The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells.

Cells of the immune system are present in the adult cochlea and respond to damage caused by noise exposure. However, the types of immune cells involved and their locations within the cochlea are unclear. We used flow cytometry and immunostaining to reveal the heterogeneity of the immune cells in the cochlea and validated the presence of immune cell gene expression by analyzing existing single-cell RNA-sequencing (scRNAseq) data. We demonstrate that cell types of both the innate and adaptive immune system are present in the cochlea. In response to noise damage, immune cells increase in number. B, T, NK, and myeloid cells (macrophages and neutrophils) are the predominant immune cells present. Interestingly, immune cells appear to respond to noise damage by infiltrating the organ of Corti. Our studies highlight the need to further understand the role of these immune cells within the cochlea after noise exposure.

Protective effect of acetyl-l-carnitine against cisplatin ototoxicity: role of apoptosis-related genes and pro-inflammatory cytokines.

OBJECTIVES: Cisplatin is an anti-neoplastic agent treatment with which causes many side effects including ototoxicity. The aim of this study was to investigate whether acetyl-L-carnitine would have protective effects on cisplatin-induced ototoxicity in vitro, and if present, to reveal roles of apoptotic gene expressions and pro-inflammatory cytokines. MATERIALS AND METHODS: House Ear Institute-Organ of Corti 1 cell line was used for this study. Apoptotic genes were evaluated with an apoptosis PCR array and pro-inflammatory cytokine levels were measured using ELISA. RESULTS: Apoptotic cell death reduced by around 22% with acetyl-L-carnitine-cisplatin treatment compared to cisplatin alone. Genes displaying increase in expression of apoptosis, related to cisplatin treatment, were Casp8, Bcl10, Bcl2, Bcl2l1, Bcl2l2, Bid, Naip1, Bnip3l, Card6, Pak7, Cd40, Trp 53inp1, Cideb and Cd70. The acetyl-L-carnitine-cisplatin combination caused reduced expression of genes Casp8, Fas, Casp1, Tnfrsf11b, Tnfrsf10b induced by cisplatin. Acetyl-L-carnitine-cisplatin also caused reduced levels of IL-6, IL-1ß and TNF-α, pro-inflammatory cytokines, induced by cisplatin. CONCLUSION: Protective mechanisms of aceytl-L-carnitine against cisplatin induced apoptosis, mainly due to activation of anti-apoptotic Bcl family members' genes, and in an Akt-related gene expression dependent manner. This is the first study to indicate that acetyl-L-carnitine can be an effective agent against cisplatin ototoxicity in auditory cells, with induction of anti-apoptotic gene expression and attenuating levels of pro-inflammatory cytokines.

An experimental study of inner ear injury in an animal model of eosinophilic otitis media.

CONCLUSION: As the periods of intratympanic injection of ovalbumin (OVA) to the middle ear became longer, marked eosinophil infiltration in the perilymphatic space was observed. Moreover severe morphological damage of the organ of Corti was observed in the 28-day antigen-stimulation side. These results indicate that eosinophilic inflammation occurred in the inner ear and caused profound hearing loss. OBJECTIVE: The purpose of the present study was to elucidate the inner ear damage in a new animal model of eosinophilic otitis media (EOM) which we recently constructed. METHODS: We constructed the animal model of EOM by intraperitoneal and intratympanic injection of OVA. Infiltrating cells and the inner ear damage were examined by histological study. RESULTS: In the inner ear, a few eosinophils were seen in the scala tympani of the organ of Corti and the dilation of capillaries of the stria vascularis was observed in the 7-day stimulation side. In the 14-day antigen stimulation side, some eosinophils and macrophages were seen in not only the scala tympani but also the scala vestibule. In the 28-day antigen-stimulation side, severe morphological damage of the organ of Corti and many eosinophils, red blood cells, and plasma cells infiltrating the perilymph were observed.

Evidence for a possible NOS back-up system in the organ of Corti of the guinea pig.

