T.J. Yoo

Morphological and functional alterations of the cochlea in apolipoprotein E gene deficient mice

The relationship between hyperlipidemia and sensorineural hearing loss remains obscure. In this study, we elucidate for the first time the cochlear morphological and auditory alterations and their relationships with hyperlipidemia, atherosclerosis, and endothelial dysfunction in apolipoprotein-E knockout (ApoE-KO) mice. Ten-week-old ApoE-KO mice were fed either atherosclerotic diet (1.25% cholesterol) or normal diet. Wild type mice (C57BL/6J) served as normal controls. Fourteen weeks later, marked hyperlipidemia, atherosclerosis, endothelial dysfunction, and hearing impairment, especially in the high frequencies, had developed in ApoE-KO mice as compared with C57BL/6J mice (P<0.001). A high positive correlation between hearing loss and the extent of atherosclerosis and plasma total cholesterol levels was found. Hearing loss, especially at high frequencies, was detected in all ApoE-KO mice. Hair cell loss mainly at the base turn, thickening of vascular intima, and lumen stenosis of the spiral modiolar artery (SMA) in cochlea were also found; these histological changes were exacerbated by the atherosclerotic diet. Furthermore, endothelial nitric oxide synthase (eNOS) in aortic wall and cochlea was distinctly reduced in ApoE-KO mice. These results demonstrate that hyperlipidemia and atherosclerosis can induce alterations in cochlear morphology and function. The stenosis of SMA, which may cause cochlear ischemia and hypoxia, endothelial dysfunction, and low eNOS activity, may contribute to hearing loss.

Antibodies against Inner-Ear Proteins in the Sera of Patients with Inner-Ear Diseases

Sera from patients with various inner-ear diseases, especially Meniere’s disease, were investigated by Western blot against guinea pig inner-ear proteins. Of 45 patients, 24 (53%) with various inner-e

House dust mite–induced sensitivity in mice

Sensitivity induced by house dust mite (HDM) extract in mice was investigated in this study. Sensitized B10.RIII mice (H-2r background) had T-cell proliferative responses to HDM extract in vitro and an HDM-specific IgE response. When mice were immunized by injection and intranasal inhalation with HDM extract, a histologic study showed eosinophils and mononuclear cell infiltration in the lung tissue and bronchial wall. Tcr alphabeta-positive cells were also found in the cell infiltration area of the lung lesions. In the control mice that were immunized by injection or intranasal inhalation (but not both), we did not observe cell accumulation in the lung tissue or in the bronchial wall. Epitope studies suggest that T cells recognize multiple epitopes. Molecular analysis of these HDM-specific T-cell hybridoma clones suggest that T-cell receptor use is restricted to members of the V alpha 8 and Vbeta 6 subfamilies.

Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen.

The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured > or = 12 mice per pedigree in > or = 2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (> or = 16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

Type II Collagen Distribution in the Ear of the Guinea Pig Fetus

By using monoclonal antibodies to type II collagen and immunohistochemical techniques, we studied the distribution of type II collagen in the developing guinea pig ear. Type II collagen appearance and disappearance corresponded to cartilage development and resorption. Type II collagen was identified in Meckels and Reicherts cartilages, the cartilage plate of the auricle and external acoustic meatus, the ossicles, eustachian tube cartilage, and the otic capsule. Type II collagen also appeared with the development of structures in noncartilaginous parts including the tympanic membrane, tympanic annulus, basilar membrane, spiral limbus, spiral ligament, and osseous spiral lamina, Rosenthals canal, the maculae of the utricle and saccule, and the semicircular canal membrane, crista ampullaris, and endolymphatic duct. Type II collagen is distributed widely in the ear after the early stages of development. Thus, type II collagen should be considered an important structural component of the ear.

Molecular Basis of Type II Collagen Autoimmune Disease: Observations of Arthritis, Auricular Chondritis and Tympanitis in Mice

A type II collagen autoimmune response results in arthritis, auricular chondritis and tympanosclerosis in humans and animals. The purpose of this study is to further define the molecular and pathogenic events involved in these lesions in rodents. Type II collagen fragment CB11-specific monoclonal antibodies induced lesions in joints, ear lobes and tympanic membranes. In immunized mice, the thickness of tympanic membranes increased to two- to fourfold normal size. Electron micrography showed that the arrangement of collagen fibers is irregular in both radial and auricular layers, containing fibroblasts, a homogeneous material resembling low-density cholesterol crystals and cell infiltration. The mice with auricular chondritis had lymphocytes expressing V beta-8 T cell receptor (TCR) in arthritic joints and lymphocytes expressing V beta-6 TCR in ear lobe lesions. A monoclonal antibody specific to the TCR V beta-8 subfamily suppressed the onset of arthritis. Sequence analyses of the V beta structure of TCR involved in the lesions confirm the immunohistologic study.

