ABR/DPOAE publications

Noise exposure represents the second most common cause of acquired sensorineural hearing loss and we observed that tumor necrosis factor α (TNFα) was involved in this context. The effect of Tnfα gene silencing on the expression profile related to the TNFα metabolic pathway in an experimental model of noise-induced hearing loss had not previously been studied. Methods: Single ears of Wistar rats were pretreated with Tnfα small interfering RNA (siRNA) by trans-tympanic administration 24 h before they were exposed to white noise (120 dBSPL for three hours). After 24 h of noise exposure, we analyzed the electrophysiological threshold and the amplitude of waves I, II, III, and IV in the auditory brain response click. In addition, qRT-PCR was performed to evaluate the TNFα metabolic pathway in the ears submitted or not to gene silencing. Results: Preservation of the electrophysiological threshold and the amplitude of waves was observed in the ears submitted to gene silencing compared to the ears not treated. Increased anti-apoptotic gene expression and decreased pro-apoptotic gene expression were found in the treated ears. Conclusion: Our results allow us to suggest that the blockade of TNFα by gene silencing was useful to prevent noise-induced hearing loss.

Age-related hearing loss (ARHL) is an irreversible, progressive neurodegenerative disorder and is presently untreatable. Previous studies using animal models have suggested mitochondrial damage and programmed cell death to be involved with ARHL. Thus, we further investigated the pathophysiologic role of mitochondria and necroptosis in aged C57BL/6J male mice. Aged mice (20 months old) exhibited a significant loss of hearing, number of hair cells, neuronal fibers, and synaptic ribbons compared to young mice. Ultrastructural analysis of aged cochleae revealed damaged mitochondria with absent or disorganized cristae. Aged mice also showed significant decrease in cochlear blood flow, and exhibited increase in gene expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) and the pseudokinase mixed-lineage kinase domain-like (MLKL). Immunofluorescence (IF) assays of cytochrome C oxidase I (COX1) confirmed mitochondrial dysfunction in aged cochleae, which correlated with the degree of mitochondrial morphological damage. IF assays also revealed localization and increased expression of RIPK3 in sensorineural tissues that underwent significant necroptosis (inner and outer hair cells and stria vascularis). Together, our data shows that the aging cochlea exhibits damaged mitochondria, enhanced synthesis of proinflammatory cytokines, and provides new evidence of necroptosis in the aging cochlea in in vivo.

AMP-activated protein kinase (AMPK) integrates the regulation of cell growth and metabolism. AMPK activation occurs in response to cellular energy decline and mitochondrial dysfunction triggered by reactive oxygen species (ROS). In aged Tg-mtTFB1 mice, a mitochondrial deafness mouse model, hearing loss is accompanied with cochlear pathology including reduced endocochlear potential (EP) and loss of spiral ganglion neurons (SGN), inner hair cell (IHC) synapses and outer hair cells (OHC). Accumulated ROS and increased apoptosis signaling were also detected in cochlear tissues, accompanied by activation of AMPK. To further explore the role of AMPK signaling in the auditory phenotype, we used genetically knocked out AMPKα1 as a rescue to Tg-mtTFB1 mice and observed: improved ABR wave I, EP and IHC function, normal SGNs, IHC synapses morphology and OHC survivals, with decreased ROS, reduced pro-apoptotic signaling (Bax) and increased anti-apoptotic signaling (Bcl-2) in the cochlear tissues, indicating that reduced AMPK attenuated apoptosis via ROS-AMPK-Bcl2 pathway in the cochlea. To conclude, AMPK hyperactivation causes accelerated presbycusis in Tg-mtTFB1 mice by redox imbalance and dysregulation of the apoptosis pathway. The effects of AMPK downregulation on pro-survival function and reduction of oxidative stress indicate AMPK serves as a target to rescue or relieve mitochondrial hearing loss.

Cisplatin is used to treat a variety of solid tumors in both children and adults. However, cisplatin has serious side-effects, some of which may permanently affect patients' quality of life following treatment, such as ototoxicity. There is currently no FDA-approved therapy for the prevention or treatment of cisplatin-induced hearing loss. Herein we examine the potential for statins to prevent cisplatin-induced ototoxicity. Statins, a class of drugs commonly used to prevent or manage hypercholesterolemia, have been of clinical utility for decades with dependable outcomes and reliable safety profiles in humans. Statins are known to be protective in animal models of noise-induced and age-related hearing loss. Moreover, studies have demonstrated an additive benefit of statins in cancer treatment. In the current study, lovastatin reduces cisplatin-induced hearing loss in adult mice. Lovastatin-mediated protection was significantly greater among female than male mice, and the dose of lovastatin required for protection was different between the sexes. Taken together our data indicate that lovastatin reduces cisplatin-induced hearing loss in mice and suggest that concurrent statin and cisplatin therapy may represent a feasible clinical strategy for reducing cisplatin-induced ototoxicity that should be explored for future clinical use.

