Sho Kanzaki

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.

Gene transfer into supporting cells of the organ of Corti

To utilize the rapidly accumulating genetic information for developing new therapeutic technologies for inner ear disease, it is necessary to design technologies for expressing transgenes in the inner ear, especially in the organ of Corti. We examined the outcome of an adenovirus gene transfer into the organ of Corti via the scala media in guinea pigs. The transgene insert is the bacterial lacZ gene driven by a cytomegalovirus promoter. We demonstrate that the inoculation is detrimental to the hair cells that surround the site of inoculation, but the supporting cells in the organ of Corti survive and retain the ability to express the reporter transgene beta-gal. The ability to deliver transgenes that are expressed in the supporting cells is an important step in the development of clinically applicable treatments that involve hair cell regeneration.

Hearing and hair cells are protected by adenoviral gene therapy with TGF-β1 and GDNF

Glial cell line-derived neurotrophic factor (GDNF) overexpression in the inner ear can protect hair cells against degeneration induced by aminoglycoside ototoxicity. The protective efficiency of GDNF increases when it is combined with co-factors such as transforming growth factor beta1 (TGF-beta1), a ubiquitous cytokine. The aim of this study was to determine whether TGF-beta1 receptors are expressed in the inner ear and whether a cocktail of GDNF and TGF-beta1 transgenes provides enhanced protection of the inner ear against ototoxic trauma. Using RT-PCR analysis, we determined that both TGF-beta1 receptors, type 1 and 2 are present in rat cochlea. We co-inoculated two adenoviral vectors, one encoding human TGF-beta1 gene (Ad.TGF-beta1) and the other encoding human GDNF gene (Ad.GDNF) into guinea pig cochleae 4 days prior to injecting an ototoxic dose of aminoglycosides. Inoculated ears had better hearing and fewer missing inner hair cells after exposure to the aminoglycoside ototoxicity, as compared with controls and ears treated only with Ad.GDNF. Cochleae with TGF-beta1 overexpression exhibited fibrosis in the scala tympani regardless of the presence of GDNF. Our results suggest that the adenovirus-mediated overexpression of GDNF and TGF-beta1 can be used in combination to protect cochlear hair cells and hearing from ototoxic trauma.

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.

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.

p27Kip1 deficiency causes organ of Corti pathology and hearing loss

Abstract p27Kip1 (p27) has been shown to inhibit several cyclin-dependent kinase molecules and to play a central role in regulating entry into the cell cycle. Once hair cells in the cochlea are formed, p27 is expressed in non-sensory cells of the organ of Corti and prevents their re-entry into the cell cycle. In one line of p27 deficient mice (p27−/−), cell division in the organ of Corti continues past its normal embryonic time, leading to continual production of cells in the organ of Corti. Here we report on the structure and function of the inner ear in another line of p27 deficient mice originating from the Memorial Sloan-Kettering Cancer Center. The deficiency in p27 expression of these mice is incomplete, as they retain expression of amino acids 52–197. We determined that mice homozygote for this mutation had severe hearing loss and their organ of Corti exhibited an increase in the number of inner and outer hair cells. There also was a marked increase in the number of supporting cells, with severe pathologies in pillar cells. These data show similarities between this p27Kip1 mutation and another, previously reported null allele of this gene, and suggest that reducing the inhibition on the cell cycle in the organ of Corti leads to pathology and dysfunction. Manipulations to regulate the time and place of p27 inhibition will be necessary for inducing functionally useful hair cell regeneration.

Mucoepidermoid Carcinoma of the Head and Neck : Clinical Analysis of 43 Patients

OBJECTIVE It is well known that mucoepidermoid carcinoma (MEC) displays a variety of biological behaviors. While the high-grade type is a highly aggressive tumor, its low-grade counterpart usually demonstrates a more benign nature and several systems have, therefore, been proposed to grade this neoplasm. METHODS This report analyzes 43 patients suffering from head and neck MEC, who were treated in our department during the period from 1989 to 2005. The relationship between clinical and pathologic characteristics and survival rate was investigated. RESULTS The 5-year overall and disease-free survival rate was 62.3 and 57.2%. Multivariate analysis demonstrated that the parameters that significantly affected survival were the patients age (P = 0.040) and treatment method (P = 0.011). CONCLUSIONS The patients age and treatment method is the prognostic parameter in this study. Although complete surgical resection is the standard treatment for MEC, we should aggressively consider adjunctive radiotherapy in those cases that have a high risk of recurrence and poor prognosis.

Bcl-2 Genes Regulate Noise-Induced Hearing Loss

Proteins of the Bcl‐2 family have been implicated in control of apoptotic pathways modulating neuronal cell death, including noise‐induced hearing loss. In this study, we assessed the expressions of anti‐ and proapoptotic Bcl‐2 genes, represented by Bcl‐xL and Bak following noise exposures, which yielded temporary threshold shift (TTS) or permanent threshold shift (PTS). Auditory brainstem responses (ABRs) were assessed at 4, 8, and 16 kHz before exposure and on days 1, 3, 7, and 10 following exposure to 100 dB SPL, 4 kHz OBN, 1 hr (TTS) or 120 dB SPL, 4 kHz OBN, 5 hr (PTS). On day 10, subjects were euthanized. ABR thresholds increased following both exposures, fully recovered following the TTS exposure, and showed a 22.6 dB (4 kHz), 42.5 dB (8 kHz), and 44.9 dB (16 kHz) mean shift on day 10 following the PTS exposure. PTS was accompanied by outer hair cell loss progressing epically and basally from the 4‐kHz region. Additional animals were euthanized for immunohistochemical assessment. BcL‐xL was robustly expressed in outer hair cells following TTS exposure, whereas Bak was expressed following PTS exposure. These results indicate an important role of the Bcl‐2 family proteins in regulating sensory cell survival or death following intense noise. Bcl‐xL plays an essential role in prevention of sensory cell death following TTS levels of noise, and PTS exposure provokes the expression of Bak and, with that, cell death.

Noninvasive in vivo delivery of transgene via adeno-associated virus into supporting cells of the neonatal mouse cochlea.

There are a number of genetic diseases that affect the cochlea early in life, which require normal gene transfer in the early developmental stage to prevent deafness. The delivery of adenovirus (AdV) and adeno-associated virus (AAV) was investigated to elucidate the efficiency and cellular specificity of transgene expression in the neonatal mouse cochlea. The extent of AdV transfection is comparable to that obtained with adult mice. AAV-directed gene transfer after injection into the scala media through a cochleostomy showed transgene expression in the supporting cells, inner hair cells (IHCs), and lateral wall with resulting hearing loss. On the other hand, gene expression was observed in Deiters cells, IHCs, and lateral wall without hearing loss after the application of AAV into the scala tympani through the round window. These findings indicate that injection of AAV into the scala tympani of the neonatal mouse cochlea therefore has the potential to efficiently and noninvasively introduce transgenes to the cochlear supporting cells, and this modality is thus considered to be a promising strategy to prevent hereditary prelingual deafness.