Recently, the two Ca(2+)/calmodulin-regulated nitric oxide synthase isoforms, nNOS and eNOS, and NO itself have been identified in the cochlea of vertebrates using specific antibodies and a new fluorescence indicator. In order to acquire more information about the quantitative and spatial distribution of these two constitutively expressed NOS isoforms (cNOS) in the organ of Corti at the cellular and subcelluar levels, ultrathin sections of London resin (LR) White-embedded cochleae of the guinea pig were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labeled secondary antibody and the amount of the immunoreactions/microm(2) was quantified for different cell types and subcellular regions. Both NOS isoforms were identified to varying degrees in the same cell types and subcellular regions. A prominent eNOS immunoreactivity was identified in nearly every cell type. In all analyzed animals the highest number of gold-coupled anti-eNOS antibodies was always seen in the cells of the reticular lamina, especially in the cuticular structures of outer and inner hair cells, pillar cells and apical Deiters' cells. Also the microtubuli-containing cytoplasmic regions of Deiters' cells were scattered with gold-coupled anti-eNOS antibodies. A clear eNOS immunoreaction was also found in the remaining cytoplasm of inner and outer hair cells and in the apical Deiters' cells. Numerous anti-nNOS antibodies were located in the outer hair cells and in the cuticular structures of the apical Deiters' cells. The amount of the gold-labeled anti-nNOS antibodies in the cuticular plates of the pillar cells and outer hair cells and in the cytoplasm of inner hair cells and apical Deiters' cells were clearly less but still above unspecific background labeling. The spatial co-localization of the two NOS isotypes in the same cell regions was proven in double-labeling experiments. The spatial distribution of the two cNOS isoforms confirmed recent findings of other authors who localized NO distribution and production sites. The cNOS co-expression with similar function in the same cell type and subcellular regions may represent a functional "back-up system" in which one NOS isoform can replace the other in case of pathophysiological malfunction.

Fas ligand expression in the organ of Corti.

We have previously demonstrated by FACS analysis and histochemistry that Fas ligand (FasL) increases on cochlear cell surfaces after immune response or stimulation with gamma-interferon (IFN-gamma). To determine whether the appearance of FasL on cochlear cell membranes is related to gene expression or to posttranslational events, cochlear cells were treated with IFN-gamma. They were evaluated for FasL gene expression by real-time PCR and for FasL protein localization by confocal microscopy of permeabilized and immunolabeled cells. Real-time PCR analysis of cDNAs generated from unstimulated or IFN-gamma-stimulated organ of Corti demonstrated no change in the transcription of the gene encoding FasL. In contrast, confocal microscopy revealed dramatic changes in the cellular distribution of FasL, consistent with movement from the endoplasmic reticulum to the cytoplasm and cell membrane. The results suggest that recruitment of preformed FasL from intracellular compartments, rather than its biosynthesis, is responsible for the increase in FasL on the cell surface following IFN-gamma stimulation. This is similar to the response of cytotoxic T lymphocytes in which gene expression is not involved in FasL surface appearance. Presumably, the use of preformed FasL increases the rapidity of this response. FasL localization to the membrane may be involved in protecting the inner ear from autoimmunity or inflammation. Alternatively it may be related to cochlear cell death in response to inflammatory stress.

Characterization of an experimentally induced inner ear immune response.

OBJECTIVE: To determine the effects of a sterile immune response on the structure and function of the cochlea. METHODS: An immune response was created in guinea pigs by systemically sensitizing the animals to keyhole limpet hemocyanin and subsequently challenging the inner ear with the protein. Animals were allowed to survive for 1 to 5 weeks, after which the cochlea was evaluated histologically. Hearing was measured by auditory brainstem response before the inner ear challenge, during the survival period, and prior to sacrifice. RESULTS: Inflammatory cells infiltrated the cochlea from the circulation. Surface preparations and plastic sections of the organ of Corti 1 and 2 weeks after the initiation of the inflammation demonstrated degeneration of the sensory and supporting cells in cochlear turns containing inflammatory cells. Good preservation of structures was seen in the more apical cochlear turns with little or no inflammatory cells. In cochleas from animals that survived 5 weeks, most of the infiltrated cells were cleared after undergoing apoptosis and the inflammatory matrix in the scala tympani began to calcify. Hearing loss was moderate to severe depending on the amount of inflammation. CONCLUSION: Although in general the immune response serves to protect an organism from infection, these results demonstrate that bystander injury associated with local immune responses in the cochlea, an organ incapable of regeneration, causes permanent cochlear destruction and hearing loss.

