Strial microvascular pathology and age-associated endocochlear potential decline in NOD congenic mice.

NOD/ShiLtJ (previously NOD/LtJ) inbred mice show polygenic autoimmune disease and are commonly used to model autoimmune-related type I diabetes, as well as Sjogren's syndrome. They also show rapidly progressing hearing loss, partly due to the combined effects of Cdh23ahl and Ahl2. Congenic NOD.NON-H2nb1/LtJ mice, which carry corrective alleles within the H2 histocompatibility gene complex, are free from diabetes and other overt signs of autoimmune disease, but still exhibit rapidly progressive hearing loss. Here we show that cochlear pathology in these congenics broadly includes hair cell and neuronal loss, plus endocochlear potential (EP) decline from initially normal values after two months of age. The EP reduction follows often dramatic degeneration of capillaries in stria vascularis, with resulting strial degeneration. The cochlear modiolus also features perivascular inclusions that resemble those in some mouse autoimmune models. We posit that cochlear hair cell/neural and strial pathology arise independently. While sensory cell loss may be closely tied to Cdh23ahl and Ahl2, the strial microvascular pathology and modiolar anomalies we observe may arise from alleles on the NOD background related to immune function. Age-associated EP decline in NOD.NON-H2nb1 mice may model forms of strial age-related hearing loss caused principally by microvascular disease. The remarkable strial capillary loss in these mice may also be useful for studying the relation between strial vascular insufficiency and strial function.

Genetic dependence of cochlear cells and structures injured by noise.

The acute and permanent effects of a single damaging noise exposure were compared in CBA/J, C57BL/6 (B6), and closely related strains of mice. Two hours of broadband noise (4-45 kHz) at 110 dB SPL led to temporary reduction in the endocochlear potential (EP) of CBA/J and CBA/CaJ (CBA) mice and acute cellular changes in cochlear stria vascularis and spiral ligament. For the same exposure, B6 mice showed no EP reduction and little of the pathology seen in CBA. Eight weeks after exposure, all mice showed a normal EP, but only CBA mice showed injury and cell loss in cochlear lateral wall, despite the fact that B6 sustained larger permanent threshold shifts. Examination of noise injury in B6 congenics carrying alternate alleles of genes encoding otocadherin (Cdh23), agouti protein, and tyrosinase (albinism) indicated that none of these loci can account for the strain differences observed. Examination of CBA x B6 F1 mice and N2 backcross mice to B6 further indicated that susceptibility to noise-related EP reduction and associated cell pathology are inherited in an autosomal dominant manner, and are established by one or a few large effect quantitative trait loci. Findings support a common genetic basis for an entire constellation of noise-related cochlear pathologies in cochlear lateral wall and spiral limbus. Even within species, cellular targets of acute and permanent cochlear noise injury may vary with genetic makeup.

Temporal and genetic influences on protection against noise-induced hearing loss by hypoxic preconditioning in mice.

The protective benefits of hypoxic preconditioning (HPC) against permanent noise-induced hearing loss (NIHL) were investigated in mice. Hypoxia induced by exposure to 8% O2 for 4 h conferred significant protection against damaging broadband noise delivered 24-48 h later in male and female CBA/J (CBA) and CBA/CaJ mice. No protection was found in C57BL/6 (B6) mice, their B6.CAST-Cdh23(CAST) (B6.CAST) congenics, or in CBAxB6 F1 hybrid mice over the same interval, suggesting that the potential for HPC depends on one or a few autosomal recessive alleles carried by CBA-related strains, and is not influenced by the Cdh23 locus. Protection against NIHL in CBA mice was associated with significant up-regulation of hypoxia-inducible factor-1alpha (HIF-1alpha) within the organ of Corti, not found in B6.CAST. In both CBA and B6.CAST mice, some hypoxia-noise intervals shorter than 24 h were associated with exacerbation of NIHL. Cellular cascades underlying the early exacerbation of NIHL by hypoxia are therefore common to both strains, and not mechanistically linked to later protection. Elucidation of the events that underlie HPC, and how these are impacted by genetics, may lead to pharmacologic approaches to mimic HPC, and may help identify individuals with elevated risk of NIHL.

Surface charge modification decreases Pseudomonas aeruginosa adherence in vitro and bacterial persistence in an in vivo implant model.

