Masatsugu Masuda

Proinflammatory cytokines expression in noise‐induced damaged cochlea

Recent studies have showed that inflammatory responses occur in inner ear under various damaging conditions including noise‐overstimulation. We evaluated the time‐dependent expression of proinflammatory cytokines in noise‐exposed rat cochlea. Among several detected cytokines, real‐time RT‐PCR showed that interleukin‐1beta (IL‐1β) and interleukin‐6 (IL‐6) were significantly induced 3 hr after noise exposure, and quickly downregulated to the basal level. Tumor necrosis factor‐alpha (TNF‐α) was also slightly upregulated immediately after noise exposure. Immunohistochemical analysis showed that IL‐6 expression was distinctively induced within the lateral side of the spiral ligament. Sequential expression analysis showed that IL‐6 immunoreactivity was initially found in the cytoplasm of lateral wall cells, including Type IV and III fibrocytes, and expanded broader throughout the lateral wall, finally to the stria vascularis. Because of the negative Iba‐1 staining, IL‐6 expression in the early‐phase was not due to macrophage or microglia activation. IL‐6 was also detected in spiral ganglion neurons at 12 and 24 hr after noise exposure. Our data demonstrates the production of proinflammatory cytokines, including TNF‐α, IL‐1β, and IL‐6, in early phase of noise overstimulated cochlea. IL‐6 expression was observed in the spiral ligament, stria vascularis, and spiral ganglion neurons. These cytokines, produced by the cochlear structure itself in response to noise exposure, may initiate an inflammatory response and have some role in the mechanism of noise‐induced cochlear damage.

Blockade of interleukin-6 signaling suppressed cochlear inflammatory response and improved hearing impairment in noise-damaged mice cochlea

Hearing impairment can be the cause of serious socio-economic disadvantages. Recent studies have shown inflammatory responses in the inner ear co-occur with various damaging conditions including noise-induced hearing loss. We reported pro-inflammatory cytokine interleukin-6 (IL-6) was induced in the cochlea 6h after noise exposure, but the pathophysiological implications of this are still obscure. To address this issue, we investigated the effects of IL-6 inhibition using the anti-IL-6 receptor antibody (MR16-1). Noise-exposed mice were treated with MR16-1 and evaluated. Improved hearing at 4kHz as measured by auditory brainstem response (ABR) was noted in noise-exposed mice treated with MR16-1. Histological analysis revealed the decrease in spiral ganglion neurons was ameliorated in the MR16-1-treated group, while no significant change was observed in the organ of Corti. Immunohistochemistry for Iba1 and CD45 demonstrated a remarkable reduction of activated cochlear macrophages in spiral ganglions compared to the control group when treated with MR16-1. Thus, MR16-1 had protective effects both functionally and pathologically for the noise-damaged cochlea primarily due to suppression of neuronal loss and presumably through alleviation of inflammatory responses. Anti-inflammatory cytokine therapy including IL-6 blockade would be a feasible novel therapeutic strategy for acute sensory neural hearing loss.

GENERATION OF HIGHLY-REACTIVE OXYGEN SPECIES IS CLOSELY RELATED TO HAIR CELL DAMAGE IN RAT ORGAN OF CORTI TREATED WITH GENTAMICIN

Reactive oxygen species (ROS) have been suggested to play a major role in aminoglycoside-induced hair cell (HC) loss, but are difficult to detect. Moreover, ROS can occur normally in cells where they have roles in metabolism, cell signaling and other processes. Two new probes, aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) are dyes which selectively detect highly-reactive oxygen species (hROS), those most associated with cellular damage. We assessed the presence of hROS in the neonatal rat organ of Corti during chronic exposure to 50 microM gentamicin in vitro, to examine the relationship between cell damage and hROS across HC type and across the three cochlear turns. hROS were initially detected at 48 hours (h), with an increase at 72 h and persistence until at least 96 h. At 48 h, hROS were restricted to outer HCs and occurred prior to loss of stereocilia. At 72 h, outer HCs showed both hROS and stereocilia loss, and hROS were noted in a few inner HCs. Basal turn HCs showed more hROS than middle turn HCs. Very little hROS accumulation or stereocilia loss was observed in the apical turn, even at 72 h. First row outer HCs were most vulnerable to gentamicin-induced hROS, followed by second and then third row outer HCs. Inner HCs behaved similarly to third row outer HCs. By 96 h stereocilia damage was extensive, but surviving HCs showed persisting fluorescence. APF consistently showed more fluorescence than HPF. The results suggest that hROS accumulation is an important initial step in gentamicin-induced HC damage, and that the differential sensitivity of HCs in the organ of Corti is closely related to differences in hROS accumulation.

