Inner ear is a target for insulin signaling and insulin resistance: evidence from mice and auditory HEI-OC1 cells.

OBJECTIVE: The mechanisms underlying the association between diabetes and inner ear dysfunction are not known yet. The aim of the present study is to evaluate the impact of obesity/insulin resistance on inner ear fluid homeostasis in vivo, and to investigate whether the organ of Corti could be a target tissue for insulin signaling using auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells as an in vitro model. METHODS: High fat diet (HFD) fed C57BL/6J mice were used as a model to study the impact of insulin resistance on the inner ear. In one study, 12 C57BL/6J mice were fed either control diet or HFD and the size of the inner ear endolymphatic fluid compartment (EFC) was measured after 30 days using MRI and gadolinium contrast as a read-out. In another study, the size of the inner ear EFC was evaluated in eight C57BL/6J mice both before and after HFD feeding, with the same techniques. HEI-OC1 auditory cells were used as a model to investigate insulin signaling in organ of Corti cells. RESULTS: HFD feeding induced an expansion of the EFC in C57BL/6J mice, a hallmark of inner ear dysfunction. Insulin also induced phosphorylation of protein kinase B (PKB/Akt) at Ser473, in a PI3-kinase-dependent manner. The phosphorylation of PKB was inhibited by isoproterenol and IBMX, a general phosphodiesterase (PDE) inhibitor. PDE1B, PDE4D and the insulin-sensitive PDE3B were found expressed and catalytically active in HEI-OC1 cells. Insulin decreased and AICAR, an activator of AMP-activated protein kinase, increased the phosphorylation at the inhibitory Ser79 of acetyl-CoA carboxylase, the rate-limiting enzyme in de novo lipogenesis. Furthermore, the activity of hormone-sensitive lipase, the rate-limiting enzyme in lipolysis, was detected in HEI-OC1 cells. CONCLUSIONS: The organ of Corti could be a target tissue for insulin action, and inner ear insulin resistance might contribute to the association between diabetes and inner ear dysfunction.

Extracellular Vesicles From Auditory Cells as Nanocarriers for Anti-inflammatory Drugs and Pro-resolving Mediators.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Preliminary Characterization of Extracellular Vesicles From Auditory HEI-OC1 Cells.

OBJECTIVES: Isolate, purify, and characterize extracellular vesicles (EVs) obtained from auditory HEI-OC1 cells, and evaluate their suitability for intracochlear transport and delivery of pharmacological drugs and/or pro-resolution mediators of acute inflammatory processes. METHODS: HEI-OC1 EVs were isolated and purified using the exoEasy Maxi Kit, and their size was evaluated by nanoparticle tracking techniques. Bottom-up proteomics of the EVs, either freshly obtained or stored for up to 4 months at -20°C, was performed by LC-ESI-MS/MS. LC-ESI-MS/MS-MRM was used to measure the loading of dexamethasone inside EVs following co-incubation at room temperature for 1 hour with and without 5 minutes sonication. RESULTS: Routinely, we were able to obtain purified fractions of >2 × 109 EVs/mL, with diameters varying between 50 and 800 nm. Bottom-up proteomics showed that among the most abundant EVs proteins, 19.2% were cytoplasmic, 17.2% were membrane localized, 12.3% were cytosolic, and 14.6% were nucleolar. No significant differences between fresh and stored EVs were detected. Importantly, co-incubation of HEI-OC1 EVs (1 × 108 EVs/mL) with dexamethasone (10 mM) resulted in the incorporation of 10.1 ± 1.9 nM dexamethasone per milliliter of EVs suspension. CONCLUSIONS: Altogether, the results suggest that EVs from HEI-OC1 cells could be advantageously used as biological nanocarriers for the delivery of specific molecules and pharmacological drugs into the inner ear.

Prevention of cisplatin-induced hearing loss by extended release fluticasone propionate intracochlear implants.