OBJECTIVE: Chronic, persistent infections complicate otologic procedures utilizing implantable devices such as cochlear implants or tympanostomy tubes. These infections are thought to be due to the establishment of microbial biofilms on implant surfaces. To address this issue, we hypothesized that surface charge modification may inhibit the formation of Pseudomonas aeruginosa biofilms on implant surfaces in vitro and in vivo. STUDY DESIGN: We evaluated the effect of surface charge modification on bacterial biofilm formation by assessing the effect of the surface charge on bacterial adhesion in vitro and bacterial persistence in vivo. METHODS: To study the effect of surface charge in vitro, the surface wells in culture plates were modified using a layer-by-layer polyelectrolyte assembly method. Bacterial adherence was measured at 30-, 60-, and 120-minute intervals. To study the effect of surface charge modification in vivo, the surface of titanium microscrews was similarly modified and then surgically implanted into the dorsal calvaria of adult rats and inoculated with bacteria. Two weeks after implantation and inoculation, the number of bacteria remaining in vivo was evaluated. RESULTS: Surface charge modification results in a significant decrease in adherence of bacteria in vitro. Surface charge modification of titanium microscrew implants also resulted in a significant decrease in P. aeruginosa recovered 2 weeks after surgical implantation. CONCLUSION: Charge modification decreases the number of bacteria adherent to a surface in vitro and decreases the risk and severity of implant infection in an in vivo rat infection model. These results have promising biomedical applications. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:1655-1661, 2017.

Cellular correlates of age-related endocochlear potential reduction in a mouse model.

Age-related degeneration of cochlear stria vascularis and resulting reduction in the endocochlear potential (EP) are the hallmark features of strial presbycusis, one of the major forms of presbycusis, or age-related hearing loss (ARHL) (Schuknecht, H.F., 1964. Further observations on the pathology of presbycusis. Archives of Otolaryngology 80, 369-382; Schuknecht, H.F., 1993. Pathology of the Ear. Lea and Febiger, Philadelphia; Schuknecht, H.F., Gacek, M.R., 1993. Cochlear pathology in presbycusis. Annals of Otology, Rhinology and Laryngology 102, 1-16). It is unclear whether there are multiple forms of strial ARHL having different sequences of degenerative events and different risk factors. Human temporal bone studies suggest that the initial pathology usually affects strial marginal cells, then spreads to other strial cell types. While inheritance studies support a moderate genetic influence, no contributing genes have been identified. Establishment of mouse models of strial ARHL may promote the identification of underlying genes and gene/environment interactions. We have found that BALB/cJ mice show significant EP reduction by 19 months of age. The reduction only occurs in a subset of animals. To identify key anatomical correlates of the EP reduction, we compared several cochlear lateral wall metrics in BALBs with those in C57BL/6J (B6) mice, which show little EP reduction for ages up to 26 months. Among the measures obtained, marginal cell density and spiral ligament thickness were the best predictors of both the EP decline in BALBs, and EP stability in B6. Our results indicate that the sequence of strial degeneration in BALBs is like that suggested for humans. Additional strain comparisons we have performed suggest that genes governing strial melanin production do not play a role.

QTL Mapping of Endocochlear Potential Differences between C57BL/6J and BALB/cJ mice.

