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.

ENU induced single mutation locus on chr 16 leads to high-frequency hearing loss in mice.

The hallmark of age-related (presbycusis) and noise-induced hearing loss is high-frequency (> 20 kHz) hearing loss. Through a collaborative study with TMGC (Tennessee Mouse Genome Consortium), seventeen ENU-induced mouse mutation strains with high-frequency hearing loss have been identified, but affected genes are yet identified. As a first step in identifying the gene/s underlying the ENU mutations, we created a F2 population between a representative mutation strain, 118 TNE and a wild type strain, CAST/EJ (CAST). Phenotypic analysis showed that there is a 3:1 ratio of segregation between normal and hearing loss in the F2 population, suggestion a single locus regulation. However, the linkage mapping identified 2 QTLs, each on chromosomes 15 and 16. Further statistical analysis of marker segregation patterns revealed that the locus on Chr 16 was ENU induced while the one on Chr 15 was derived from the parental strain, CAST.

Induction of tolerance by oral administration of beta-tubulin in an animal model of autoimmune inner ear disease.

Induction of peripheral tolerance by oral administration of low-dose beta-tubulin antigen may be an effective, antigen-specific method to suppress experimental autoimmune hearing loss. Five groups of mice were fed with phosphate-buffered saline (PBS), ovalbumin (OVA), 20, 30 or 200 microg of beta-tubulin, respectively. All mice were then immunized by beta-tubulin. Hearing thresholds were measured before and after immunization. Inner ear histology and cytokine profile were examined. Mice fed with 20 or 30 microg of beta-tubulin showed less hearing loss and less inner ear damage compared to the groups treated with PBS, OVA or 200 microg of beta-tubulin. Interferon-gamma (IFN-gamma) was decreased while interleukin-4 (IL-4), IL-5, IL-13 and TGF-beta were increased in both sera and in cell culture supernatants of the mice fed with 20 or 30 microg of beta-tubulin. However, no cytokine profile change was found in the group treated with 200 microg of tubulin. These results suggest that a low dose of beta-tubulin is active orally in an antigen-specific fashion and capable of inhibiting the autoimmune reactions in the inner ear by suppressing Th1 (IFN-gamma) and increasing Th2 and Th3 (IL-4, IL-5, IL-13 and TGF-beta) cytokines. Oral antigen tolerance may be used to treat autoimmune inner ear disease.

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.

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.

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.

Distribution of beta-tubulin in guinea pig inner ear.

Our previous research had suggested that beta-tubulin might be an autoantigen for autoimmune inner ear disease. In this study, the expression of beta-tubulin in inner ears of normal and tubulin-immunized guinea pigs was examined by immunohistochemical staining. Strong immunoreactivity to beta-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 beta-tubulin, staining intensity was diminished in the stria vascularis, the spiral ligament, and the neurons of the spiral ganglion. The results suggest that beta-tubulin is distributed to most structures of guinea pig inner ear. A challenge to the inner ear by tubulin could change the beta-tubulin distribution and cause degeneration in the spiral ganglion. The results support the hypothesis that beta-tubulin might be an autoantigen for autoimmune inner ear disease.

TUNEL-positive labeling in mouse inner ear caused by tubulin immunization is not apoptosis.

Apoptosis is involved in all fundamental processes of the immune system. To study whether apoptosis plays any role in the pathogenesis of autoimmune inner ear disease, we immunized Balb/c mice with tubulin. The inner ears were examined with TUNEL (in situ terminal dUTP nick-end labeling) and immunocytochemistry of the apoptosis regulatory proteins Bcl-2 and Bax. TUNEL-positive cells are found in the tubulin-immunized inner ears but not in the control inner ears. The positive cells are the marginal cells in the stria vascularis, and the hair cells in Corti's organ and the saccule. However, under morphological analysis by light microscope, these cells lack the features characteristic of apoptosis. Moreover, no cells staining positive for Bcl-2 and Bax are found in any structures of the inner ears. These results suggest that positive TUNEL staining in this model does not indicate apoptosis and apoptosis may be not involved in autoimmune inner ear disease.