Oxidative stress and inflammation cause auditory system damage via glial cell activation and dysregulated expression of gap junction proteins in an experimental model of styrene-induced oto/neurotoxicity.

BACKGROUND: Redox imbalance and inflammation have been proposed as the principal mechanisms of damage in the auditory system, resulting in functional alterations and hearing loss. Microglia and astrocytes play a crucial role in mediating oxidative/inflammatory injury in the central nervous system; however, the role of glial cells in the auditory damage is still elusive. OBJECTIVES: Here we investigated glial-mediated responses to toxic injury in peripheral and central structures of the auditory pathway, i.e., the cochlea and the auditory cortex (ACx), in rats exposed to styrene, a volatile compound with well-known oto/neurotoxic properties. METHODS: Male adult Wistar rats were treated with styrene (400 mg/kg daily for 3 weeks, 5/days a week). Electrophysiological, morphological, immunofluorescence and molecular analyses were performed in both the cochlea and the ACx to evaluate the mechanisms underlying styrene-induced oto/neurotoxicity in the auditory system. RESULTS: We showed that the oto/neurotoxic insult induced by styrene increases oxidative stress in both cochlea and ACx. This was associated with macrophages and glial cell activation, increased expression of inflammatory markers (i.e., pro-inflammatory cytokines and chemokine receptors) and alterations in connexin (Cxs) and pannexin (Panx) expression, likely responsible for dysregulation of the microglia/astrocyte network. Specifically, we found downregulation of Cx26 and Cx30 in the cochlea, and high level of Cx43 and Panx1 in the ACx. CONCLUSIONS: Collectively, our results provide novel evidence on the role of immune and glial cell activation in the oxidative/inflammatory damage induced by styrene in the auditory system at both peripheral and central levels, also involving alterations of gap junction networks. Our data suggest that targeting glial cells and connexin/pannexin expression might be useful to attenuate oxidative/inflammatory damage in the auditory system.

Hearing dysfunction heralds an increase in non-motor burden and a worse quality of life in Parkinson's disease: new insights from non-motor spectrum.

BACKGROUND: Sensorial non-motor symptoms (NMSs) in Parkinson's disease (PD) still lack appropriate investigation in clinical practice. This study aimed to assess if and to what extent auditory dysfunction is associated with other NMSs in PD and its impact on patient's quality of life (QoL). METHODS: We selected patients with idiopathic PD, without other concomitant neurological diseases, dementia, or diagnosis of any audiological/vestibular disease. Demographic and clinical data were collected. Patients underwent otoscopic examination, audiological testing with pure tone audiometry (PTA) and distortion product otoacoustic emissions (DPOAEs) and completed Non-Motor Symptoms Scale (NMSS) and Parkinson's Disease Questionnaires-39 (PDQ-39). ANCOVA and partial correlation analysis have been used for statistical analysis. RESULTS: 60 patients were enrolled and completed PTA and DPOAEs. 32 patients with hearing impairment (HI), assessed by PTA, (hearing threshold ≥ 25 dB) showed similar disease duration, motor impairment, and staging, compared to patients without HI, but higher scores both in NMSS and in PDQ-39, except for cardiovascular (CV), gastrointestinal (GI), urogenital (U) and sexual function (SF) of NMSS. In addition, DPOAEs showed a significant correlation with higher scores both in NMSS and PDQ-39, except for CV, SF, GI, U and perceptual problem subdomains of NMSS. CONCLUSION: This study demonstrated that PD patients with HI have a greater burden of NMS and lower related QoL and functioning. Our results highlight the importance to reconsider HI as a NMS, in parallel with the others. HI evaluation, even in asymptomatic patients, may reveal a wider pathology with a worse QoL.

Crucial 3-D viscous hydrodynamic contributions to the theoretical modeling of the cochlear response.

