Ai Matsubara

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.

Measurement of nasal resistance by rhinomanometry in 892 Japanese elementary school children

OBJECTIVE The normal value of nasal resistance in adults has been reported (0.25 Pa/cm³/s), but that in children has not. In this study, we measured nasal resistance in Japanese school children by employing rhinomanometry. METHODS An otolaryngologist examined 939 Japanese school children with regard to the presence or absence of nasal diseases and tonsil size. Nasal resistance was measured by rhinomanometry employing the active anterior method in 892 children. A questionnaire concerning the condition during sleep, such as the presence or absence of snoring and sleep apnea syndrome, was performed. RESULTS The mean nasal resistance was 0.43 ± 0.50 Pa/cm³/s: 0.46 ± 0.65 and 0.39 ± 0.22 Pa/cm³/s in boys and girls, respectively. Of the 892 children, Grade 3 and 4 tonsil hypertrophy was noted in 84 (9%), but the presence of tonsil hypertrophy did not influence nasal resistance. Nasal diseases were noted in 335 children (38%) and the nasal condition was normal (the normal group) in 557 (62%). Nasal resistance was 0.56 ± 0.75 Pa/cm³/s in the nasal disease group and 0.36 ± 0.21 Pa/cm³/s in the normal group, showing that the resistance was significantly higher in the nasal disease group. The resistance tended to decrease as the school grade increased. In the normal group, 290 children (33%) experienced no problem regarding the upper airway, such as snoring and sleep apnea syndrome, based on a questionnaire, and nasal resistance was 0.35 ± 0.17 Pa/cm³/s. CONCLUSION This normal nasal resistance value may be adopted for the objective evaluation of nasal obstruction and effects of treatment in pediatric nasal diseases.

The Detailed Localization Pattern of Na+/K+/2Cl− Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na+/K+/2Cl− cotransporter type 2 (NKCC-2) and Na+/K+-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na+/K+-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na+/K+-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules.

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.

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.

Cystic fibrosis transmembrane conductance regulator in the endolymphatic sac of the rat

OBJECTIVE Na(+) and Cl(-) are dominant ions in the endolymphatic fluid in the endolymphatic sac and are important for volume regulation in the endolymphatic sac. An epithelial sodium channel (ENaC) and other Na(+) transporters have been identified in the endolymphatic sac epithelia, and they are involved in the regulation of endolymph. Although the presence of Cl(-) channels in the endolymphatic sac epithelia has been speculated, no Cl(-) channels have been identified. In this study, we confirmed the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the endolymphatic sac by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemical staining. METHODS Pure mRNA from endolymphatic sac epithelia was prepared using laser capture microdissection (LCM) and examined using RT-PCR. Localization of CFTR and ENaC in the endolymphatic sac was examined using immunohistochemistry. RESULTS mRNA of the CFTR was expressed in the endolymphatic sac. Immunohistochemical analysis showed the expression of the CFTR on apical side of the endolymphatic sac epithelia and co-localization with the ENaC. CONCLUSION RT-PCR and immunohistochemistry were used to identify the expression of CFTR in the endolymphatic sac epithelia, which gives us a clue for understanding Cl(-) transport in the endolymphatic sac. These results suggest a pathway for Cl(-), possibly through interaction with the ENaC, which may regulate the endolymph in the endolymphatic sac.

The mRNA of claudins is expressed in the endolymphatic sac epithelia

OBJECTIVE Claudins are a family of membrane proteins which localize to tight junctions (TJs). Recent studies have shown that claudins can form pores for ions in the TJs and regulate the permeability of epithelial paracellular ion transport. The endolymphatic sac (ES) is a part of the inner ear, absorbing the endolymphatic fluid. ES dysfunction may result in endolymphatic hydrops. In this study, we focused on the paracellular transport and examined claudin mRNA expression in the ES epithelia. MATERIALS AND METHODS Total RNA was isolated from whole ES epithelia of rats by laser capture microdissection. RT-PCR was used to evaluate the expression of claudins. The expression of each claudin mRNA in the epithelial cells of rat ES was confirmed by in situ hybridization. RESULTS RT-PCR indicated the expression of cldn2, cldn4, cldn6, cldn7, cldn9, cldn11, cldn12, and cldn14. The expression of these claudin mRNAs in the epithelial cells of rat ES was confirmed by in situ hybridization. CONCLUSION We demonstrated mRNA expression of multiple claudins in the rat ES epithelia. These results in the ES epithelia were consistent with a role of claudins in paracellular ion transport.

Presence of FXYD6 in the endolymphatic sac epithelia

A homeostasis of the electrochemical properties and volume of the endolymph in the inner ear is essential for hearing and equilibrium sensing and is maintained by ion-transport across an epithelial tissue, the endolymphatic sac. One of the key proteins in the maintenance is Na(+), K(+)-ATPase. Although we previously found that the Na(+), K(+)-ATPase in the sac plays a pivotal role in the control of the endolymphatic volume, the mechanism remains unclear. Therefore, in this study, we examined the expression of FXYD6, a functional modulator of the Na(+), K(+)-ATPase, in the epithelial cells of the endolymphatic sac using various approaches. Laser capture microdissection RT-PCR was used to identify FXYD6 mRNA in the endolymphatic sac. Immunolabeling with the specific antibody showed that FXYD6 was predominantly expressed in the intermediate portion of the endolymphatic sac, and it was colocalized with the Na(+), K(+)-ATPase. Because the Na(+), K(+)-ATPase in this region is known to exhibit a high level of activity, an interaction of FXYD6 with this transporter may be critically involved in the regulation of the characteristics of the endolymph.

The difference in endolymphatic hydrostatic pressure elevation induced by isoproterenol between the ampulla and the cochlea

OBJECTIVE The purpose of the study was to investigate the difference in the responses of endolymphatic hydrostatic pressure to isoproterenol, β-adrenergic receptor agonist, between pars superior and pars inferior. METHODS The hydrostatic pressure of endolymph and perilymph and endolymphatic potential in the ampulla and the cochlea during the intravenous administration of isoproterenol were recorded using a servo-null system in guinea pigs. RESULTS The hydrostatic pressure of endolymph and perilymph in the ampulla and cochlea was similar in magnitude. Isoproterenol significantly increased hydrostatic pressure of ampullar and cochlear endolymph and perilymph with no change in the ampullar endolymphatic potential and endocochlear potential, respectively. The isoproterenol-induced maximum change of endolymphatic hydrostatic pressure in ampulla was significantly (p<0.01) smaller than that in the cochlea. In ears with an obstructed endolymphatic sac, the action of isoproterenol on endolymphatic hydrostatic pressure in the ampulla disappeared like that in the cochlea. CONCLUSION Isoproterenol elevates endolymphatic hydrostatic pressure in different manner between the vestibule and the cochlea.