Regulation of Endothelial Nitric Oxide Synthase in the Reticular Lamina of the Organ of Corti by a Nitric Oxide Donor.

In the vertebrate cochlea, the reticular lamina seals the organ of Corti against the endolymph filled scala media. After noise exposure, fast alterations in the endothelial nitric oxide synthase (eNOS) expression level were identified in this cochlear structure. Minor amounts of nitric oxide (NO) produced by eNOS or applied by NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP) might protect this vulnerable part of the organ of Corti, on the line of gap junctions of supporting cells and cochlear microcirculation. In n=5 anesthetized guinea pigs, SNAP was intravenously applied in two concentrations. Six untreated animals served as controls. The cochleae were removed and prepared for immunoelectron microscopy using specific gold-labeled anti-eNOS antibodies. The density of the gold particles was quantified for seven cellular regions in the reticular lamina at the ultrastructural level. Following SNAP application, a significant increase in eNOS expression (+176%) was detected compared with controls (p=0.012). The increase occurred mainly in actin-rich cuticular structures and the prominent microtubules bundles. Correlation analysis revealed three clear and five moderate cellular associations for controls, whereas only one clear and one moderate after SNAP application. Thus, application of the NO donor SNAP resulted in an increase in eNOS expression in distinct regions of the reticular lamina.

The endocochlear potential as an indicator of reticular lamina integrity after noise exposure in mice.

The endocochlear potential (EP) provides part of the electrochemical drive for sound-driven currents through cochlear hair cells. Intense noise exposure (110 dB SPL, 2 h) differentially affects the EP in three inbred mouse strains (C57BL/6 [B6], CBA/J [CBA], BALB/cJ [BALB]) (Ohlemiller and Gagnon, 2007, Hearing Research 224:34-50; Ohlemiller et al., 2011, JARO 12:45-58). At least for mice older than 3 mos, B6 mice are unaffected, CBA mice show temporary EP reduction, and BALB mice may show temporary or permanent EP reduction. EP reduction was well correlated with histological metrics for injury to stria vascularis and spiral ligament, and little evidence was found for holes or tears in the reticular lamina that might 'short out' the EP. Thus we suggested that the genes and processes that underlie the strain EP differences primarily impact cochlear lateral wall, not the organ of Corti. Our previous work did not test the range of noise exposure conditions over which strain differences apply. It therefore remained possible that the relation between exposure severity and acute EP reduction simply has a higher exposure threshold in B6 mice compared to CBA and BALB. We also did not test for age dependence. It is well established that young adult animals are especially vulnerable to noise-induced permanent threshold shifts (NIPTS). It is unknown, however, whether heightened vulnerability of the lateral wall contributes to this condition. The present study extends our previous work to multiple noise exposure levels and durations, and explicitly compares young adult (6-7 wks) and older mice (>4 mos). We find that the exposure level-versus-acute EP relation is dramatically strain-dependent, such that B6 mice widely diverge from both CBA and BALB. For all three strains, however, acute EP reduction is greater in young mice. Above 110 dB SPL, all mice exhibited rapid and severe EP reduction that is likely related to tearing of the reticular lamina. By contrast, EP-versus-noise duration examined at 104 dB suggested that different processes contribute to EP reduction in young and older mice. The average EP falls to a constant level after ∼7.5 min in older mice, but progressively decreases with further exposure in young mice. Confocal microscopy of organ of Corti surface preparations stained for phalloidin and zonula occludens-1 (ZO-1) indicated this corresponds to rapid loss of outer hair cells (OHCs) and formation of both holes and tears in the reticular lamina of young mice. In addition, when animals exposed at 119 dB were allowed to recover for 1 mo, only young B6 mice showed collapse of the EP to ≤5 mV. Confocal analysis suggested novel persistent loss of tight junctions in the lateral organ of Corti. This may allow paracellular leakage that permanently reduces the EP. From our other findings, we propose that noise-related lateral wall pathology in young CBA and BALB mice promotes hair cell loss and opening of the reticular lamina. The heightened vulnerability of young adult animals to noise exposure may in part reflect special sensitivity of the organ of Corti to acute lateral wall dysfunction at younger ages. This feature appears genetically modifiable.

Aminoglycoside Increases Permeability of Osseous Spiral Laminae of Cochlea by Interrupting MMP-2 and MMP-9 Balance.

