Distribution and change of peroxynitrite in the guinea pig cochlea following noise exposure.

Nitric oxide (NO)-mediated pathology depends on the formation of reactive intermediates, such as the peroxynitrite (ONOO-). ONOO- can nitrate free tyrosine and tyrosine residues of proteins. Therefore, increases in tyrosine nitration reflect the amount of ONOO- produced by oxidative stress. The distribution of 3-nitrotyrosine (3-NT), an ONOO- marker, in the organ of corti and the cochlear lateral wall tissue from the guinea pig were examined using fluorescence immunohistochemistry. The immunoactivity of 3-NT in the normal guinea pig was compared with animals exposed to 122dBA broadband noise, 4 h/day, for 2 consecutive days. In the normal animals, 3-NT immunoreactivity was found in the outer hair cells (OHCs), inner hair cells (IHCs), pillar cells (PCs), spiral ganglion cells (SPCs) and the marginal cells of stria vascularis in the lateral wall. Sound exposure increased the 3-NT signal in all of the cells and resulted in extensive outer hair cell loss. A quantitative analysis of the 3-NT change in OHCs and marginal cells of lateral wall showed that immunolabeling was significant (P<0.01, n=10) in the noise exposure group compared with that of the control group. Anti-3-NT and propidium iodide double labeling showed that 3-NT was distributed mainly in the apical end of OHCs. In addition, 3-NT was distributed outside of the nucleus of the OHCs and marginal cells. In conclusion, the data indicate that noise exposure leads to a significant production of ONOO- in the cochlear lateral wall and organ of corti. This is consistent with the known increase of NO production by loud sound stress and suggests that NO-derived free radicals participate in the cochlear pathophysiology of noise-induced hearing loss.

A mutation in SLC22A4 encoding an organic cation transporter expressed in the cochlea strial endothelium causes human recessive non-syndromic hearing loss DFNB60.

The high prevalence/incidence of hearing loss (HL) in humans makes it the most common sensory defect. The majority of the cases are of genetic origin. Non-syndromic hereditary HL is extremely heterogeneous. Genetic approaches have been instrumental in deciphering genes that are crucial for auditory function. In this study, we first used NADf chip to exclude the implication of known North-African mutations in HL in a large consanguineous Tunisian family (FT13) affected by autosomal recessive non-syndromic HL (ARNSHL). We then performed genome-wide linkage analysis and assigned the deafness gene locus to ch:5q23.2-31.1, corresponding to the DFNB60 ARNSHL locus. Moreover, we performed whole exome sequencing on FT13 patient DNA and uncovered amino acid substitution p.Cys113Tyr in SLC22A4, a transporter of organic cations, cosegregating with HL in FT13 and therefore the cause of ARNSHL DFNB60. We also screened a cohort of small Tunisian HL families and uncovered an additional deaf proband of consanguineous parents that is homozygous for p.Cys113Tyr carried by the same microsatellite marker haplotype as in FT13, indicating that this mutation is ancestral. Using immunofluorescence, we found that Slc22a4 is expressed in stria vascularis (SV) endothelial cells of rodent cochlea and targets their apical plasma membrane. We also found Slc22a4 transcripts in our RNA-seq library from purified primary culture of mouse SV endothelial cells. Interestingly, p.Cys113Tyr mutation affects the trafficking of the transporter and severely alters ergothioneine uptake. We conclude that SLC22A4 is an organic cation transporter of the SV endothelium that is essential for hearing, and its mutation causes DFNB60 form of HL.

Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba(2+) 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K(+), while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K(+) typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba(2+)-sensitive inward rectifier K(+) (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.

Noise-induced nitrotyrosine increase and outer hair cell death in guinea pig cochlea.

BACKGROUND: Modern research has provided new insights into the biological mechanisms of noise-induced hearing loss, and a number of studies showed the appearance of increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) during and after noise exposure. This study was designed to investigate the noise exposure induced nitrotyrosine change and the mechanism of outer hair cells death in guinea pig cochlea. METHOD: Thirty guinea pigs were used in this study. The experimental animals were either exposed for 4 hours per day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused cochleae with 5 mg/ml of the SIN1 solutions, an exogenous NO and superoxide donor, for 30 minutes. Then the cochleae of the animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. The distribution of nitrotyrosine (NT) in the organ of Corti and the cochlear lateral wall tissue from the guinea pigs were examined using fluorescence immunohistochemistry method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. RESULTS: Either after noise exposure or after SIN1 perfusion, outer hair cells (OHCs) death with characteristics of both apoptotic and necrotic degradation appeared. Nitrotyrosine immunolabeling could be observed in the OHCs from the control animals. After noise exposure, NT immunostaining became much greater than the control animals in OHCs. The apoptotic OHC has significant increase of nitrotyrosine in and around the nucleus following noise exposure. In the normal later wall of cochleae, relatively weak nitrotyrosine immunolabeling could be observed. After noise exposure, nitrotyrosine immunoactivity became stronger in stria vascularis. CONCLUSION: Noise exposure induced increase of nitrotyrosine production is associated with OHCs death suggesting reactive nitrogen species participation in the cochlear pathophysiology of noise-induced hearing loss.

[The primary evaluation of minimal prepared ceramic veneer in anterior teeth for clinical application].