This study uses a 3-D representation of the cochlear fluid to extend the results of a recent paper [Sisto, Belardinelli, and Moleti (2021b). J. Acoust. Soc. Am. 150, 4283-4296] in which two hydrodynamic effects, pressure focusing and viscous damping of the BM motion, both associated with the sharp increase in the wavenumber in the peak region, were analyzed for a 2-D fluid, coupled to a standard 1-D transmission-line WKB approach to cochlear modeling. The propagation equation is obtained from a 3-D fluid volume conservation equation, yielding the focusing effect, and the effect of viscosity is represented as a correction to the local 1-D admittance. In particular, pressure focusing amplifies the BM response without modifying the peak admittance, and viscous damping determines the position of the response peak counteracting focusing, as sharp gradients of the velocity field develop. The full 3-D WKB formalism is necessary to represent satisfactorily the behavior of the fluid velocity field near the BM-fluid interface, strictly related to viscous losses. As in finite element models, a thin layer of fluid is effectively attached to the BM due to viscosity, and the viscous force associated with the vertical gradient of the fluid vertical velocity acts on the BM through this layer.

Low-passed outer hair cell response and apical-basal transition in a nonlinear transmission-line cochlear model.

The low-pass characteristic of the outer hair cell (OHC) voltage response to mechanical stimulation could be considered a serious problem for cochlear models aiming at explaining high-frequency active amplification by introducing instantaneous nonlinear terms because active gain would dramatically decrease at high frequency. Evidence from experimental studies by Nam and Fettiplace [(2012). PloS One 7, e50572] suggests that the local cutoff frequency significantly increases approaching the cochlear base, somehow mitigating this problem. In this study, low-pass filtering of an internal force term, derived from a physiologically plausible OHC schematization by Lu, Zhak, Dallos, and Sarpeshkar [(2006). Hear. Res. 214, 45-67] is included in a simple one-dimensional (1-D) two-degrees-of-freedom transmission-line model by Sisto, Shera, Altoè, and Moleti [(2019). J. Acoust. Soc. Am. 146, 1685-1695] The frequency dependence of the low-pass filter phase-shift naturally yields a transition from sharp tuning and wide dynamical gain range in the basal cochlea to low tuning and poor dynamical range in the apical region. On the other hand, the frequency-dependent attenuation of low-pass filtering makes it more difficult to obtain the high gain (40-50 dB) of the basal basilar membrane response that is experimentally measured in mammals at low stimulus levels. Pressure focusing in the short-wave resonant region, which is not accounted for in this 1-D model, may help in acquiring the additional gain necessary to match the experimental data.

Fluid focusing and viscosity allow high gain and stability of the cochlear response.

This paper discusses the role of two-dimensional (2-D)/three-dimensional (3-D) cochlear fluid hydrodynamics in the generation of the large nonlinear dynamical range of the basilar membrane (BM) and pressure response, in the decoupling between cochlear gain and tuning, and in the dynamic stabilization of the high-gain BM response in the peak region. The large and closely correlated dependence on stimulus level of the BM velocity and fluid pressure gain [Dong, W., and Olson, E. S. (2013). Biophys. J. 105(4), 1067-1078] is consistent with a physiologically oriented schematization of the outer hair cell (OHC) mechanism if two hydrodynamic effects are accounted for: amplification of the differential pressure associated with a focusing phenomenon, and viscous damping at the BM-fluid interface. The predictions of the analytical 2-D Wentzel-Kramers-Brillouin (WKB) approach are compared to solutions of a 3-D finite element model, showing that these hydrodynamic phenomena yield stable high-gain response in the peak region and a smooth transition among models with different effectiveness of the active mechanism, mimicking the cochlear nonlinear response over a wide stimulus level range. This study explains how an effectively anti-damping nonlinear outer hair cells (OHC) force may yield large BM and pressure dynamical ranges along with an almost level-independent admittance.

Cochlear tuning estimates from level ratio functions of distortion product otoacoustic emissions.

Objective: Distortion product otoacoustic emission (DPOAE) levels plotted as a function of stimulus frequency ratio demonstrate a bandpass shape. This bandpass shape is narrower at higher frequencies compared to lower frequencies and thus has been thought to be related to cochlear mechanical tuning.Design: However, the frequency- and level-dependence of these functions above 8 kHz is largely unknown. Furthermore, how tuning estimates from these functions are related to behavioural tuning is not fully understood.Study Sample: From experiment 1, we report DPOAE level ratio functions (LRF) from seven normal-hearing, young-adults for f2 = 0.75-16 kHz and two stimulus levels of 62/52 and 52/37 dB FPL. We found that LRFs became narrower as a function of increasing frequency and decreasing level.Results: Tuning estimates from these functions increased as expected from 1-8 kHz. In experiment 2, we compared tuning estimates from DPOAE LRF to behavioural tuning in 24 normal-hearing, young adults for 1 and 4 kHz and found that behavioural tuning generally predicted DPOAE LRF estimated tuning.Conclusions: Our findings suggest that DPOAE LRFs generally reflect the tuning profile consistent with basilar membrane, neural, and behavioural tuning. However, further investigations are warranted to fully determine the use of DPOAE LRF as a clinical measure of cochlear tuning.