The spiral ganglion neurons (SGNs) located in the Rosenthal's canal of cochlea are essential target for cochlear implant. Previous studies found that the canaliculi perforantes, small pores on the surface of the osseous spiral lamina (OSL) of the scala tympanic (ST) of cochlea, may provide communication between the cochlear perilymph and SGNs. In this study, we found that chronic treatment of aminoglycosides antibiotics, which is well known to cause sensory cell damage in the cochlea, induced significant damage of bone lining cells on the OSLs and increased the permeability of the Rosenthal's canal. The pores among the bone lining cells became significantly wider after chronic treatment of amikacin (100 mg/kg/day for 3-7 days). Injection of Evans Blue in the ST resulted in significant increase in its migration in the modulus in the amikacin-treated cochlea compared to the control ears, suggesting increased permeability of these passages. Treatment of amikacin with oxytetracycline, an inhibitor of matrix metalloproteases (MMPs), significantly reduced the amount of dye migrated from the ST to the modiolus. These results suggest that amikacin enhanced the permeability between the ST and SGNs by increasing MMPs. Aggregating the permeability of the bone lining cells on the OSLs may benefit gene and stem cell delivery to the SGNs in the cochlea.

Expression of myelin basic protein in the human auditory nerve - an immunohistochemical and comparative study.

OBJECTIVE: The aim of this study is to analyse the expression and distribution of myelin basic protein (MBP or Myelin A1 protein) in the human spiral ganglion and auditory nerve. MATERIALS AND METHODS: Cryostat sections were made from freshly fixed human cochlear specimens removed at surgery in patients with life-threatening petro-clival meningiomas compressing the brain stem. The sections were subjected to immunohistochemistry using antibodies against MBP, S-100 and Tubulin. The immunoreaction was documented using laser confocal microscopy. RESULTS: Type I spiral ganglion nerve somata (SGN) were surrounded by so-called "satellite glial cells" (SGCs) that lacked expression of MBP consistent with earlier light and electron microscopic findings indicating that these cells are non-myelinating. S-100 labeling showed that the SGCs form a continuous network in the apical region. CONCLUSIONS: The pattern of myelination in human spiral ganglion is different from that in other species' spiral ganglion. The striking differences in myelin outline should be investigated further in combination with its influence on signal coding and preservation properties in man.

Fast alterations of vascular endothelial growth factor (VEGF) expression and that of its receptors (Flt-1, Flk-1 and Neuropilin) in the cochlea of guinea pigs after moderate noise exposure.

Vascular endothelial growth factor (VEGF) is a vascular permeability regulating, proangiogenic factor with neuroprotective properties. Its expression in the inner ear has been demonstrated, but little is known concerning its subcellular distribution or potential involvement in sound perception and adaptation to noise. Therefore, we determined the expression patterns and levels of VEGF and the three VEGF-receptors FLK, FLT and Neuropilin in the cochlea of guinea pigs, and examined the alterations occurring after noise exposure. After 70 dB exposure, VEGF expression was found to be reduced in all cell types of the organ of Corti, in the stria vascularis and in spiral ganglion cells. Additional down-regulation was observed in the spiral ligament and in interdental cells after 90 dB. In contrast, VEGF showed an in tendency increased level after both intensities in nerve fibers of the osseous spiral lamina. Expression of FLT was affected similarly, showing down-regulation after 70 and 90 dB on spiral ganglion cells, the nerve fibers of the osseous spiral lamina and on Deiters cells. Additionally, down-regulation was observed in the remaining cell types of the organ of Corti, the stria vascularis, the spiral ligament and the interdental cells. The Neuropilin levels remained unchanged by our experiments; apart from the blood vessel endothelium, there was no detectable expression in any of the cell types investigated. The FLK expression pattern was likewise unaffected by exposure to 70 or 90 dB, with the notable exception of an increased level occurring in Schwann cells after 90 dB. We postulate that modulation of VEGF and its receptors may be part of a neuroprotective mechanism in response to noise.

[Distribution of calcitonin gene-related peptide in guinea pig cochlea].

OBJECTIVE: To observe the distribution of CGRP in guinea pig cochlea. METHOD: CGRP was labeled by immunostaning technique. Distribution of CGRP immunoreactivity was observed with light microscopy. RESULT: CGRP immunoreactivity was located in SGN, nerve fibers at osseous spiral lamina, stria vascularis, IHC, adjacent area of OHC and Deiters' cells. CONCLUSION: CGRP distributes extensively in guinea pig cochlea. CGRP may play an important role as neurotransmitter and/or neuromodulator to hearing function by modulating HC, supporting cells, SGN and other neurons along the process of auditory nerve. It may have effect on on cochlear blood flow, too.

Changes in cytochemistry of sensory and nonsensory cells in gentamicin-treated cochleas.