OBJECTIVE: Cisplatin is a chemotherapeutic drug known to induce hearing loss. Although corticosteroids may help to mitigate the ototoxic side effects of cisplatin, there are complications associated with their systemic and prolonged use. The goal of this study is to test the efficacy of extended-release fluticasone propionate intracochlear implant particles to protect against cisplatin-induced hearing loss. METHODS: We used guinea pigs (n = 9) injected with cisplatin (IP, 12 mg/kg weight). Fluticasone particles were delivered to the cochlear scala tympani through the round window membrane into the right ears of the guinea pigs (left ears being used as a control) two weeks prior to cisplatin administration, and hearing function was evaluated by ABR and DPOAE before implantation, immediately before cisplatin administration, and 2 weeks after the challenge with cisplatin. Data was statistically evaluated using paired t-test analysis. RESULTS: No significant differences were observed in ABR threshold between control and implanted ears on day 14 (23.9 ±â€¯2.3 dB vs. 25.6 ±â€¯1.3 dB, P = 0.524), whereas the significant cisplatin-induced hearing loss in control animals (23.9 ±â€¯2.3 dB at day 14 vs. 40.7 ±â€¯2.5 dB at day 28, P ≤ 0.0001) was prevented in implanted animals (25.6 ±â€¯1.3 dB at day 14 vs. 25.0 ±â€¯3.1 at day 28, P ≥ 0.85). A similar, though not statistically significant, trend was observed in DPOAE responses in untreated ears (7.9 ±â€¯5.8 dB at day14 vs. -0.5 ±â€¯5.3 dB at day 28, P = 0.654) as compared to treatment (11.1 ±â€¯3.4 dB at day 14 vs. 13.6 ±â€¯4.8 dB at day 28, P = 0.733). CONCLUSION: These results suggest that fluticasone intracochlear implants are safe and able to provide effective otoprotection against cisplatin-induced hearing loss in the guinea pig model.

A Polymer-Based Extended Release System for Stable, Long-term Intracochlear Drug Delivery.

OBJECTIVE: Investigate a new polymer-based drug coating suitability for safe intracochlear delivery and ability to maintain long-term physiologically active levels of the corticosteroid fluticasone propionate. STUDY DESIGN: In vitro dissolution study to evaluate release profiles of polymer-coated drug particles and in vivo studies using a guinea pig model to measure perilymph drug concentrations at specific time points after implantation with polymer-coated drug particles and evaluate their effect on hearing function. METHODS: Polymer-coated fluticasone propionate (FP) particles were surgically implanted in guinea pigs through the round window membrane into the cochlear scala tympani. In the pilot study, pre- and post-op hearing thresholds were conducted on days 7, 14, and 42. In a second study, post-op hearing thresholds were conducted on days 90, 120, and 180. Perilymph drug concentrations were measured on the same time points. RESULTS: In 15 of 16 animals from day 7 through day 90, drug levels were within the targeted range, with no initial burst release detected. Drug was present in all animals on day 90 and was detected in some animals at 120 and 180 days. Hearing was tested and compared with non-implanted ears. Very good hearing preservation was observed in ears implanted with intracochlear particles when compared with contralateral ears. CONCLUSIONS: The polymer-based extended release system is effective in providing long-term, stable drug delivery for at least 90 days with good hearing outcomes. The results of this study support the potential for achieving long-term drug delivery with a single intracochlear administration.

Resolution of Cochlear Inflammation: Novel Target for Preventing or Ameliorating Drug-, Noise- and Age-related Hearing Loss.

A significant number of studies support the idea that inflammatory responses are intimately associated with drug-, noise- and age-related hearing loss (DRHL, NRHL and ARHL). Consequently, several clinical strategies aimed at reducing auditory dysfunction by preventing inflammation are currently under intense scrutiny. Inflammation, however, is a normal adaptive response aimed at restoring tissue functionality and homeostasis after infection, tissue injury and even stress under sterile conditions, and suppressing it could have unintended negative consequences. Therefore, an appropriate approach to prevent or ameliorate DRHL, NRHL and ARHL should involve improving the resolution of the inflammatory process in the cochlea rather than inhibiting this phenomenon. The resolution of inflammation is not a passive response but rather an active, highly controlled and coordinated process. Inflammation by itself produces specialized pro-resolving mediators with critical functions, including essential fatty acid derivatives (lipoxins, resolvins, protectins and maresins), proteins and peptides such as annexin A1 and galectins, purines (adenosine), gaseous mediators (NO, H2S and CO), as well as neuromodulators like acetylcholine and netrin-1. In this review article, we describe recent advances in the understanding of the resolution phase of inflammation and highlight therapeutic strategies that might be useful in preventing inflammation-induced cochlear damage. In particular, we emphasize beneficial approaches that have been tested in pre-clinical models of inflammatory responses induced by recognized ototoxic drugs such as cisplatin and aminoglycoside antibiotics. Since these studies suggest that improving the resolution process could be useful for the prevention of inflammation-associated diseases in humans, we discuss the potential application of similar strategies to prevent or mitigate DRHL, NRHL and ARHL.