Suppression tuning curves in a two-degrees-of-freedom nonlinear cochlear model.

A two-degrees-of-freedom nonlinear cochlear model [Sisto, Shera, Altoè, and Moleti (2019). J. Acoust. Soc. Am. 146, 1685-1695] correctly predicts that the reticular lamina response is nonlinear over a wide basal region. Numerical simulations of suppression tuning curves agree with a recent experiment [Dewey, Applegate, and Oghalai (2019). J. Neurosci. 39, 1805-1816], supporting the idea that the strong susceptibility of the reticular lamina response to suppression by high-frequency tones does not imply that the total traveling wave energy builds-up in correspondingly basal regions. This happens because the reticular lamina is the lightest element of a coupled-oscillators system, only indirectly coupled to the differential pressure.

Distortion product otoacoustic emission sensitivity to different solvents in a population of industrial painters.

Objective: To evaluate the ototoxic effect of the exposure to different organic solvents and noise using distortion product otoacoustic emissions (DPOAEs).Design: The exposure to different solvents was evaluated by measuring, before and at the end of the work-shift, the urinary concentrations of solvent metabolites used as dose biomarkers. The urinary concentrations of DNA and RNA oxidation products were also measured as biomarkers of oxidative damage. The simultaneous exposure to noise was also evaluated. DPOAEs and pure tone audiometry (PTA) were used as outcome variables, and were correlated to the exposure variables using mixed effect linear regression models.Study sample: Seventeen industrial painters exposed to a solvent mixture in a naval industry. A sample size of 15 was estimated from previous studies as sufficient for discriminating small hearing level and DPOAE level differences (5 dB and 2 dB, respectively) at a 95% confidence level.Results: Statistically significant associations were found between the DPOAE level and the urinary dose biomarkers and the oxidative damage biomarkers. DPOAE level and the logarithm of the metabolite concentration showed a significant negative correlation.Conclusions: DPOAE are sensitive biomarkers of exposure to ototoxic substances and can be effectively used for the early detection of hearing dysfunction.

Environmental ototoxicants, a potential new class of chemical stressors.

Hearing loss is an injury that can develop over time, and people may not even be aware of it until it becomes a severe disability. Ototoxicants are substances that may damage the inner ear by either affecting the structures in the ear itself or by affecting the nervous system. We have examined the possibility that ototoxicants may present a health hazard in association with environmental exposures, adding to existing knowledge of their proven hazards under medical therapeutic conditions or occupational activities. In addition to the already described human environmental ototoxicants, mainly organochlorines such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), hexachlorocyclohexane (HCH) and hexachlorobenzene (HCB), we have examined the ubiquitous chemical stressors phthalates, bisphenol A/S/F/, PFCs, flame retardants (FRs) and cadmium for potential ototoxic properties, both as single substances or as chemical mixtures. Our literature review confirmed that these chemicals may disturb thyroid hormones homeostasis, activate aryl hydrocarbon receptor (AhR), and induce oxidative stress, which in turn may initiate a chain of events resulting in impairment of cochlea and hearing loss. With regard to auditory plasticity, diagnostics of a mixture of effects of ototoxicants, potential interactions of chemical and physical agents with effects on hearing, parallel deterioration of hearing due to chemical exposures and ageing, metabolic diseases or obesity, even using specific methods as brainstem auditory evoked potentials (BAEP) or otoacoustic emissions (OAEs) registration, may be difficult, and establishment of concentration-response relationships problematic. This paper suggests the establishment of a class of environmental oxotoxicants next to the established classes of occupational and drug ototoxicants. This will help to properly manage risks associated with human exposure to chemical stressors with ototoxic properties and adequate regulatory measures.

Constraints imposed by zero-crossing invariance on cochlear models with two mechanical degrees of freedom.