OBJECTIVE: To evaluate the clinical application value of minimal prepared ceramic veneer in anterior teeth, by analyzing the esthetic effects and success rates. METHODS: Forty-four anterior teeth in 30 patients with minor esthetic defect were included in this study. Less than 0.5 mm preparation or no preparaion technique was used in the clinical procedure. Glass ceramics veneers were delivered and 3M Relyx Veneer were used as the adhesive. The final appearance of each restoration was evaluated by patients on visual analogue scales (VAS) and by professional prosthodontists. Evaluation criteria included margin effects, color, shape and translucency. The success rate of all the restoration were analyzed in 6, 12 and 24 month after the treatment. RESULTS: The patients' degree of satisfaction was 9.2 ± 0.4, while the excellent rate of esthetic effect of margin effect, color, shape and translucency was 89% (39/44), 91% (40/44), 98% (43/44) and 93% (41/44) by professional prosthodontist. The success rate of 6, 12 and 24 month were 100% (44/44), 98% (43/44) and 91% (40/44). CONCLUSIONS: The minimal prepared venneers have a good esthetic effect and a satisfactory success rate, and is a suitable technique in esthetic treatment under the critical indications.

[The influence of toothguide training box on shade matching veracity].

PURPOSE: The aim of the study was to evaluate the influence of toothguide training box(TTB) on shade matching veracity, and compare the differences of influence on shade matching veracity of tabs of shadeguide and on that of tabs out of shadeguide. METHODS: 31 graduated students who had 1 to 5-year clinical experience without color blindness were trained by TTB once a week for 3 weeks. Elapsed time and scores from the training system were recorded each time. And everyone was tested by 29 tabs of Vita 3D-Master shadeguide and 7 tabs out of Vita 3D-Master shadeguide before the first training as the base line and tested again after the last training as the test after training. SPSS 10.0 software package was used for analysis. The variances of scores and elapsed time were analyzed by ANOVA, and the differences of veracity between tabs of shadeguide and that of tabs out of shadeguide by Wilcoxon signed ranks test. RESULTS: The scores of the tests were rising via training. There was significant difference (P<0.01) between the first (900.29+/-51.68) and the third training (933.81+/-32.94). The elapsed time was turning shorter via training. There is significant difference (P<0.01) between the first [(46.29+/-13.29)min] and the second training [(32.68+/-8.81)min], and also between the first and the third training (30.00+/-7.07)min. The veracity of test after training was higher than the veracity of base line. A significant difference (P<0.01) of shade matching veracity was demonstrated between base line and test after training in both tabs of shadeguide (60.37%+/-18.33%,46.08%+/-22.04%) and the total (37.10%+/-11.58%,28.34%+/-11.73%), but that difference in tabs out of shadeguide (13.82+/-14.98%,10.60%+/-11.65%) was not significant(P>0.01). CONCLUSION: TTB can improve the shade matching ability of prosthodontists, but has limited influence on the shade matching ability of tabs out of shadeguide.

[Caspase 3 activation and apoptosis inducing factor translocation in noise exposure induced out hair cells apoptosis].

OBJECTIVE: To investigate the pathway and mechanism of noise exposure induced out hair cells (OHC) apoptosis. METHODS: The cochleae of control and noise exposure group were dissected. The activity of caspase 3, an important mediator of apoptosis, in OHC, was examined with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The apoptosis inducing factor (AIF) translocation from mitochondria in OHC were further examined by immunohistology method. The nuclei were labeled with PI and the mitochondrion was labeled with Mito-tracker. Whole mount organ of Corti was prepared. Morphological and fluorescent change was observed use confocal microscope. RESULTS: In the normal OHC, AIF is distributed where the mitochondria were located and no activated caspase 3 was observed. After the animals exposed to broadband noise at 122 dB in 4 h/day for 2 days, both apoptosis and necrosis were appeared in OHC. AIF translocated from mitochondrion to nuclei in apoptotic and necrotic OHC following noise exposure. The noise exposure triggered activation of caspase 3 in apoptic hair cells. But no caspase 3 activation appeared in necrotic OHC. CONCLUSIONS: These findings indicated that the caspase-dependent pathway is an important pathway in noise exposure induced apoptosis. And AIF also involves OHC death pathway following noise exposure.

Dihydropyridines inhibit acetylcholine-induced hyperpolarization in cochlear artery via blockade of intermediate-conductance calcium-activated potassium channels.

Acetylcholine (ACh) induces hyperpolarization and dilation in a variety of blood vessels, including the cochlear spiral modiolar artery (SMA) via the endothelium-derived hyperpolarization factor (EDHF). We demonstrated previously that the ACh-induced hyperpolarization in the SMA originated in the endothelial cells (ECs) by activating a Ca(2+)-activated K(+) channel (K(Ca)); the hyperpolarization in smooth muscle cells was mainly an electrotonic spread via gap junction coupling. In the present study, using intracellular recording, immunohistology, and vascular diameter tracking techniques on in vitro SMA preparations, we found that 1) ACh-induced hyperpolarization was suppressed by intermediate-conductance K(Ca) (IK) blockers clotrimazole (IC(50) = 116 nM) and nitrendipine and by the calmodulin antagonist trifluoperazine, but it was not suppressed by the big-conductance K(Ca) blocker iberiotoxin. The immunoreactivity to anti-SK4/IK1 antibody was localized mainly in ECs. 2) The three dihydropyridines--nifedipine, nitrendipine, and nimodipine--all concentration-dependently inhibited the ACh-induced hyperpolarization, with an IC(50) value of 455, 34, and 3.2 nM, respectively. 3) Among other L-type Ca(2+) channel (I(L)) blockers, 10 microM verapamil exerted a 20% inhibition on ACh-induced hyperpolarization, whereas diltiazem and the metal ion Ca(2+) channel blockers Cd(2+) and Ni(2+) had no effect. 4) Nitrendipine and charybdotoxin abolished ACh-induced dilation in the SMA. We conclude that ACh-induced hyperpolarization in the SMA is generated mainly by activation of the IK in the ECs, and dihydropyridines suppress the EDHF-mediated hyperpolarization by blocking the IK channel, not the I(L) channel. The clinical relevance of this dihydropyridine action is discussed.