Nuclear factor-kappa B nuclear translocation in the cochlea of mice following acoustic overstimulation.

There is increasing evidence to suggest that the expression of many molecules in the lateral wall of the cochlea plays an important role in noise-induced stress responses. In this study, activation of the nuclear transcription factor nuclear factor-kappa B (NF-kappaB) was investigated in the cochlea of mice treated with intense noise exposure (4 kHz, octave band, 124 dB, for 2 h). The present noise exposure led to remarkable auditory brainstem response threshold shifts and cochlear damage on surface preparations. To assess the effects of noise exposure on NF-kappaB/DNA binding activity in the cochlea, we prepared nuclear extracts from the cochlea at different time points after noise exposure and carried out an electrophoretic mobility shift assay using a probe specific to NF-kappaB. NF-kappaB/DNA binding was significantly enhanced in the cochlea 2-6 h after noise exposure and returned to basal levels after 12 h. Supershift analysis using antibodies against p65 and p50 proteins, which are components of NF-kappaB, demonstrated that enhancement of NF-kappaB/DNA binding was at least in part due to nuclear translocation of p65. An immunohistochemical study also showed that nuclear translocation of both p65 and p50 was observed in the lateral wall after noise exposure and that there may be a possible close association between p65 and enhanced inducible nitric oxide synthase expression. These results suggest that NF-kappaB may have a detrimental role in the response to acoustic overstimulation in the cochlea of mice.

Correlations of inflammatory biomarkers with the onset and prognosis of idiopathic sudden sensorineural hearing loss

Hypothesis We investigated whether inflammatory biomarkers and stress are involved in the pathophysiology of idiopathic sensorineural hearing loss (ISHL). Study Design Individual cohort study. Setting Two tertiary centers. Patients Forty-three ISHL and 10 non-ISHL patients seen in our ENT departments from 2004 to 2010 within a week from the onset of new symptoms and without steroid administration before visiting our departments. Intervention Multiple audiologic evaluations, blood tests including leukocyte counts, natural killer cell activity (NKCA), interleukin 6 (IL-6), tumor necrosis factor, high-sensitivity CRP (hCRP), and the General Health Questionnaire were used to evaluate the systemic stress and inflammatory response. Main Outcome Measures Correlations between biomarkers and ISHL severity and prognosis were evaluated by statistical analysis. Results In the ISHL patients, a neutrophil count above the reference range was associated with severe hearing loss and poor prognosis, and was accompanied by low NKCA and high IL-6. In the non-ISHL patients, these associations were not present. The abnormal neutrophil count was independent of preexisting vascular diseases. The abnormal counts responded to treatment and decreased into the reference range. Conclusion Neutrophil counts above the reference range of a facility will be a useful indicator of poor prognosis of ISHL. Synchronism of different types of NF-&kgr;B activation pathways could be required to cause severe ISHL. An NKCA decrease, an acute neutrophil count increase, and an IL-6 increase can induce NF-&kgr;B activation in the cochlea and cause severe ISHL. Further epidemiologic surveys should be conducted to evaluate whether stressful life events increase the risk of severe ISHL onset.

Hearing in the MRL/lpr mouse as a possible model of immune-mediated sensorineural hearing loss

Abstract In order to clarify the possible mechanism of hearing loss in immune-mediated sensorineural hearing loss, basic research needed includes animal model studies. In the present investigation, we examined hearing thresholds and cochlear histologies of the MRL/lpr mouse which is now well-known as a model for pathology consistent with systemic lupus erythematosis (SLE). Present findings demonstrated that there were no statistically significant differences in auditory brainstem response (ABR) thresholds between 4- to 6-week-old “young” and 20- to 25-week-old “old” MRL mice. These differences were not sex-dependent. Under light microscopy, there were no abnormal morphological findings in the cochleas of either young or old MRL mice. With immunohistochemistry, mouse IgG was detected around the capillary walls in the stria vascularis in both young and old MRL mice. Serum IgG level of the MRL mice significantly decreased after predonisolone (PSL) administration. However, expression of mouse IgG in the stria vascularis was not observed in the MRL mice after PSL administration. From these results, we speculate that the hearing of the MRL mouse does not always deteriorate, and the deposition of mouse IgG on the capillary wall in the stria vascularis is not a sufficient factor to induce hearing loss. At this point, we conclude that the MRL mouse should not be considered a useful model for immune-mediated sensorineural hearing loss.