The zero crossings of basilar-membrane (BM) responses to clicks are nearly independent of stimulus intensity. This work explores the constraints that this invariance imposes on one-dimensional nonlinear cochlear models with two degrees of freedom (2DoF). The locations of the poles and zeros of the BM admittance, calculated for a set of linear models in which the strength of the active force is progressively decreased, provides a playground for evaluating the behavior of a corresponding nonlinear model at increasing stimulus levels. Mathematical constraints on the model parameters are derived by requiring that the poles of the admittance move horizontally in the s-plane as the active force is varied. These constraints ensure approximate zero-crossing invariance over a wide stimulus level range in a nonlinear model in which the active force varies as a function of the local instantaneous BM displacement and velocity. Two different 2DoF models are explored, each capable of reproducing the main qualitative characteristics of the BM response to tones (i.e., the tall and broad activity pattern at low stimulus levels, the large gain dynamics, and the partial decoupling between gain and phase). In each model, the motions of the two masses are compared with response data from animal experiments.

Intensimetric detection of distortion product otoacoustic emissions with ear canal calibration.

Distortion product otoacoustic emissions (DPOAEs) have been accurately measured with an intensimetric technique, involving simultaneous measure of pressure and velocity in the ear canal, which allows one to correctly calibrate both the input stimuli and the otoacoustic emission (OAE) level. Suitable combinations of standard intensimetric quantities, active intensity and power density [Stanzial, Shiffrer, and Sacchi, J. Acoust. Soc. Am. 131, 269-280 (2012)], are used to equalize the stimuli transmitted to the middle ear, and to estimate the DPOAE level emitted by the eardrum. The DPOAE intensimetric spectra are consistent with those recorded with a high-quality conventional otoacoustic probe with state-of-the-art calibration of both stimulus and OAE response [Charaziak and Shera, J. Acoust. Soc. Am. 141, 515-525 (2017)], demonstrating the applicability of the intensimetric method to OAE measurements.

PCB exposure and cochlear function at age 6 years.

Epidemiological studies have documented adverse associations between exposure to polychlorinated biphenyls (PCBs) and otological outcomes. Previously, we documented decreased distortion product otoacoustic emission (DPOAE) levels in children exposed to PCBs, up to the age of 45 months, amongst a cohort of children in eastern Slovakia. The objective of the present study is to evaluate cochlear dysfunction at 72 months of age in 214 children from this same cohort and to compare the otoacoustic test sensitivity to that of pure tone audiometry (PTA). The association between DPOAE, PTA, and PCBs was estimated by means of multivariate ANOVA (MANOVA) and linear regression models. ROC curves were computed to estimate the DPOAE-test power in children. The DPOAE level at 72 months was related to PCB-153 serum levels. The DPOAE Input/Output function test at mid-frequency (2kHz) has shown instead nonmonotonic dependence on PCB exposure, for the left ears of children, over the whole growth curve. No significant association was found between PTA hearing levels and PCB-153 concentration. High diagnostic power of the DPOAE-test was found in children, similar to that found by the same authors in adults. In conclusions the DPOAE-PCB correlation obtained at 72 months is similar to that at 45 months suggesting a permanent and stable ototoxic effect of the PCB exposure. The lack of statistical significance of the PCB-PTA correlation suggests that DPOAEs are sensitive biomarkers of cochlear damage.

Estimating cochlear tuning dependence on stimulus level and frequency from the delay of otoacoustic emissions.

An objective technique based on the time-frequency analysis of otoacoustic emissions is proposed to get fast and stable estimates of cochlear tuning. Time-frequency analysis allows one to get stable measurements of the delay/frequency function, which is theoretically expected to be a function of cochlear tuning. Theoretical considerations and numerical solutions of a nonlinear cochlear model suggest that the average phase-gradient delay of the otoacoustic emission single-reflection components, weighted, for each frequency, by the amplitude of the corresponding wavelet coefficients, approximately scales as the square root of the cochlear quality factor. The application of the method to human stimulus-frequency and transient-evoked otoacoustic emissions shows that tuning decreases approximately by a factor of 2, as the stimulus level increases by 30 dB in a moderate stimulus level range. The results also show a steady increase of tuning with increasing frequency, by a factor of 2 between 1 and 5 kHz. This last result is model-dependent, because it relies on the assumption that cochlear scale-invariance breaking is only due to the frequency dependence of tuning. The application of the method to the reflection component of distortion product otoacoustic emissions, separated using time-frequency filtering, is complicated by the necessity of effectively canceling the distortion component.

