Yutaka Takumi

Discrete cellular and subcellular localization of glutamine synthetase and the glutamate transporter GLAST in the rat vestibular end organ.

Glial cells play an important role in the removal and metabolism of synaptically released glutamate in the central nervous system (CNS). It is not clear how glutamate is handled at peripheral glutamate synapses, which are not associated with glia. Glutamate is a likely transmitter in the synapse between the hair cells and afferent dendrites of the vestibular end organ. Immunocytochemistry was performed to investigate the distribution at this site of the high affinity glutamate transporter GLAST and glutamate metabolizing enzyme glutamine synthetase. Confocal microscopy revealed that GLAST and glutamine synthetase were co-localized in supporting cells apposed to the immunonegative hair cells. Postembedding immunoelectron microscopy revealed that GLAST was heterogeneously distributed along the plasma membranes of the supporting cells, with higher concentrations basally (at the level of the afferent synapses) than apically. Both immunoreactivities were also present in non-neuronal cells in the vestibular ganglion. The present findings suggest that glutamate released at the afferent synapse of vestibular hair cells may be taken up by adjacent supporting cells and converted into glutamine. Thus, at this peripheral synapse, the supporting cells may carry out functions similar to those of glial cells in the CNS.

Achievement of hearing preservation in the presence of an electrode covering the residual hearing region

Abstract Conclusions: With full insertion with a long electrode, hearing preservation can be achieved even in the presence of a long electrode covering the residual hearing region. Objectives: Advances in developing new atraumatic concepts of electrode design as well as surgical technique have enabled hearing preservation after cochlear implantation surgery, and EAS (electric acoustic stimulation) accompanied with hearing preservation is a new trend for patients with residual hearing at the lower frequencies. However, full insertion with a long/medium electrode and hearing preservation is still a challenging field that calls for discussion. Method: In this study, round window insertion, an atraumatic electrode, and dexamethasone administration were used and atraumaticity (hearing preservation and conservation of vestibular function) was evaluated with full insertion of the electrode. Results: Postoperative evaluation after full insertion of the electrodes showed that hearing at low frequencies was well preserved in all five cases. Combined postoperative imaging with the referential tonotopic map confirmed achievement of full insertion and indicated the corresponding frequencies and the depth of the electrode. Achievement of atraumaticity of round window insertion in the present cases was confirmed from the viewpoint of the minimal drilling time as well as the preserved vestibular function.

The responsible genes in Japanese deafness patients and clinical application using Invader assay

Discovery of deafness genes has progressed but clinical application lags because of the genetic heterogeneity. To establish clinical application strategy, we reviewed the frequency and spectrum of mutations found in Japanese hearing loss patients and compared them to those in populations of European ancestry. Screening revealed that in Japanese, mutations in GJB2, SLC26A4, and CDH23, and the mitochondrial 12S rRNA are the major causes of hearing loss. Also, mutations in KCNQ4, TECTA, COCH, WFS1, CRYM, COL9A3, and KIAA1199 were found in independent autosomal dominant families. Interestingly, spectrums of GJB2, SLC26A4, and CDH23 mutations in Japanese were quite different from those in Europeans. Simultaneous screening of multiple deafness mutations based on the mutation spectrum of a corresponding population using an Invader panel revealed that approximately 30% of subjects could be diagnosed. This assay will enable us to detect deafness mutations in an efficient and practical manner in the clinical platform. We conclude that specific racial populations may have unique deafness gene epidemiologies; therefore, ethnic background should be considered when genetic testing is performed. Simultaneous examination of multiple mutations based on a populations spectrum may be appropriate and effective for detecting deafness genes, facilitating precise clinical diagnosis, appropriate counseling, and proper management.

Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs.

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1-4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.

Comparison of the diagnostic value of 3 T MRI after intratympanic injection of GBCA, electrocochleography, and the glycerol test in patients with Meniere's disease

Abstract Conclusion. 3 T MRI after intratympanic injection of gadolinium-based contrast agent (GBCA) is more useful for the diagnosis of endolymphatic hydrops compared with the glycerol test and electrocochleography (ECoG). Objective: To investigate the relationship between 3 T MRI after intratympanic injection of GBCA, the glycerol test, and ECoG in patients with Menieres disease (MD). Methods: A total of 20 patients with MD were evaluated. Diluted gadodiamide (a gadolinium-based contrast agent) was administered to the bilateral tympanic cavity by injection through the tympanic membrane. After 24 h, the endolymphatic hydrops was evaluated by a 3.0 T MR scanner. To investigate cochlear hydrops, the glycerol test and ECoG were carried out in all patients. Results: A positive result was observed in 11 patients (55%) in the glycerol test and in 12 patients (60%) by ECoG. The incidence of positive findings when evaluating the same patients with both the glycerol test and ECoG increased to 75%. Nineteen of 20 (95%) patients showed positive results for 3 T MRI.