We reported earlier that the endocochlear potential (EP) differs between C57BL/6J (B6) and BALB/cJ (BALB) mice, being lower in BALBs by about 10 mV (Ohlemiller et al. Hear Res 220: 10-26, 2006). This difference corresponds to strain differences with respect to the density of marginal cells in cochlear stria vascularis. After about 1 year of age, BALB mice also tend toward EP reduction that correlates with further marginal cell loss. We therefore suggested that early sub-clinical features of the BALB stria vascularis may predispose these mice to a condition modeling Schuknecht's strial presbycusis. We further reported (Ohlemiller et al. J Assoc Res Otolaryngol 12: 45-58, 2011) that the acute effects of a 2-h 110 dB SPL noise exposure differ between B6 and BALB mice, such that the EP remains unchanged in B6 mice, but is reduced by 40-50 mV in BALBs. In about 25 % of BALBs, the EP does not completely recover, so that permanent EP reduction may contribute to noise-induced permanent threshold shifts in BALBs. To identify genes and alleles that may promote natural EP variation as well as noise-related EP reduction in BALB mice, we have mapped related quantitative trait loci (QTLs) using 12 recombinant inbred (RI) strains formed from B6 and BALB (CxB1-CxB12). EP and strial marginal cell density were measured in B6 mice, BALB mice, their F1 hybrids, and RI mice without noise exposure, and 1-3 h after broadband noise (4-45 kHz, 110 dB SPL, 2 h). For unexposed mice, the strain distribution patterns for EP and marginal cell density were used to generate preliminary QTL maps for both EP and marginal cell density. Six QTL regions were at least statistically suggestive, including a significant QTL for marginal cell density on chromosome 12 that overlapped a weak QTL for EP variation. This region, termed Maced (Marginal cell density QTL) supports the notion of marginal cell density as a genetically influenced contributor to natural EP variation. Candidate genes for Maced notably include Foxg1, Foxa1, Akap6, Nkx2-1, and Pax9. Noise exposure produced significant EP reductions in two RI strains as well as significant EP increases in two RI strains. QTL mapping of the EP in noise-exposed RI mice yielded four suggestive regions. Two of these overlapped with QTL regions we previously identified for noise-related EP reduction in CBA/J mice (Ohlemiller et al. Hear Res 260: 47-53, 2010) on chromosomes 5 and 18 (Nirep). The present map may narrow the Nirep interval to a ~10-Mb region of proximal Chr. 18 that includes Zeb1, Arhgap12, Mpp7, and Gjd4. This study marks the first exploration of natural gene variants that modulate the EP. Their orthologs may underlie some human hearing loss that originates in the lateral wall.

FleQ, a Transcriptional Activator, Is Required for Biofilm Formation In Vitro But Does Not Alter Virulence in a Cholesteatomas Model.

HYPOTHESIS: Bacterial biofilm formation within cholesteatomas is responsible for increased persistence and tissue destruction and Pseudomonas aeruginosa deficient in biofilm formation (PAO1 ΔfleQ) are less virulent than the parent bacteria. BACKGROUND: Infected aural cholesteatomas have been demonstrated to be more destructive than uninfected cholesteatomas and infections are more persistent. The chronicity and persistence of infections within cholesteatomas may be because of the presence of biofilm formation. METHODS: Twenty-seven mutant strains of PAO1 were screened for surface adherence. These strains were also screened for static biofilm formation. The biofilms were quantified by staining with crystal violet. Aural cholesteatomas were then induced in Mongolian gerbils by ligation of the ear canal. At the time of ligation, the ear canals were inoculated with wild-type PAO1 and a biofilm deficient PAO1 ΔfleQ strain of P. aeruginosa. A 7 weeks course of ciprofloxacin (20 mg/kg/day) was started on postoperative day 7. Eight weeks after induction of cholesteatomas, the cholesteatoma size, levels of bone destruction, and levels of bone remodeling were evaluated using microCT imaging. RESULTS: PAO1 ΔfleQ was identified as a poorly adherent and deficient biofilm forming mutant strain of P. aeruginosa. Infected cholesteatomas had more growth, bone destruction and bone remodeling than uninfected cholesteatomas. However, there was no difference observed between cholesteatomas infected with PAO1 (biofilm competent strain) and PAO1 ΔfleQ (biofilm deficient strain). CONCLUSION: We demonstrate that the biofilm phenotype is not an important virulence factor in cholesteatomas infected with P. aeruginosa.

Cellular correlates of progressive hearing loss in 129S6/SvEv mice.

Several strains of mice hear well initially but show progressive sensorineural hearing loss. Affected cochlear cell types include all those known to be affected in human age-related hearing loss (ARHL), or presbycusis. Thus these mice have been offered as models of human ARHL. At present, however, few mouse ARHL models are sufficiently well described to serve as the basis for specific hypotheses about human ARHL. We examined 1-month-old and 15-month-old 129S6/SvEv (129S6) mice and compared them with BALB/cJ and CBA/J mice. Age-related elevation of compound action potential thresholds was interpreted in the light of endocochlear potentials and changes in hair cells, afferent neurons, fibrocytes in spiral limbus and ligament, and supporting cells within the organ of Corti. Aging in 129S6 mice was associated with high-frequency hearing loss. Four components of age-related cochlear degeneration emerged from quantitative analyses, including 1) basal loss of outer hair cells; 2) basal loss of type IV fibrocytes in the spiral ligament; 3) apical loss of fibrocytes in spiral limbus, and 4) anomalies of supporting cells in the cochlear base. Although neuronal loss was not consistently found, two mice showed loss of afferent dendrites and cell bodies in the cochlear apex without inner hair cell loss. Despite multifaceted degeneration, hearing loss in 129S6 mice appears to be best explained by degenerative changes in outer hair cells and in the organ of Corti, conforming to human sensory ARHL. Age-related changes in the apical spiral limbus may promote pathology of the medial organ of Corti and eventual loss of afferent neurons, with possible implications for human neural ARHL.