Working with Auditory HEI-OC1 Cells.

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 °C to stimulate cell proliferation and incubation at 39 °C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

HEI-OC1 cells as a model for investigating drug cytotoxicity.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few, and arguable the most used, mouse auditory cell line available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, it has been used to investigate, among other topics, apoptotic pathways, autophagy and senescence, mechanism of cell protection, inflammatory responses, cell differentiation, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. However, the use of different techniques with different goals resulted in apparent contradictions on the actual response of these cells to some specific treatments. We have now performed studies to characterize the actual response of HEI-OC1 cells to a battery of commonly used pharmacological drugs. We evaluated cell toxicity, apoptosis, viability, proliferation, senescence and autophagy in response to APAP (acetaminophen), cisplatin, dexamethasone, gentamicin, penicillin, neomycin, streptomycin, and tobramycin, at five different doses and two time-points (24 and 48 h), by flow cytometry techniques and caspase 3/7, MTT, Cytotoxicity, BrdU, Beclin1, LC3 and SA-ß-galactosidase assays. We also used HEK-293 and HeLa cells to compare some of the responses of these cells to those of HEI-OC1. Our results indicate that every cell line responds to the each drug in a different way, with HEI-OC1 cells showing a distinctive sensitivity to at least one of the mechanisms under study. Altogether, our results suggest that the HEI-OC1 might be a useful model to investigate biological responses associated with auditory cells, including auditory sensory cells, but a careful approach would be necessary at the time of evaluating drug effects.

HEI-OC1 cells as a model for investigating prestin function.

The House Ear Institute-Organ of Corti 1 (HEI-OC1) is a mouse auditory cell line that endogenously express, among other several markers of cochlear hair cells, the motor protein prestin (SLC26A5). Since its discovery fifteen years ago, and because of the difficulties associated with working with outer hair cells, prestin studies have been performed mostly by expressing it exogenously in non-specific systems such as HEK293 and TSA201, embryonic kidney cells from human origin, or Chinese Hamster Ovary (CHO) cells. Here, we report flow cytometry and confocal laser scanning microscopy studies on the pattern of prestin expression, as well as nonlinear capacitance (NLC) and whole cell-patch clamping studies on prestin motor function, in HEI-OC1 cells cultured at permissive and non-permissive conditions. Our results indicate that both total prestin expression and plasma membrane localization increase in a time-dependent manner when HEI-OC1 cells differentiate under non-permissive culture conditions. In addition, we demonstrate that HEI-OC1 cells have a robust NLC associated to prestin motor function, which decreases when the density of prestin molecules present at the plasma membrane increases. Altogether, our results show that the response of endogenously expressed prestin in HEI-OC1 cells is different from the response of prestin expressed exogenously in non-auditory cells, and suggest that the HEI-OC1 cell line may be an important additional tool for investigating prestin function.

PKCα-Mediated Signals Regulate the Motile Responses of Cochlear Outer Hair Cells.

