Akiko Taura

Recovery of hair cell function after damage induced by gentamicin in organ culture of rat vestibular maculae

Here, we report the functional and morphological evidence of hair cell recovery after damages induced by gentamicin (GM) in cultured explants of rat vestibular maculae. We evaluated mechano-electrical transduction (MET) function in hair cells, by measuring Ca(2+) responses in the explants with fura-2 when hair bundles were stimulated. After the MET testing, hair bundles were observed in high resolution by scanning electron microscopy, or by fluorescence microscopy after staining with phalloidin-FITC (fluorescent isothiocyanate). In the control culture, the number of hair bundles on the explants gradually decreased, and the percentage of explants showing Ca(2+) responses decreased and disappeared after 17 days in culture. Following GM (1-2 mM) treatment, most of the hair bundles were eliminated initially, but the hair bundles gradually increased in number during culture. Short hair bundle-like structures emerged in the areas where hair bundles had been completely lost. Consistent with the morphological observations, Ca(2+) responses disappeared after GM treatment, and they gradually recovered to a peak 13-17 days after treatment and were even induced at 17 days or more in culture. Furthermore, cells accumulated FM1-43, a dye permeable through the MET channel, when Ca(2+) responses recovered after GM treatment. Application of steroid hormone increased the percentage of explants showing MET activity, and enhanced the recovery of MET after GM treatment. We investigated Ki-67 immunoreactivity to detect cell proliferation and TUNEL staining to detect apoptotic cell death. Ki-67 immunoreactivity was negative after GM treatment, however TUNEL staining was positive and the positivity was GM dose dependent. Therefore, this functional recovery of transduction activity was not owing to the proliferation of hair cells but was likely the self-repair of the hair bundle.

Hydrogen protects vestibular hair cells from free radicals

Abstract Conclusion: Hydrogen gas effectively protected against the morphological and functional vestibular hair cell damage by reactive oxygen species (ROS). Objective: ROS are generally produced by oxidative stress. In the inner ear, ROS levels increase as a result of noise trauma and ototoxic drugs and induce damage. It is thus important to control ROS levels in the inner ear. The protective effects of hydrogen gas in cochlear hair cells have been reported previously. Methods: This study examined the effects of hydrogen gas on mouse vestibular hair cell damage by ROS using antimycin A. Results: In the group *exposed to hydrogen gas, vestibular hair cells were morphologically well preserved and their mechano-electrical transduction activities were relatively well maintained when compared with controls. Hydroxyphenyl fluorescein (HPF) fluorescence in vestibular tissue was also reduced by hydrogen gas.

One-third of vertiginous episodes during the follow-up period are caused by benign paroxysmal positional vertigo in patients with Meniere's disease.

Abstract Conclusion: In the present study, about one-third of patients with Meniere’s disease developed benign paroxysmal positional vertigo (BPPV)-like attacks. Additionally, more than one-third of all vertigo attacks were BPPV-like attacks. Thus, vertigo attacks in patients with Meniere’s disease must be carefully treated because the therapy for such vertigo attacks is totally different from the therapy for BPPV. Objective: Physicians sometimes encounter patients with previously diagnosed Meniere’s disease who develop BPPV attacks during the course of clinical follow-up. In this study, we explored the frequency with which BPPV was involved in all vertiginous episodes among patients with Meniere’s disease. Methods: This retrospective study involved 296 patients with Meniere’s disease who visited Kyoto University Hospital. The diagnosis of Meniere’s disease was based on the guidelines for the diagnosis of Meniere’s disease proposed by the Committee on Hearing and Equilibrium. We judged the cause of vertigo as one of the following five types: (1) definite Meniere’s disease attack, (2) suspicious Meniere’s disease attack, (3) definite BPPV attack, (4) suspicious BPPV attack, or (5) unknown. Results: In all, 96 patients (32.8%) developed BPPV-like attacks, and 187 vertiginous episodes (37.9%) were caused by BPPV. The lateral semicircular canal was the most frequently involved canal.

Insulin-like growth factor 1 protects vestibular hair cells from aminoglycosides.

This study investigated the therapeutic potential of insulin-like growth factor-1 (IGF-1) for vestibular hair cells using explant cultures of mouse utricles. After incubation with the ototoxic drug gentamicin, explants from neonatal mouse utricles were cultured in medium containing IGF-1 at various concentrations. Histological evaluation revealed significant increases in the number of surviving hair cells cultured with IGF-1 at concentrations reflecting a clinical setting. Immunostaining for trio-binding protein and espin showed the maintenance of functional structures in hair bundles at the apex of surviving hair cells. An FM1-43 assay indicated the presence of mechanoelectrical transduction channels in surviving hair cells. These findings indicate that IGF-1 may protect the functionality of vestibular hair cells against drug-induced injury.

Hair cell stereociliary bundle regeneration by espin gene transduction after aminoglycoside damage and hair cell induction by Notch inhibition.

Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. Although this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin-bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. Although γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 h after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1-43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs.

Clinical study of vertigo in the outpatient clinic of Kyoto University Hospital.