Apical-to-basal gradients in age-related cochlear degeneration and their relationship to "primary" loss of cochlear neurons.

The predominant conceptual framework for understanding human age-related hearing loss (ARHL, or presbycusis) holds that three different cochlear elements (organ of Corti, afferent neurons, and stria vascularis) can degenerate independently, and exert independent influences on hearing. Within this framework, temporal bones from subjects with ARHL may be classified as exemplifying sensory (referring to organ of Corti), "primary" neural (loss of afferent neurons without loss of their hair cell targets), strial, or mixed ARHL. While there is general agreement as to the types of cochlear cells most affected by aging, there is less agreement about how to classify ARHL, and whether contributions of particular structures to hearing loss can be isolated. The cochlear apex of humans and animals is particularly prone to apparent primary loss of neurons that may represent an aspect of neural ARHL. We recently reported that in 129S6/SvEv mice apical neuronal loss is often accompanied by abnormalities of spiral limbus, pillar cells, and Reissner's membrane (Ohlemiller and Gagnon [2004] J Comp Neurol 469:377-390). We proposed that the initial pathology occurs within limbus, leading to disruption of perilymphatic ion homeostasis, and eventual loss of neurons as one consequence. We have now examined this issue quantitatively in young and old mice of four different strains (129S6/SvEv, CBA/J, C57BL/6, and BALB/c). Abnormalities of apical spiral limbus were found to correlate only weakly with neuronal loss. Strong correlations were found between neuronal loss and abnormalities of both pillar cells and Reissner's membrane, however. Apical neuronal loss and apical-to-basal progression of pathology of limbus, pillar cells, and Reissner's membrane run counter to most reported age-related cochlear trends. Our findings suggest that these changes share a common triggering influence.

Inactivation of specific Pseudomonas aeruginosa biofilm factors does not alter virulence in infected cholesteatomas.

HYPOTHESIS: When experimental cholesteatomas are infected with Pseudomonas aeruginosa (PA) mutants lacking factors associated with the formation of biofilms, host defenses are more effective against these strains when compared with wild-type strains (PAO1 and OPPA8) in preventing tissue destruction. BACKGROUND: Previous studies have identified biofilms within chronically infected aural cholesteatomas. These infected cholesteatomas are associated with increased tissue destruction. Because biofilms are highly resistant to host defenses leading to prolonged infection, we propose that the biofilm phenotype of P. aeruginosa may be a virulence factor leading to persistence of infection and increased tissue destruction. METHODS: Aural cholesteatomas were induced in Mongolian gerbils. At the time of induction, the ear canals were inoculated with wild-type (PAO1 and OPPA8) and biofilm-deficient (PAO1 ΔpilA, PAO1 algD::aacC1 and PAO1 galU::aacC1) strains of P. aeruginosa. After 8 weeks, the size of the cholesteatomas and levels of bone destruction and deposition were measured using microCT scanning and double fluorochrome bone labeling. RESULT: Infected cholesteatomas resulted in increased growth, bone destruction, and bone deposition when compared with vehicle-only controls. We observed no differences between the wild-type (biofilm forming) and the biofilm-deficient strains of P. aeruginosa. CONCLUSION: Our hypothesis that biofilm formation is a virulence factor in cholesteatomas infected with P. aeruginosa was not supported. A number of interpretations of these data are reasonable. It is possible that biofilms are not critical in infected cholesteatomas. Alternatively, the mutants that are deficient in generating biofilms in vitro may be able to form effective biofilms in vivo using alternative pathways.

Biofilm formation by otopathogenic strains of Pseudomonas aeruginosa is not consistently inhibited by ethylenediaminetetraacetic acid.