Distortion product otoacoustic emission generation mechanisms and their dependence on stimulus level and primary frequency ratio.

In this study, a systematic analysis of the dependence on stimulus level and primary frequency ratio r of the different components of human distortion product otoacoustic emissions has been performed, to check the validity of theoretical models of their generation, as regards the localization of the sources and the relative weight of distortion and reflection generation mechanisms. 2f1 - f2 and 2f2 - f1 distortion product otoacoustic emissions of 12 normal hearing ears from six human subjects have been measured at four different levels, in the range [35, 65] dB sound pressure level, at eight different ratios, in the range [1.1, 1.45]. Time-frequency filtering was used to separate distortion and reflection components. Numerical simulations have also been performed using an active nonlinear cochlear model. Both in the experiment and in the simulations, the behavior of the 2f1 - f2 distortion and reflection components was in agreement with previous measurements and with the predictions of the two-source model. The 2f2 - f1 response showed a rotating-phase component only, whose behavior was in general agreement with that predicted for a component generated and reflected within a region basal to the characteristic place of frequency 2f2 - f1, although alternative interpretations, which are also discussed, cannot be ruled out.

Decoupling the level dependence of the basilar membrane gain and phase in nonlinear cochlea models.

In animal experiments, the strong dependence on stimulus level of the basilar membrane gain and tuning is not matched by a corresponding change in the phase slope in the resonant region. Linear models, in which the gain dependence on the stimulus level has to be schematized by explicitly changing the tuning parameters of the resonant model, do not easily match this feature of the experimental data. Nonlinear models predict a phase slope that is relatively decoupled from tuning. In addition, delayed-stiffness and feed-forward models also show a significant intrinsic decoupling between gain and tuning, which helps in matching the experimental data.

On the spatial distribution of the reflection sources of different latency components of otoacoustic emissions.

The experimental observation of long- and short-latency components in both stimulus-frequency and transient-evoked otoacoustic emissions admits a comprehensive explanation within the coherent reflection mechanism, in a linear active transmission-line cochlear model. A local complex reflectivity function associated with roughness was defined and analyzed by varying the tuning factor of the model, systematically showing, for each frequency, a multiple-peak spatial structure, compatible with the observed multiple-latency structure of otoacoustic emissions. Although this spatial pattern and the peak relative intensity changes with the chosen random roughness function, the multiple-peak structure is a reproducible feature of different "digital ears," in good agreement with experimental data. If one computes the predicted transmission delays as a function of frequency and position for each source, one gets a good match to the latency-frequency patterns that are directly computed from synthesized otoacoustic spectra using time-frequency analysis. This result clarifies the role of the spatial distribution of the otoacoustic emission sources, further supporting the interpretation of different-latency otoacoustic components as due to reflection sources localized at different places along the basilar membrane.

Exposure to airborne culturable microorganisms and endotoxin in two Italian poultry slaughterhouses.

Even if slaughterhouses' workers handle large amounts of organic material and are potentially exposed to a wide range of biological agents, relatively little and not recent data are available. The main objective of this study was to characterize indoor concentrations of airborne bacteria, fungi, and endotoxin mod = Im (endotoxin∼Gram-negative*plant*filter) in two Italian poultry slaughterhouses. Air samples near air handling units inlets were also collected. Since there are not standardized protocols for endotoxin sampling and extraction procedures, an additional aim of the study was to compare the extraction efficiency of three different filter.. The study was also aimed at determining the correlation between concentrations of Gram-negative bacteria and endotoxin. In Plant A bacterial levels ranged from 17.5 to 2.6×10(3) CFU/m3. The highest concentrations were observed in evisceration area of chickens, between the automatic detachment of the neck and washing offal, and near birds coupling before hair-chilling. The highest mean value of Gram-negative (266.5 CFU/m3) was found near the washing offal of turkeys. In Plant B bacterial concentration ranged from 35 to 8×10(3) CFU/m3. The highest concentration. with the highest value of Gram-negative (248 CFU/m3), was found after defeathering. Fungal concentrations were overall lower than those found for bacteria (range: 0-205 CFU/m3 in Plant A and 0-146.2 CFU/m3 in Plant B). The microbial flora was dominated by Gram-negative and coagulase-negative staphylococci for bacteria and by species belonging to Cladosporium, Penicillium and Aspergillus genera for molds. The highest endotoxin concentrations were measured in washing offal for Plant A (range: 122.7-165.9 EU/m3) and after defeathering for Plant B (range: 0.83-38.85 EU/m3). In this study airborne microorganisms concentrations were lower than those found in similar occupational settings and below the occupational limits proposed by some authors. However, these microorganisms may exert adverse effects on exposed workers, in particular for those engaged in the early slaughtering stages, as evidenced by the presence of pathogenic species. The detection of pathogenic bacteria near AHU inlet may constitute a risk to public health and environmental pollution.

