Patricia M. Gagnon

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.

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 Reissners 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 Reissners membrane, however. Apical neuronal loss and apical‐to‐basal progression of pathology of limbus, pillar cells, and Reissners membrane run counter to most reported age‐related cochlear trends. Our findings suggest that these changes share a common triggering influence. J. Comp. Neurol. 479:103–116, 2004.

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.

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.

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. J. Comp. Neurol. 469:377–390, 2004.

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 Sjogrens 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.

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.

D-amino acids do not inhibitPseudomonas aeruginosabiofilm formation: D-Amino Acids do not InhibitP. AeruginosaBiofilms

Pseudomonas aeruginosa, a known biofilm‐forming organism, is an opportunistic pathogen that plays an important role in chronic otitis media, tracheitis, cholesteatoma, chronic wounds, and implant infections. Eradication of biofilm infections has been a challenge because the biofilm phenotype provides bacteria with a protective environment from the immune system and antibiotics; thus, there has been great interest in adjunctive molecules that may inhibit biofilm formation or cause biofilm dispersal. There are reports that D‐amino acids may inhibit biofilms. In this study, we test the ability of various D‐amino acids to inhibit P. aeruginosa biofilm formation in vitro.

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 &Dgr;pilA, 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.

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

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.