HYPOTHESIS: Biofilm formation in otopathogenic of Pseudomonas aeruginosa (OPPA) strains is inhibited by ethylenediaminetetraacetic acid (EDTA). BACKGROUND: EDTA, a widely used chelating agent, has been shown to inhibit biofilm formation in a number of bacteria. Because EDTA may be a well-tolerated reagent to inhibit biofilm formation in cases of suppurative otitis media, we asked if it might be effective in all OPPA strains isolated from chronically infected cholesteatomas. METHODS: OPPA strains were isolated from patients with infected cholesteatomas. These strains were grown into log phase then were placed in minimal media with varying concentrations of EDTA and incubated for varying periods. Biofilm production was measured colorimetrically by staining with crystal violet. RESULTS: Without added EDTA, most otopathogenic PA exhibited a distinct, but varying, time course of biofilm formation and dissolution with peak production at 12 to 18 hours. Addition of 1 mM EDTA resulted in a delay in the time to peak biofilm formation for most strains, although the amount of biofilm was not decreased. In contrast, some strains showed greater biofilm production with 1 mM EDTA compared with the untreated bacteria. Addition of 10 mM EDTA resulted in a similar effect. Some strains increased biofilm production over controls. Moreover, EDTA inhibited planktonic growth of all OPPA strains at the concentrations studied. CONCLUSION: Our hypothesis was disproven: EDTA tends to delay biofilm development, although it consistently inhibits planktonic growth. Because EDTA does not cause suppression of biofilm production in all isolates of OPPA, usefulness as an antimicrobial is questioned.

Nutrient-enhanced diet reduces noise-induced damage to the inner ear and hearing loss.

Oxidative stress has been implicated broadly as a cause of cell death and neural degeneration in multiple disease conditions; however, the evidence for successful intervention with dietary antioxidant manipulations has been mixed. In this study, we investigated the potential for protection of cells in the inner ear using a dietary supplement with multiple antioxidant components, which were selected for their potential interactive effectiveness. Protection against permanent threshold shift (PTS) was observed in CBA/J mice maintained on a diet supplemented with a combination of ß-carotene, vitamins C and E, and magnesium when compared with PTS in control mice maintained on a nutritionally complete control diet. Although hair cell survival was not enhanced, noise-induced loss of type II fibrocytes in the lateral wall was significantly reduced (P < 0.05), and there was a trend toward less noise-induced loss in strial cell density in animals maintained on the supplemented diet. Taken together, our data suggest that prenoise oral treatment with the high-nutrient diet can protect cells in the inner ear and reduce PTS in mice. The demonstration of functional and morphologic preservation of cells in the inner ear with oral administration of this antioxidant supplemented diet supports the possibility of translation to human patients and suggests an opportunity to evaluate antioxidant protection in mouse models of oxidative stress-related disease and pathology.

Different cellular and genetic basis of noise-related endocochlear potential reduction in CBA/J and BALB/cJ mice.

The acute and permanent effects of noise exposure on the endocochlear potential (EP) and cochlear lateral wall were evaluated in BALB/cJ (BALB) inbred mice, and compared with CBA/J (CBA) and C57BL/6 (B6) mice. Two-hour exposure to broadband noise (4-45 kHz) at 110 dB SPL leads to a approximately 50 mV reduction in the EP in BALB and CBA, but not B6. EP reduction in BALB and CBA is reliably associated with characteristic acute cellular pathology in stria vascularis and spiral ligament. By 8 weeks after exposure, the EP in CBA mice has returned to normal. In BALBs, however, the EP remains depressed by an average approximately 10 mV, so that permanent EP reduction contributes to permanent threshold shifts in these mice. We recently showed that the CBA noise phenotype in part reflects the influence of a large effect quantitative trait locus on Chr. 18, termed Nirep (Ohlemiller et al., Hear Res 260:47-53, 2010b). While CBA "EP susceptibility" alleles are dominant to those in B6, examination of (B6 × BALB) F1 hybrid mice and (F1 × BALB) N2 backcross mice revealed that noise-related EP reduction and associated cell pathology in BALBs are inherited in an autosomal recessive manner, and are dependent on multiple genes. Moreover, while N2 mice formed from B6 and CBA retain strong correspondence between acute EP reduction, ligament pathology, and strial pathology, N2s formed from B6 and BALB include subsets that dissociate pathology of ligament and stria. We conclude that the genes and cascades that govern the very similar EP susceptibility phenotypes in BALB and CBA mice need not be the same. BALBs appear to carry alleles that promote more pronounced long term effects of noise on the lateral wall. Separate loci in BALBs may preferentially impact stria versus ligament.