Effects of a single local dose of gentamicin upon sensory and nonsensory cells throughout the cochlea were assessed by changes in immunostaining patterns for a broad array of functionally important proteins. Cytochemical changes in hair cells, spiral ganglion cells, and cells of the stria vascularis, spiral ligament, and spiral limbus were found beginning 4 days post administration. The extent of changes in immunostaining varied with survival time and with cell type and was not always commensurate with the degree to which individual cell types accumulated gentamicin. Outer hair cells, types I and II fibrocytes of the spiral ligament, and fibrocytes in the spiral limbus showed marked decreases in immunostaining for a number of constituents. In contrast, inner hair cells, type III fibrocytes and root cells of the spiral ligament, cells of the stria vascularis, and interdental cells in the spiral limbus showed less dramatic decreases, and in some cases they showed increases in immunostaining. Results indicate that, in addition to damaging sensory cells, local application of gentamicin results in widespread and disparate disruptions of a variety of cochlear cell types. Only in the case of ganglion cells was it apparent that the changes in nonsensory cells were secondary to loss or damage of hair cells. These results indicate that malfunction of the ear following gentamicin treatment is widespread and far more complex than simple loss of sensory elements. The results have implications for efforts directed toward detecting, preventing, and treating toxic effects of aminoglycosides upon the inner ear.

Expression of gap junction protein connexin43 in the adult rat cochlea: comparison with connexin26.

To elucidate whether the two different gap junction proteins connexin43 (Cx43) and connexin26 (Cx26) are expressed and localized in a similar manner in the adult rat cochlea, we performed three-dimensional confocal microscopy using cryosections and surface preparations. In the cochlear lateral wall, Cx43-positive spots were localized mainly in the stria vascularis and only a few spots were present in the spiral ligament, whereas Cx26-positive spots were detected in both the stria vascularis and the spiral ligament. In the spiral limbus, Cx43 was widely distributed, whereas Cx26 was more concentrated on the side facing the scala vestibuli and in the basal portion. In the organ of Corti, Cx43-positive spots were present between the supporting cells but they were fewer and much smaller than those of Cx26. These data demonstrated distinct differences between Cx43 and Cx26 in expression and localization in the cochlea. In addition, the area of overlap of zonula occludens-1 (ZO-1) immunolabeling with Cx43-positive spots was small, whereas it was fairly large with Cx26-positive spots in the cochlear lateral wall, suggesting that the differences are not associated with the structural difference between carboxyl terminals, i.e., those of Cx43 possess sequences for binding to ZO-1, whereas those of Cx26 lack these binding sequences.

Mechanism of endothelin 1 production in the cochlea of rats.

The purpose of this study was to elucidate the mechanism of endothelin 1 (ET1) production in the cochlea of rats. Animals were anesthetized with pentobarbital sodium intraperitoneally and sacrificed by cardiac perfusion with warm saline followed by periodate-lysine-paraformaldehyde. The temporal bones and kidneys were fixed overnight in the same fixative, decalcified with 5% EDTA and embedded in paraffin. Immunohistochemical staining was performed using the labeled streptavidin biotin method to detect the localization of big endothelin 1 (BET1), ET1 and endothelin-converting enzyme 1 (ECE1). Immunoreactivity for BET1, ET1 and ECE1 was localized to the small vessels of the bony labyrinth adjacent to the spiral ligament. Immunohistochemistry for ET1 and BET1 was also localized to the spiral ligament. These results suggest that ET1 may be produced in the small vessels of the bony labyrinth adjacent to the spiral ligament and may play an important role in the cochlear function.

Microtubule-associated proteins in adult human sensory organs.

The distribution of microtubule-associated proteins MAP-1 and MAP-2 was analysed with immunomorphological techniques in the serially sectioned adult human membranous labyrinth. In the organ of Corti, monoclonal antibodies to MAP-1 did not stain. Positivity for MAP-2 occurred in the entire outer hair cell cytoplasm, in the inner hair cells (?), in the nerve fibres and in the cytoplasm of epithelial cells of the spiral prominence. In addition, staining for MAP-2 was identified in many (but not all) cells or Reissner's membrane. Immunofluorescence for MAP-1 occurred in the supporting cells of the cristae and maculae interpreted to be localized in the apical region adjacent to the sensory cells. Thus, the distribution of MAP-1 and MAP-2 in the adult human membranous labyrinth was the same as described for several animal species with regard to the cochlea. In contrast to such a pattern, both MAP-1 and MAP-2 were identified in the human vestibular organs, thus identifying a subpopulation of centrally located nerve calyces and possibly also the apical portion of vestibular hair cells.

Autoradiographic demonstration of deoxyglucose uptake in guinea pig cochlea.

The 2-deoxyglucose autoradiographic method was applied to whole-body cryosectioning to include the cochlea. The highest levels of 2-deoxyglucose uptake were observed in the vascular stria, spiral ligament and spiral prominence. The cochlear nerve showed the next highest level of uptake, while the organ of Corti and the spiral ganglion showed low levels. The functional significance of the results was briefly discussed.