerase chain reaction using laser capture microdissection (LCM) and immunohistochemical analysis. Expression of α1A-, α1B-, α2A-, α2B-, β1-, β2- and β3-ARs was observed in LCM samples of ES epithelia. Immunohistochemical analysis using specific antibodies showed immunofluorescence of β2- and β3-ARs in epithelial cells of the ES intermediate portion, and no specific staining results were obtained for α1-, α2A-, α2B- and β1-ARs. The presence of β2-AR with no clear immunostaining of β1-AR in ES epithelial cells is in accordance with previous electrophysiological and pharmacological results, which suggests that β2-AR mediates the action of catecholamines on the ESP. The presence of β3-AR in the ES epithelial cells and its absence in the stria vascularis implies that β3-AR plays a specific role in the ES.

The expression of P2Y1, 2, 4, and 6 receptors in rat endolymphatic sac epithelia.

Earlier studies have suggested that activation of the purinergic receptor causes Na+ absorption from the endolymph through nonselective cation channels in endolymphatic sac (ES) epithelia. In this study, the mRNA expression patterns of the P2Y1, 2, 4, and 6 receptors in the rat ES were examined by conventional reverse-transcription PCR. Their cellular localization was investigated by high-specificity reverse-transcription PCR using laser capture microdissection and in-situ hybridization. Our experiments showed that the mRNA of these receptors is expressed in ES epithelia. These results indicate that extracellular nucleotides may regulate ion transport by several purinergic pathways that operate through these receptors in the ES and that some of these receptors may be responsible for regulating Na+ absorption through the activation of nonselective cation channels.

Tinnitus retraining therapy using portable music players

OBJECTIVE We aimed to perform acoustic analysis of environmental sounds used in sound therapy for tinnitus retraining therapy (TRT) and to evaluate the efficacy of TRT performed by using a portable music player (PMP) with recorded environmental sounds as the sound generator. METHODS Acoustic analysis of environmental sounds was performed using a sound analyzer. The subjects were 23 patients with chronic tinnitus. Patients who had bilateral hearing loss and required hearing assistance were fitted with hearing aids (HAs). Patients with normal hearing or unilateral hearing loss were fitted with a tinnitus control instrument (TCI) or a PMP. The patients were divided into the PMP group, TCI group, and HA group. All subjects underwent audiometric evaluations prior to TRT and completed the tinnitus handicap inventory (THI). The THI scores were evaluated before treatment and 1 month, 3 months, 6 months, and 12 months after treatment. RESULTS The sound spectrogram of the murmur of a stream showed a wide-frequency band with a constant strength, whereas that of a wave sound showed a wide-frequency band with variable strength. The THI score clearly decreased after 1 month, and this decrease tended to continue over 12 months. The TRT efficacy ratios in the PMP group, TCI group, and HA group at 12 months after treatment were 71%, 67%, and 70%, respectively. CONCLUSIONS TRT using a PMP had efficacy similar to those of TCI and HA. The murmur of a stream was one of the most effective sounds in TRT. TRT using a PMP as the sound generator can provide the most cost-effective treatment option for tinnitus patients.

Expression and localization of 11β-hydroxysteroid dehydrogenase (11βHSD) in the rat endolymphatic sac

Conclusions: 11β-Hydroxysteroid dehydrogenase type 2 (11βHSD-2) enables aldosterone to bind to mineralocorticoid receptors (MRs) selectively by converting cortisol (corticosterone) into inactive metabolites. Its expression in the endolymphatic sac (ES) suggests that aldosterone may selectively act on the ES through its binding to MRs by the action of 11βHSD-2, and supports the notion that ES is an aldosterone target organ. We propose that 11βHSD-2 is a dominant isoform of 11βHSDs in the ES, and the ES (especially the intermediate portion of the ES) may be the main aldosterone target in the inner ear. Objective: The purpose of this study was to examine 11βHSD isoform expression in the rat inner ear, mainly 11βHSD-2 in the ES. Materials and methods: In the ES and whole cochlea, 11βHSDs were examined by RT-PCR using highly specific ES RNA by laser capture microdissection. In addition, 11βHSD-2 localization in the rat ES was determined by immunohistochemistry. Results: RT-PCR demonstrated 11βHSD-2 expressed in the rat ES. In addition, its localization was observed mainly in the intermediate portion and a faint immune positive signal was observed in other parts of the ES. In contrast, 11βHSD-1 was undetectable in the ES by RT-PCR. Both types of 11βHSDs were expressed in rat cochlea.

Hormonal changes following a low-salt diet in patients with Ménière's disease

OBJECTIVE A low-salt diet has been the main treatment modality for Ménières disease (MD) since the 1930s, although the mechanisms behind this therapy have not yet been elucidated. Salt reduction is associated with a physiological increase in plasma aldosterone concentration. Several experimental reports have suggested that aldosterone may increase endolymph absorption in the inner ear, particularly in the endolymphatic sac. Therefore, aldosterone elevations due to a low-salt diet may increase endolymph absorption in the endolymphatic sac. In this study, urinary sodium excretion, plasma aldosterone, and other hormones were measured during low-salt diet therapy in patients with MD. METHODS We included 13 patients with unilateral definite MD diagnosed at the Kagawa University Hospital. A national registered dietitian provided nutritional guidance initially for 14 enrolled patients with MD and prescribed them a low-salt diet (2g Na/day). Twenty-four hour urine was sampled at baseline, at 2, 4, 6, and 8 weeks, and at 6, 12, 18, and 24 months after initiating the low-salt diet. Urine osmotic pressure, and Na, K, and Cl levels were measured, and 24-h urinary Na, K, and Cl excretion was estimated. Aldosterone, cortisol, hormones (including anti-diuretic hormone), Na, K, and Cl in the blood were measured, alongside plasma osmotic pressure. A total of 13 patients followed the low salt diet therapy for more than 2 years, while one patient dropped out. RESULTS Group 1 (n=7) included patients with a mean urinary sodium excretion amount lower than 3g/day and Group 2 (n=6) included those with more than 3g/day. Vertiginous states of all Group 1 patients comprised complete control (Class A, 100%), while Group 2 patients included Class A (four patients, 66%), Class C (one patient, 17%), and Class D (one patients, 17%). Plasma aldosterone concentrations significantly increased during the 2-year low-salt diet; concentrations in Group 1 tended to be higher than that in Group 2. Hearing improvements after 2 years in Group 1 were significantly better than that in Group 2. The plasma concentration of the hormones except aldosterone was not significantly changed during 2-year low-salt diet. CONCLUSION A low-salt diet was an effective treatment for patients with Ménières disease. This treatment will have a greater effect, when sodium intake is reduced to less than 3g/day. A low-salt diet may induce an increase in the plasma aldosterone concentration that can activate ion transport and absorbing endolymph in the endolymphatic sac.