Protection by low-dose kanamycin against noise-induced hearing loss in mice: dependence on dosing regimen and genetic background.

We recently demonstrated that sub-chronic low-dose kanamycin (KM, 300 mg/kg sc, 2×/day, 10 days) dramatically reduces permanent noise-induced hearing loss (NIHL) and hair cell loss in 1 month old CBA/J mice (Fernandez et al., 2010, J. Assoc. Res. Otolaryngol. 11, 235-244). Protection by KM remained for at least 48 h after the last dose, and appeared to involve a cumulative effect of multiple doses as part of a preconditioning process. The first month of life lies within the early 'sensitive period' for both cochlear noise and ototoxic injury in mice, and CBA/J mice appear exquisitely vulnerable to noise during this period (Ohlemiller et al., 2011; Hearing Res. 272, 13-20). From our initial data, we could not rule out 1) that less rigorous treatment protocols than the intensive one we applied may be equally-or more-protective; 2) that protection by KM is tightly linked to processes unique to the sensitive period for noise or ototoxins; or 3) that protection by KM is exclusive to CBA/J mice. The present experiments address these questions by varying the number and timing of fixed doses (300 mg/kg sc) of KM, as well as the age at treatment in CBA/J mice. We also tested for protection in young C57BL/6J (B6) mice. We find that nearly complete protection against at least 2 h of intense (110 dB SPL) broadband noise can be observed in CBA/J mice at least for ages up to 1 year. Reducing dosing frequency to as little as once every other day (a four-fold decrease in dosing frequency) appeared as protective as twice per day. However, reducing the number of doses to just 1 or 2, followed by noise 24 or 48 h later greatly reduced protection. Notably, hearing thresholds and hair cells in young B6 mice appeared completely unprotected by the same regimen that dramatically protects CBA/J mice. We conclude that protective effects of KM against NIHL in CBA/J mice can be engaged by a wide range of dosing regimens, and are not exclusive to the sensitive period for noise or ototoxins. While we cannot presently judge the generality of protection across genetic backgrounds, it appears not to be universal, since B6 showed no benefit. Classical genetic approaches based on CBA/J × B6 crosses may reveal loci critical to protective cascades engaged by kanamycin and perhaps other preconditioners. Their human analogs may partly determine who is at elevated risk of acquired hearing loss.

Divergent aging characteristics in CBA/J and CBA/CaJ mouse cochleae.

Two inbred mouse strains, CBA/J and CBA/CaJ, have been used nearly interchangeably as 'good hearing' standards for research in hearing and deafness. We recently reported, however, that these two strains diverge after 1 year of age, such that CBA/CaJ mice show more rapid elevation of compound action potential (CAP) thresholds at high frequencies (Ohlemiller, Brain Res. 1277: 70-83, 2009). One contributor is progressive decline in endocochlear potential (EP) that appears only in CBA/CaJ. Here, we explore the cellular bases of threshold and EP disparities in old CBA/J and CBA/CaJ mice. Among the major findings, both strains exhibit a characteristic age (∼18 months in CBA/J and 24 months in CBA/CaJ) when females overtake males in sensitivity decline. Strain differences in progression of hearing loss are not due to greater hair cell loss in CBA/CaJ, but instead appear to reflect greater neuronal loss, plus more pronounced changes in the lateral wall, leading to EP decline. While both male and female CBA/CaJ show these pathologies, they are more pronounced in females. A novel feature that differed sharply by strain was moderate loss of outer sulcus cells (or 'root' cells) in spiral ligament of the upper basal turn in old CBA/CaJ mice, giving rise to deep indentations and void spaces in the ligament. We conclude that CBA/CaJ mice differ both quantitatively and qualitatively from CBA/J in age-related cochlear pathology, and model different types of presbycusis.

A major effect QTL on chromosome 18 for noise injury to the mouse cochlear lateral wall.