There is strong evidence that changes in the actin/spectrin-based cortical cytoskeleton of outer hair cells (OHCs) regulate their motile responses as well as cochlear amplification, the process that optimizes the sensitivity and frequency selectivity of the mammalian inner ear. Since a RhoA/protein kinase C (PKC)-mediated pathway is known to inhibit the actin-spectrin interaction in other cell models, we decided to investigate whether this signaling cascade could also participate in the regulation of OHC motility. We used high-speed video microscopy and confocal microscopy to explore the effects of pharmacological activation of PKCα, PKCßI, PKCßII, PKCδ, PKCε, and PKCζ with lysophosphatidic acid (LPA) and their inhibition with bisindolylmaleimide I, as well as inhibition of RhoA and Rho-associated protein kinase (ROCK) with C3 and Y-27632, respectively. Motile responses were induced in isolated guinea pig OHCs by stimulation with an 8 V/cm external alternating electrical field as 50 Hz bursts of square wave pulses (100 ms on/off). We found that LPA increased expression of PKCα and PKCζ only, with PKCα, but not PKCζ, phosphorylating the cytoskeletal protein adducin of both Ser-726 and Thr-445. Interestingly, however, inhibition of PKCα reduced adducin phosphorylation only at Ser-726. We also determined that LPA activation of a PKCα-mediated signaling pathway simultaneously enhanced OHC electromotile amplitude and cell shortening, and facilitated RhoA/ROCK/LIMK1-mediated cofilin phosphorylation. Altogether, our results suggest that PKCα-mediated signals, probably via adducin-mediated inhibition of actin-spectrin binding and cofilin-mediated depolymerization of actin filaments, play an essential role in the homeostatic regulation of OHC motility and cochlear amplification.

Biology and pathobiology of lipid droplets and their potential role in the protection of the organ of Corti.

The current review article seeks to extend our understanding on the role of lipid droplets within the organ of Corti. In addition to presenting an overview of the current information about the origin, structure and function of lipid droplets we draw inferences from the collective body of knowledge about this cellular organelle to build a conceptual framework to better understanding their role in auditory function. This conceptual model considers that lipid droplets play a significant role in the synthesis, storage, and release of lipids and proteins for energetic use and/or modulating cell signaling pathways. We describe the role and mechanism by which LD play a role in human diseases, and we also review emerging data from our laboratory revealing the potential role of lipid droplets from Hensen cells in the auditory organ. We suggest that lipid droplets might help to develop rapidly and efficiently the resolution phase of inflammatory responses in the mammalian cochlea, preventing inflammatory damage of the delicate inner ear structures and, consequently, sensorineural hearing loss.

Acetaminophen and NAPQI are toxic to auditory cells via oxidative and endoplasmic reticulum stress-dependent pathways.

Pain relievers containing N-acetyl-para-aminophenol, also called APAP, acetaminophen or paracetamol, in combination with opioid narcotics are top-selling pharmaceuticals in the U.S. Individuals who abuse these drugs for as little as sixty days can develop tinnitus and progressive bilateral sensorineural hearing loss. Recently published studies indicate that APAP and its metabolic product N-acetyl-p-benzoquinoneimine (NAPQI) are the primary ototoxic agents in this type of pain relievers. However, the mechanisms underlying the deleterious effects of these drugs on auditory cells remain to be fully characterized. In this study, we report cellular, genomic, and proteomic experiments revealing that cytotoxicity by APAP and NAPQI involves two different pathways in Immortomouse-derived HEI-OC1 cells, implicating ROS overproduction, alterations in ER morphology, redistribution of intra-cisternal chaperones, activation of the eIF2α-CHOP pathway, as well as changes in ER stress and protein folding response markers. Thus, both oxidative and ER stress are part of the cellular and molecular mechanisms that contribute to the cytotoxic effects of APAP and NAPQI in these cells. We suggest that these in vitro findings should be taken into consideration when designing pharmacological strategies aimed at preventing the toxic effects of these drugs on the auditory system.

In vitro assessment of antiretroviral drugs demonstrates potential for ototoxicity.

Several studies have reported an increased incidence of auditory dysfunction among HIV/AIDS patients. We used auditory HEI-OC1 cells in cell viability, flow cytometry and caspases 3/7-activation studies to investigate the potential ototoxicity of fourteen HIV antiretroviral agents: Abacavir, AZT, Delavirdine, Didenosine, Efavirenz, Emtricitabine, Indinavir, Lamivudine, Nefinavir, Nevirapine, Tenofovir, Ritonavir, Stavudine and Zalcitabine, as well as combinations of these agents as used in the common anti-HIV cocktails Atripla™, Combivir™, Epzicom™, Trizivir™, and Truvada™. Our results suggested that most of the single assayed anti-HIV drugs are toxic for HEI-OC1 auditory cells. The cocktails, on the other hand, decreased auditory cells' viability with high significance, with the following severity gradient: Epzicom âˆ¼ Trizivir >> Atripla âˆ¼ Combivir > Truvada. Interestingly, our results suggest that Trizivir- and Epzicom-induced cell death would be mediated by a caspase-independent mechanism. l-Carnitine, a natural micronutrient known to protect HEI-OC1 cells against some ototoxic drugs as well as to decrease neuropathies associated with anti-HIV treatments, increased viability of cells treated with Lamivudine and Tenofovir as well as with the cocktail Atripla, but had only minor effects on cells treated with other drugs and drug combinations. Altogether, these results suggest that some frequently used anti-HIV agents could have deleterious effects on patients' hearing, and provide arguments in favor of additional studies aimed at elucidating the potential ototoxicity of current as well as future anti-HIV drugs.