Fluid Focusing Contributes to the BM Vibration Amplification by Boosting the Pressure.

Two hydrodynamic effects are introduced in the standard transmission-line formalism, the focusing of the pressure and fluid velocity fields near the basilar membrane and the viscous damping at the fluid-basilar membrane interface, which significantly affect the cochlear response in the short-wave region. In this region, in which the wavelength is shorter than the cochlear duct height, only a layer of fluid of order of the wavelength is effectively involved in the traveling wave. This has been interpreted [8] as a reduced fluid contribution to the system inertia in the peak region, which is a viewpoint common to the 3-D FEM solutions. In this paper we propose an alternative approach, from a slightly different physical viewpoint. Invoking the fluid flux conservation along the traveling wave propagation direction, we can derive a rigorous propagation equation for the pressure integrated along the vertical axis. Consequently, the relation between the average pressure and the local pressure [4] at the fluid-BM interface can be written. The local pressure is amplified by a factor dependent on the local wavenumber with respect to the average pressure, a phenomenon we refer to as "fluid focusing", which plays a relevant role in the BM total amplification gain. This interpretation of the hydrodynamic boost to the pressure provides a physical justification to the strategy [10] of fitting the BM admittance with a polynomial containing both a conjugated pole and a zero. In the short-wave region, the sharp gradients of the velocity field yield a second important effect, a damping force on the BM motion, proportional to the local wavenumber, which stabilizes active models and shifts the peak of the response towards the base, with respect to the resonant place. This way, the peaked BM response is not that of a proper resonance, corresponding to a sharp maximum of the admittance, but rather a focusing-driven growth toward the resonant place, which is "aborted" before reaching it by the sharply increasing viscous losses. The large values of the wavenumber that ensure strong focusing are ultimately fueled, against viscosity, by the nonlinear OHC mechanism, hence the otherwise puzzling observation of a wide nonlinear gain dynamics with almost level-independent admittance.

Maternal exposure to zinc oxide nanoparticles causes cochlear dysfunction in the offspring.

Introduction: Zinc oxide nanoparticles (ZnO NPs) have been engineered and are largely used in material science and industry. This large and increasing use justifies a careful study about the toxicity of this material for human subjects. The concerns regard also the reproductive toxicity and the fetotoxicity. Materials and methods: The effect of the exposure to ZnO NPs on the cochlear function was studied in a group of pregnant CD1 mice and in their offspring. This study is part of a larger toxicological study about the toxicity of ZnO NPs during pregnancy. Four groups were analyzed and compared, exposed and non-exposed dams and their offspring. The cochlear function was quantitatively assessed by means of Distortion Product Otoacoustic Emissions (DPOAEs). Results and discussion: A large statistically significant difference was found between the non-exposed dams offspring and the exposed dams offspring (p = 1.6 · 10-3), whose DPOAE levels were significantly lower than those of non-exposed dams offspring and comparable to those of the adults. The DPOAE levels of the exposed and non-exposed dams were very low and not significantly different. This occurrence is related to the fact that these mice encounter a rapid aging process. Conclusion: Our findings show that maternal exposure to ZnO NPs does not reflect in overt toxicity on fetal development nor impair offspring birth, however it may damage the nervous tissue of the inner ear in the offspring. Other studies should confirm this result and identify the mechanisms through which ZnO NPs may affect ear development.