We recently demonstrated a striking difference among inbred mouse strains in the effects of a single noise exposure, whereby CBA/J and CBA/CaJ (CBA) mice show moderate reversible reduction in the endocochlear potential (EP) while C57BL/6J (B6) mice do not (Ohlemiller, K.K., Gagnon, P.M., 2007. Genetic dependence of cochlear cells and structures injured by noise. Hear. Res. 224, 34-50). Acute EP reduction in CBA was reliably associated with characteristic pathology of the spiral ligament and stria vascularis, both immediately after noise and 8weeks later. Analysis of B6xCBA F1 hybrid mice indicated that EP reduction and its anatomic correlates are co-inherited in an autosomal dominant manner. Further analysis of N2 mice resulting from the backcross of F1 hybrids to B6 mice led us to suggest that the EP reduction phenotype principally reflects the influence of a small number of quantitative trait loci (QTLs). Here we report the results of QTL mapping of the EP reduction phenotype in CBA/J using 106 N2 mice from a (CBAxB6)xB6 backcross. Correlation of acute post-noise EP with 135 markers distributed throughout the genome revealed a single major effect QTL on chromosome 18 (12.5 cM, LOD 3.57) (Nirep, for noise-induced reduction in EP QTL), and two marginally significant QTLs on chromosomes 5 and 16 (LOD 1.43 and 1.73, respectively). Our results underscore that fact that different cochlear structures may possess different susceptibilities to noise through the influence of non-overlapping genes. While Nirep and similar-acting QTLs do not appear to influence the extent of permanent hearing loss from a single noise exposure, they could reduce the homeostatic 'reserve' of the lateral wall in protracted or continual exposures, and thereby influence long term threshold stability.

Protection against noise-induced hearing loss in young CBA/J mice by low-dose kanamycin.

Animal studies indicate that a combination of kanamycin (KM) and noise produces a synergistic effect, whereby the threshold shift from the combination is greater than the sum of the shifts caused by either agent alone. Most such studies have focused on adult animals, and it has remained unclear whether younger, presumably more susceptible, animals show an even greater synergistic effect. The present study tested the hypothesis that young CBA/J mice receiving a low dose of KM (300 mg/kg, 2x/day, s.c.) from 20 to 30 days post-gestational age followed by brief noise exposure (110 dB SPL; 4-45 kHz, 30 s) would show greater noise-induced permanent threshold shifts (NIPTS) than mice receiving either treatment alone. Noise exposure produced 30-40 dB of NIPTS and moderate hair cell loss in young saline-treated mice. KM alone at this dose had no effect on thresholds. Surprisingly, mice receiving KM plus noise were protected from NIPTS, showing ABR thresholds not significantly different from unexposed controls. Mice receiving KM prior to noise exposure also showed significantly less outer hair cell loss than saline-treated mice. Additional experiments indicated protection by KM when the noise was applied either 24 or 48 h after the last KM injection. Our results demonstrate a powerful protective effect of sub-chronic low-dose kanamycin against NIPTS in young CBA/J mice. Repeated kanamycin exposure may establish a preconditioned protective state, the molecular bases of which remain to be determined.

Absence of strial melanin coincides with age-associated marginal cell loss and endocochlear potential decline.

Cochlear stria vascularis contains melanin-producing intermediate cells that play a critical role in the production of the endocochlear potential (EP) and in maintaining the high levels of K(+) that normally exist in scala media. The melanin produced by intermediate cells can be exported to the intrastrial space, where it may be taken up by strial marginal cells and basal cells. Because melanin can act as an antioxidant and metal chelator, evidence for its role in protecting the stria and organ of Corti against noise, ototoxins, and aging has long been sought. While some evidence supports a protective role of melanin against noise and ototoxins, no evidence yet presented has demonstrated a clear role for melanin in maintaining the EP during aging. We tested this by comparing basal turn EPs and a host of cochlear cellular metrics in aging C57BL/6 (B6) mice and C57BL/6-Tyr(c-2J) mice. The latter mice carry a naturally occurring inactivating mutation of the tyrosinase locus, and produce no strial melanin. Because these two strains are coisogenic, and because pigmented B6 mice show essentially no age-related EP decline, they provide an ideal test of importance of melanin in the aging stria. Pigmented and albino B6 mice showed identical rates of hearing loss and sensory cell loss. However, after two years of age, basal turn EPs significantly diverged, with 42% of albinos showing EPs below 100 mV versus only 18% of pigmented mice. The clearest anatomical correlate of this EP difference was significantly reduced strial thickness in the albinos that was highly correlated with loss of marginal cells. Combined with findings in human temporal bones, plus recent work in BALB/c mice and gerbils, the present findings point to a common etiology in strial presbycusis whereby EP reduction is principally linked to marginal cell loss or dysfunction. For any individual, genetic background, environmental influences, and stochastic events may work together to determine whether marginal cell density or function falls below some critical level, and thus whether EP decline occurs.