Expression and dexamethasone-induced nuclear translocation of glucocorticoid and mineralocorticoid receptors in guinea pig cochlear cells.

Glucocorticoids (GC) are powerful anti-inflammatory agents frequently used to protect the auditory organ against damage associated with a variety of conditions, including noise exposure and ototoxic drugs as well as bacterial and viral infections. In addition to glucocorticoid receptors (GC-R), natural and synthetic GC are known to bind mineralocorticoid receptors (MC-R) with great affinity. We used light and laser scanning confocal microscopy to investigate the expression of GC-R and MC-R in different cell populations of the guinea pig cochlea, and their translocation to different cell compartments after treatment with the synthetic GC dexamethasone. We found expression of both types of receptors in the cytoplasm and nucleus of sensory inner and outer hair cells as well as pillar, Hensen and Deiters cells in the organ of Corti, inner and outer sulcus cells, spiral ganglion neurons and several types of spiral ligament and spiral limbus cells; stria vascularis cells expressed mostly MC-R whereas fibrocytes type IV were positive for GC-R only. GC-R and MC-R were also localized at or near the plasma membrane of pillar cells and outer hair cells, whereas GC-R were found at or near the plasma membrane of Hensen cells only. We investigated the relative levels of receptor expression in the cytoplasm and the nucleus of Hensen cells treated with dexamethasone, and found they varied in a way suggestive of dose-induced translocation. These results suggest that the oto-protective effects of GC could be associated with the concerted activation of genomic and non-genomic, GC-R and MC-R mediated signaling pathways in different regions of the cochlea.

Microdomains shift and rotate in the lateral wall of cochlear outer hair cells.

Outer hair cell (OHC) electromotility, a response consisting of reversible changes in cell length and diameter induced by electrical stimulation, confers remarkable sensitivity and frequency resolution to the mammalian inner ear. Looking for a better understanding of this mechanism, we labeled isolated guinea pig OHCs with microspheres and, using high-speed video recording, investigated their movements at the apical, mid, and basal regions of osmotically and electrically stimulated cells. After hypoosmotic challenge, OHCs shortened and their diameter increased, with microspheres moving always toward the central plane; iso-osmolarity returned OHCs to their original shape and microspheres to their original positions. Under electrical stimulation, microspheres exhibited robust movements, with their displacement vectors changing in direction from random to parallel to the longitudinal axis of the cells with peak reorientation speeds of up to 6 rad/s and returning to random after 5 min without stimulation. Alterations in plasma-membrane cholesterol levels as well as cytoskeleton integrity affected microsphere responses. We concluded that microspheres attach to different molecular microdomains, and these microdomains are able to shift and rotate in the plane of the OHC lateral wall with a dynamics tightly regulated by membrane lipid composition and the cortical cytoskeleton.

Deiters cells tread a narrow path--the Deiters cells-basilar membrane junction.

Deiters cells extend from the basilar membrane to the reticular lamina and, together with pillar cells and outer hair cells, structurally define the micro-architecture of the organ of Corti. Studying vibrotome sections of the mouse organ of Corti with confocal and scanning electron microscopy we found that the basal pole of every Deiters cell, independently of their position in the organ of Corti and along the cochlear spiral, attached to the basilar membrane within a 15.1 ± 0.3 µm-wide stripe running the length of the cochlear spiral adjacent to the row of outer pillar cells. All Deiters cells' basal poles had similar diameter and general morphology, and distributed on the stripe in a precise arrangement with a center-to-center distance of 7.1 ± 0.3 µm between neighbor cells of the same row and 5.9 ± 0.4 µm for neighbor cells in adjacent rows. Complete detachment of Deiters cells revealed an elliptical imprint on the top surface of the basilar membrane consisting of a smaller central structure with a very smooth surface surrounded by a rougher area, suggesting the presence of two different anchoring junctions. These previously unidentified morphological features of Deiters cells could be critical for the mechanical response of the organ of Corti.