Abstract Conclusion: The epidemiology of vertigo remains unclear. This study might contribute to an understanding of the mechanisms underlying vestibular disease. Objective: To investigate the epidemiological features of patients with vertigo. Methods: A retrospective study on 612 patients with vertigo who visited Kyoto University Hospital. Results: Elderly individuals comprised > 30% of patients presenting with vertigo, with more female patients than male patients.

Effects of mouse utricle stromal tissues on hair cell induction from induced pluripotent stem cells

BackgroundHair cells are important for maintaining our sense of hearing and balance. However, they are difficult to regenerate in mammals once they are lost. Clarification of the molecular mechanisms underlying inner ear disorders is also impeded by the anatomical limitation of experimental access to the human inner ear. Therefore, the generation of hair cells, possibly from induced pluripotent stem (iPS) cells, is important for regenerative therapy and studies of inner ear diseases.ResultsWe generated hair cells from mouse iPS cells using an established stepwise induction protocol. First, iPS cells were differentiated into the ectodermal lineage by floating culture. Next, they were treated with basic fibroblast growth factor to induce otic progenitor cells. Finally, the cells were co-cultured with three kinds of mouse utricle tissues: stromal tissue, stromal tissue + sensory epithelium, and the extracellular matrix of stromal tissue. Hair cell-like cells were successfully generated from iPS cells using mouse utricle stromal tissues. However, no hair cell-like cells with hair bundle-like structures were formed using other tissues.ConclusionsHair cell-like cells were induced from mouse iPS cells using mouse utricle stromal tissues. Certain soluble factors from mouse utricle stromal cells might be important for induction of hair cells from iPS cells.

Clinical and epidemiological study on inpatients with vertigo at the ENT Department of Kyoto University Hospital

Abstract Conclusion: The number of studies on inpatients with vertigo is limited. This study provides useful information for clarifying the underlying causes of vertigo. Objective: To investigate the epidemiological features of patients with vertigo. Methods: This retrospective study investigated 78 patients who had been emergently hospitalized in the Otolaryngology Department of Kyoto University Hospital with vertigo. Results: The number of female patients was significantly higher than the number of male patients. Menieres disease was the most common underlying pathology among hospitalized patients. Mean patient age was 56.1 years, with no significant difference between male and female patients.

An attempt to measure the diametric relationship between slow and quick phases of nystagmus

Abstract Objective: To investigate whether our original method can precisely evaluate the angle between slow and quick phases of nystagmus (vector angle) and to determine whether vector angle analysis is helpful in differentiating between horizontal nystagmus and mixed nystagmus with horizontal and vertical components. Methods: We included 20 healthy volunteers, 17 patients with horizontal nystagmus, and 15 patients with mixed nystagmus. Caloric nystagmus was recorded in healthy volunteers; positional nystagmus was recorded in each patient. We extracted the velocity of nystagmus from eye movement of each subject and analysed the vector angle. Results: In caloric nystagmus, the vector angle approached 180 degrees as slow-phase velocity increased, suggesting that our vector angle measurement is more reliable with faster nystagmus. Importantly, in horizontal nystagmus from peripheral vestibular disease, the vector angle similarly approached 180 degrees as slow-phase velocity increased; in contrast, the vector angle in cases of mixed nystagmus from vertebrobasilar insufficiency or spinocerebellar degeneration significantly differed from the angle of caloric nystagmus. Conclusions: Vector angle analysis using our original algorithm can precisely evaluate the diametric relationship in vestibular nystagmus; it may be helpful in diagnosis of non-peripheral vestibular disorders.

Sensory preservation in neck dissection: outcomes of a sub-sternocleidomastoid approach

Abstract Objective: Cutaneous anesthesia in early postoperative period is common after neck dissection even if the cervical nerve (CN) rootlets are preserved. The aim of this study was to evaluate if the preservation of the terminal branches of CNs using sub-sternocleidomastoid (SCM) approach combined with medially placed skin incision can prevent early postoperative anesthesia. Material and methods: A retrospective chart review was performed on 129 neck dissections in 87 head and neck cancer patients. Results: The early postoperative sensory preservation rates for the ear tab, submandibular, lateral neck, and sub-clavicular areas of CN rootlet-preserved necks (n = 86) were 75.6%, 20.9%, 74.4%, and 86.0%, respectively, compared with 37.2%, 2.3%, 2.3%, and 4.7%, respectively, in CN rootlet-resected necks (n = 43). In CN rootlet-preserved necks, the sub-SCM approach (n = 54) showed 81.5%, 27.8%, 92.6%, and 94.4% preservation rates, respectively, compared with 65.6%, 9.4%, 43.8%, and 71.9%, respectively, using the conventional subplatysmal approach (n = 32). The rates were significantly better in the submandibular, lateral neck, and sub-clavicular areas after sub-SCM approach. Conclusions: Preservation of CN rootlets is a required element for sensory preservation in neck dissection. The sub-SCM approach can effectively prevent early postoperative cutaneous anesthesia following CN-preserving neck dissection.