Effects of Simvastatin on Plasma Lipoproteins and Hearing Loss in Apolipoprotein E Gene-Deficient Mice

Apolipoprotein E (ApoE) gene knockout (KO) mice develop hearing loss, as well as hair cell damage in the inner ear. Furthermore, the hearing loss and hair cell damage may be caused and exacerbated by hyperlipidemia and atherosclerosis. We are therefore seeking an effective treatment to protect the inner ear in ApoE-KO mice. ApoE-KO mice fed with an atherosclerotic diet were treated with simvastatin or with Strauss Heartdrops. The auditory brainstem responses were recorded, and plasma lipid levels and inner ear histology were examined. ApoE-KO and wild-type C57BL/6J mice fed with a normal chow diet served as controls. Compared to ApoE-KO mice fed only the atherosclerotic diet, ApoE-KO mice treated with simvastatin had no significant hearing loss and less severe atherosclerosis and hair cell damage in the inner ear. However, ApoE-KO mice treated with Strauss Heartdrops developed significant hearing loss and severe atherosclerosis and hair cell damage in the inner ear. These results demonstrate that statins may prevent hearing loss and inner ear damage in ApoE-KO mice by reducing the atherosclerotic lesions and levels of glucose, cholesterol, low-density lipoprotein, and triglyceride. Statins could be used to treat hearing loss associated with hyperlipidemia.

Impaired Stria Vascularis in the Inner Ear of Apolipoprotein E Gene Knockout Mice

Purpose: To study apoptotic cell death, the expression of endothelial nitric oxide synthase (eNOS) and the capillary density in the cochleae of apolipoprotein E gene knockout (ApoE KO) mice. Methods: Cochleae of ApoE KO mice were stained by HE, anti-caspase-3 and anti-eNOS antibodies and TUNEL (in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling). Capillary density was measured in the stria vascularis and in the modiolus. Results: Apoptotic cell death was found in the stria vascularis of ApoE KO mice, but not in the organ of Corti. The expression of eNOS and the capillary density were decreased in the stria vascularis and in the modiolus of ApoE KO mice. Conclusion: These results suggest that cells in the inner ear of ApoE KO mice take different pathways to undergo cell death, and apoptotic cell death is only involved in the stria vascularis. These pathological changes may play a role in the hearing loss that has been found in ApoE KO mice.

Distribution of Beta-Tubulin in Guinea Pig Inner Ear

Our previous research had suggested that β-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of β-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to β-tubulin monoclonal antibody was found in stria vascularis, neurons of the spiral ganglion, cochlear nerve fibers and spiral ligament. Diffuse staining was found in the stria vascularis and the neurons of the spiral ganglion, while dense network staining was found in the spiral ligament, the nerve fibers and the vestibular end organs. The semicircular canals, endolymphatic duct and sac were also positively stained. In inner ears of guinea pigs challenged with β-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that β-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the β-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that β-tubulin might be an autoantigen for autoimmune inner ear disease.

Experimental autoimmune hearing loss is exacerbated in IL-10-deficient mice and reversed by IL-10 gene transfer.

Interleukin-10 (IL-10) has an important role in the homeostatic regulation of autoreactive T-cell repertoire. We hypothesized that endogenous IL-10 would regulate the severity of β-tubulin-induced experimental autoimmune hearing loss (EAHL) and that exogenous IL-10 would abrogate it. BALB/c wild-type (WT) and homozygous IL-10-deficient mice (IL-10−/−) underwent β-tubulin immunization to develop EAHL; some IL-10 mice with EAHL were administered IL-10 DNA at the peak of EAHL. Auditory brainstem responses were examined over time. EAHL developed progressively in both WT and IL-10−/− mice. However, the severity of hearing loss in the IL-10−/− mice was significantly greater than that in WT animals. Moreover, disease severity was associated with a significantly enhanced interferon-γ level and loss of hair cells in IL-10−/− mice. IL-10 administered to EAHL IL-10−/− mice promoted IL-10 expression. Consequently, hearing significantly improved by protecting hair cells in established EAHL. Importantly, IL-10 treatment suppressed proliferation of antigen-specific T-helper type 1 (Th1) cells, and the suppression can be attributed to inducing IL-10-secreting regulatory T cells that suppressed autoreactive T cells. We demonstrated that the lack of IL-10 exacerbated hearing loss, and the exogenous administration of IL-10 improved hearing. Mechanistically, our results indicate that IL-10 is capable of controlling autoimmune reaction severity by suppressing Th1-type proinflammatory responses and inducing IL-10-secreting regulatory T cells.