In vitro and in vivo models of drug ototoxicity: studying the mechanisms of a clinical problem.

INTRODUCTION: Drug ototoxicity represents one of the main preventable causes of deafness. Ototoxicity is a trait shared by aminoglycoside and macrolide antibiotics, antimalarial medications, loop diuretics, platinum-based chemotherapeutic agents, some NSAIDs and most recently described, acetaminophen when abused with narcotic medication. These medications are prescribed despite their side effects, which includes inner ear toxicity, because they are life-saving drugs or there is a lack of better treatment. AREAS COVERED: This review will discuss in vitro and in vivo models of ototoxicity highlighting recently published ototoxicity research. The reader will learn the strengths and limitations of different ototoxicity models and what molecular insights have been gained from their application. A better understanding of the cellular mechanisms of these ototoxins will help in the discovery of ways to prevent and treat hearing loss associated with ototoxic medications. EXPERT OPINION: There are benefits to both in vitro and in vivo models of ototoxicity. Research of a particular medication and its ototoxic mechanisms should draw from several models, enabling a better answer to the clinical question of prevention and treatment of inner ear drug toxicity.

Investigating outer hair cell motility with a combination of external alternating electrical field stimulation and high-speed image analysis.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy. OHCs are able to change shape -elongate, shorten and bend- in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals. OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells). Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research. The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF). This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

Linking LIMK1 deficiency to hyperacusis and progressive hearing loss in individuals with Williams syndrome.

Williams syndrome (a.k.a. Williams-Beuren Syndrome) is a multisystem disorder caused by the hemizygous deletion of a 1.6 Mb region at 7q11.23 encompassing about 26 genes, including that encoding LIM kinase 1 (LIMK1). Individuals with Williams Syndrome manifest hyperacusis and progressive hearing loss, and hyperacusis early onset suggests that it could be associated with one of the deleted genes. Based on our results about the critical role of LIM kinases in the regulation of the motile responses of cochlear outer hair cells (OHC) and cochlear amplification, we propose here that a reduced expression of LIMK1 in OHC would be the major underlying cause of the hyperacusis and progressive hearing loss observed in patients with Williams Syndrome. Moreover, we propose a novel model of gain-control for cochlear amplification based on LIMK-mediated regulation of OHC's slow motility.

Motile responses of cochlear outer hair cells stimulated with an alternating electrical field.

The goal of the present study was to evaluate and characterize the motile responses of guinea pig OHCs, stimulated at frequencies varying from 50 Hz to 4 kHz, using high-definition, high-speed video recording and fully automatic image analysis software. Cells stimulated in continuous, burst and sweeping modes with an external alternating electrical field showed robust fast and slow motility, which were dependent on frequency, mode and intensity of stimulation. In response to continuous stimulation, electromotile amplitude ranged from 0.3% to 3.2% of total cell length, whereas cell length usually decreased in amounts varying from 0.1% to 4.3%. Electromotile amplitude in OHCs stimulated with square wave's sweeps was near constant up to 200 Hz, progressively decreased between 200 Hz and 2 kHz, and then remained constant up to 4 kHz. In continuous and burst modes electromotility followed cycle-by-cycle the electrical stimulus, but it required 1-2 s to fully develop and reach maximal amplitude. Instead, slow cell length changes started about 0.6 s after the beginning and continuously developed up to 3 s after the end of electrical stimulation. Incubation of OHCs with 10 mM salicylate affected electromotility but not slow motility, whereas incubation with 3 mM gadolinium affected both. Thus, combination of external electrical stimulation, high-speed video recording and advanced image analysis software provides information about OHC motile responses at acoustic frequencies with an unprecedented detail, opening new areas of research in the field of OHC mechanics.