Natural killer cell response in the inner ear.

Heterologous tumor cells (human chronic myelogenous leukemia K-562 cells) were injected into the perilymph and skin in guinea pigs in order to study the induction of NK cell activity in the inner ear. An in vitro transmission electron microscopic and immunohistochemical study showed that K-562 cells were attacked by guinea pig large granular lymphocytes. K-562 cells injected through the round window membrane were found to be targeted by NK cells emerging from surrounding venules after 7 to 9 days. During this time morhological changes occurred in the organ of Corti and stria vacularis. These findings suggest that the inner ear response to foreign cells induces activation and invasion of NK cells which occur relatively late compared with those in other organs such as skin.

Monoclonal antibody induced hearing loss.

Monoclonal antibodies KHRI-3 and KHRI-5 identify antigens expressed on inner ear supporting cells and auditory hair cells respectively. To determine if these antibodies affect inner ear function groups of syngeneic Balb/c mice were inoculated with hybridomas KHRI-3, KHRI-5 and other Ig-secreting hybridomas. Hybridomas UM-A9, UM-7F11, the non-secreting SP2/0 myeloma and mice with no hybridoma were used as controls. Animals were tested for auditory brainstem responses (ABR) for frequencies of 4, 8, 16 and 24 kHz, before the inoculation of the hybridomas and at intervals of 6 to 10 days thereafter or daily once tumors became palpable. In normal mice there were no changes in ABR thresholds over the course of the experiment. Other control animals showed little change in ABR even when the growth of the hybridoma or myeloma tumors were far advanced. Of the KHRI-5 hybridoma bearing animals only one of seven animals exhibited threshold shifts greater than 15 dB. In contrast, most mice bearing the KHRI-3 hybridoma exhibited high frequency threshold shifts of 40-50 dB that coincided temporally with the growth of the hybridoma, the presence of circulating KHRI-3 antibody, and greatly increased immunoglobulin titers. Ears from KHRI-3-bearing mice that developed high frequency hearing loss also had a novel type of lesion in the basal turn of the cochlea that was characterized by loss of outer hair cells and absence of typical supporting cell scars. Such changes were not found in control hybridoma-bearing mice. These findings suggest that KHRI-3 antibody has an effect on hearing that is secondary to damage to the organ of Corti and loss of outer hair cells. Our results have important implications for antibody-mediated mechanisms of hearing loss and provide an animal model in which to study this phenomenon.

The localization and specificity of guinea pig inner ear antigenic epitopes.

In this study, we investigated the relative localization of some antigenic epitopes in the inner ear. The inner ear protein antigens were extracted from various parts of the guinea pig inner ear. Brain, kidney, lung, heart and liver extracts were also obtained. We found by SDS-polyacrylamide gel electrophoresis that total inner ear extracts separated into three high concentration polypeptide bands with molecular weights of approximately 30, 42, 58 kd and three low density bands of 20, 25 and 35 kd. The 30 kd band was found mainly in the extract of the spiral ganglion and the acoustic nerve in the modiolus. The 42 and 58 kd bands were detected in the extract of the spiral ligament and the stria vascularis. The Organ of Corti and the basilar membrane extract gave rise to three bands of 30, 42 and 58 kd. Twenty-eight of the 75 sera from patients with inner ear disease reacted with the 30 and 58 kd bands of the inner ear protein extracts by immunoblotting. Sixteen of these 28 positive sera were then used to probe immunoblots of the brain, kidney, lung, heart and liver extracts. The 58 kd band was also found in protein extracts of the brain, the lung and the liver. This study suggests that the 30 kd antigenic epitope may be mainly related to the acoustic nerve and that the 58 kd antigenic epitope is not cochlear specific.

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti.

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.