WFS1 and GJB2 mutations in patients with bilateral low-frequency sensorineural hearing loss

Evaluating the prevalence of specific gene mutations associated with a certain audiometric configuration facilitates clinical assessment of patients with sensorineural hearing loss (SNHL). WFS1 is responsible for autosomal dominant nonsyndromic deafness 6/14/38 and is the most frequent genetic cause of low‐frequency SNHL (LFSNHL); however, the exact prevalence of WFS1 mutations in LFSNHL is unknown. Therefore, we evaluated genetic mutations and clinical features in patients with nonsyndromic bilateral LFSNHL, focusing on the WFS1.

A novel frameshift variant of COCH supports the hypothesis that haploinsufficiency is not a cause of autosomal dominant nonsyndromic deafness 9.

COCH (coagulation factor C homology) encodes cochlin, and certain mutations of COCH cause autosomal dominant nonsyndromic deafness 9 (DFNA9). Hearing loss due to COCH mutation begins in adulthood, and 17 missense mutations and two in-frame mutations have been reported. Studies with animal and cellular models have suggested that the underlying biological mechanism of DFNA9 is the dominant-negative effect of mutated COCH and not haploinsufficiency. However, no human cases of DFNA9 that support this hypothesis have been reported. The proband of the present case was an 18-year-old male with congenital or infantile hearing loss. Targeted next-generation sequencing analysis detected a heterozygous novel frameshift mutation of COCH (c.146dupT, p.C50LfsX8) in the proband, whose hearing loss began earlier than what is typical for DFNA9. His mother also carried the mutation but had normal hearing. Consequently, the mutation was not considered to be the cause of the probands hearing loss. This family is the first case of a truncating COCH variant and supports the hypothesis that COCH haploinsufficiency is not the cause of hearing loss in humans.

Assessment of hyperacusis with a newly produced Japanese version of the Khalfa hyperacusis questionnaire

Abstract Objectives: The purpose of this study was to determine the validity and reliability of a Japanese version of the Khalfa hyperacusis questionnaire (KHQ) and proposed a threshold KHQ score for classifying hyperacusis. Methods: In total, 112 patients with hyperacusis (group A) and 103 patients without hyperacusis (group B). The patients in group A were further classified into the following subgroups: subjects with hyperacusis as their chief complaint (n = 26, group A1) and subjects with hyperacusis accompanied by chief complaints of tinnitus and/or hearing loss (n = 86, group A2). Results: The average total questionnaire score for patients in group A was 11.8 ± 9.7, which was statistically significantly higher than that of patients in group B, 5.7 ± 4.8. Cronbach’s coefficients for internal consistency were high for the total score (0.92). The average total scores for groups A1 and A2 were 18.1 ± 11.1 and 9.9 ± 8.4, respectively, and the difference between the groups was statistically significant. Conclusions: We developed a Japanese version of the KHQ. It showed high reliability and validity; suggesting its usefulness in clinical practice. We propose that a total KHQ score of 16 is an appropriate cutoff for classifying hyperacusis

The regulation of gene expression in hair cells.

No genes have been discovered for which expression is limited only to inner ear hair cells. This is hardly surprising, since the number of mammalian genes is estimated to be 20-25,000, and each gene typically performs many tasks in various locations. Many genes are expressed in inner ear sensory cells and not in other cells of the labyrinth. However, these genes are also expressed in other locations, often in other sensory or neuronal cell types. How gene transcription is directed specifically to hair cells is unclear. Key transcription factors that act during development can specify cell phenotypes, and the hair cell is no exception. The transcription factor ATOH1 is well known for its ability to transform nonsensory cells of the developing inner ear into hair cells. And yet, ATOH1 also specifies different sensory cells at other locations, neuronal phenotypes in the brain, and epithelial cells in the gut. How it specifies hair cells in the inner ear, but alternate cell types in other locations, is not known. Studies of regulatory DNA and transcription factors are revealing mechanisms that direct gene expression to hair cells, and that determine the hair cell identity. The purpose of this review is to summarize what is known about such gene regulation in this key auditory and vestibular cell type.