CRYM mutations cause deafness through thyroid hormone binding properties in the fibrocytes of the cochlea

Background: In a search for mutations of μ-crystallin (CRYM), a taxion specific crystalline which is also known as an NADP regulated thyroid hormone binding protein, two mutations were found at the C-terminus in patients with non-syndromic deafness. Objective: To investigate the mechanism of hearing loss caused by CRYM mutations Methods: T3 binding activity of mutant μ-crystallin was compared with that of wild-type μ-crystallin, because μ-crystallin is known to be identical to T3 binding protein. To explore the sites within the cochlea where μ-crystallin is functioning, its localisation in the mouse cochlea was investigated immunocytochemically using a specific antibody. Results: One mutant was shown to have no binding capacity for T3, indicating that CRYM mutations cause auditory dysfunction through thyroid hormone binding properties. Immunocytochemical results indicated that μ-crystallin was distributed within type II fibrocytes of the lateral wall, which are known to contain Na,K-ATPase. Conclusions:CRYM mutations may cause auditory dysfunction through thyroid hormone binding effects on the fibrocytes of the cochlea. μ-Crystallin may be involved in the potassium ion recycling system together with Na,K-ATPase. Future animal experiments will be necessary to confirm a causal relation between Na,K-ATPase, T3, and deafness.

Type IX collagen is crucial for normal hearing

cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.

Distribution and frequencies of CDH23 mutations in Japanese patients with non-syndromic hearing loss

Mutations in the CDH23 gene are known to be responsible for both Usher syndrome type ID (USH1D) and non‐syndromic hearing loss (DFNB12), and the molecular confirmation of the CDH23 gene has become important in the diagnosis of these conditions. The present study was performed to find whether the CDH23 mutations are also responsible for non‐syndromic hearing loss in patients in the Japanese population. A total of 51 sequence variants were found in 64 Japanese probands with non‐syndromic sensorineural hearing impairment from autosomal recessive families. Among them, at least four missense mutations in six patients from five families were confirmed to be responsible for deafness by segregation study. All mutations detected were missense mutations, corroborating the previous reports regarding DFNB12. The present data confirmed that CDH23 mutations are frequently found and significantly responsible in Japanese. Interestingly, the CDH23 mutation spectrum in Japanese is very different from that found in Caucasians. This Japanese spectrum may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of DFNB12 and USH1D.

Semi-quantitative evaluation of endolymphatic hydrops by bilateral intratympanic gadolinium-based contrast agent (GBCA) administration with MRI for Meniere's disease

Conclusion: Bilateral intratympanic administration of a gadolinium-based contrast agent (GBCA) in MRI was successfully performed and proved to be beneficial in the semi-quantitative evaluation of endolymphatic hydrops. Such image-based diagnosis will lead to re-revaluation and reclassification of the diagnostic criteria for Menieres disease (MD). Objective: To visualize endolymphatic hydrops semi-quantitatively in patients with MD, by using bilateral intratympanic GBCA administration with MRI. Patients and methods: A total of 13 patients were evaluated, including 12 with MD and one with acute low-tone sensorineural hearing loss. Diluted gadodiamide (a kind of GBCA) was administered to the bilateral tympanic cavity by injection through the tympanic membrane. After 24 h, the endolymphatic hydrops was evaluated with a 3.0 T MR scanner. The areas enhanced by gadodiamide were measured semi-quantitatively. Results: Three-dimensional, fluid-attenuated inversion recovery (3D-FLAIR) MRI showed that the gadodiamide successfully penetrated the round window membrane, entering the perilymphatic space and delineating the gadodiamide-enhanced perilymphatic and gadodiamide-negative endolymphatic spaces of the inner ear. All the patients with MD showed a reduced gadodiamide-enhanced area representing the perilymphatic space, and the quantitative ratio was 0.15 to 0.85. Furthermore, endolymphatic hydrops was also demonstrated in the patient with atypical MD who had fluctuating low frequency sensorineural hearing loss without vertigo.

TRANSPLANTED HUMAN AMNIOTIC EPITHELIAL CELLS EXPRESS CONNEXIN 26 AND NA-K-ADENOSINE TRIPHOSPHATASE IN THE INNER EAR

Cochlear fibrocytes are the crucial component of the inner ear homeostasis and its defect by various causes; GJB2 (connexin [Cx] 26) mutation, for example, leads to hearing loss. In the present study, we investigated the potential use of human amniotic epithelial cells, proposed to possess pluripotential properties, as a source of transplantation therapy in inner ear disease. The mRNA of the gap junction protein Cx26 and Na-K-adenosine triphosphatase, the immunohistologic expression of these proteins, and the cells’ intercellular communication capacity were detected in vitro. Their transplantation into the guinea pig cochlea revealed the survival and expression of the proteins even 3 weeks after transplantation. Transplanted human amniotic epithelial cells were localized at the site where the proteins function, strongly indicating their cooperation in the regional potassium ion recycling. This technology suggests the therapeutic potential for the treatment of hearing loss.