It is commonly assumed that difficulty in listening to speech in noise is at least partly due to deficits in neural temporal processing. Given that noise reduces the temporal fidelity of the auditory brainstem response (ABR) to speech, it has been suggested that the speech ABR may serve as an index of such neural deficits. However, the temporal fidelity of ABRs, to both speech and non-speech sounds, is also known to be influenced by the cochlear origin of the response, as responses from higher-frequency cochlear regions are faster and more synchronous than responses from lower-frequency regions. Thus, if noise caused a reweighting of response contributions from higher- to lower-frequency cochlear regions, the temporal fidelity of the aggregate response should be reduced even in the absence of any changes in neural processing. This ‘place mechanism’ has been demonstrated for non-speech ABRs. The aim of this study was to test whether it also applies to speech ABRs. We used the so-called ‘derived-band’ method to isolate response contributions from frequency-limited cochlear regions. Broadband and derived-band speech ABRs were measured both in quiet and in noise. Whilst the noise caused significant changes to the temporal properties of the broadband response, its effects on the derived-band responses were mostly restricted to the response amplitudes. Importantly, the amplitudes of the higher-frequency derived-band responses were much more strongly affected than those of the lower-frequency responses, suggesting that the noise indeed caused a reweighting effect. Our results indicate that, as for non-speech ABRs, the cochlear place mechanism can represent a potentially substantial confound to speech-ABR-in-noise measurements.

Background Ototoxicity is one of the major side effects of platinum-based chemotherapy, especially cisplatin therapy. To date, no FDA approved agents to alleviate or prevent this ototoxicity are available. However, ototoxicity is generally believed to be produced by excessive generation of reactive oxygen species (ROS) in the inner ear, thus leading to the development of various antioxidants, which act as otoprotective agents. Astaxanthin (ATX) is an interesting candidate in the development of new therapies for preventing and treating oxidative stress-related pathologies, owing to its unique antioxidant capacity. Methods and results In this study, we aimed to evaluate the potential antioxidant properties of ATX in the inner ear by using the HEI-OC1 cell line, zebrafish, and guinea pigs. Because ATX has poor solubility and cannot pass through round window membranes (RWM), we established lipid-polymer hybrid nanoparticles (LPN) for loading ATX. The LPN enabled ATX to penetrate RWM and maintain concentrations in the perilymph in the inner ear for 24 h after a single injection. ATX-LPN were found to have favorable biocompatibility and to strongly affect cisplatin-induced generation of ROS, on the basis of DCFHDA staining in HEI-OC1 cells. JC-1 and MitoTracker Green staining suggested that ATX-LPN successfully reversed the decrease in mitochondrial membrane potential induced by cisplatin in vitro and rescued cells from early stages of apoptosis, as demonstrated by FACS stained with Annexin V-FITC/PI. Moreover, ATX-LPN successfully attenuated OHC losses in cultured organ of Corti and animal models (zebrafish and guinea pigs) in vivo. In investigating the protective mechanism of ATX-LPN, we found that ATX-LPN decreased the expression of pro-apoptotic proteins (caspase 3/9 and cytochrome-c) and increased expression of the anti-apoptotic protein Bcl-2. In addition, the activation of JNK induced by CDDP was up-regulated and then decreased after the administration of ATX-LPN, while P38 stayed unchanged. Conclusions To best of our knowledge, this is first study concluded that ATX-LPN as a new therapeutic agent for the prevention of cisplatin-induced ototoxicity.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39 . However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Hgf . Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness. SIGNIFICANCE STATEMENT Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3′UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 bp deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

Tsukushi (TSK)—a small, secreted, leucine-rich-repeat proteoglycan—interacts with and regulates essential cellular signaling cascades. However, its functions in the mouse inner ear are unknown. In this study, measurement of auditory brainstem responses, fluorescence microscopy, and scanning electron microscopy revealed that TSK deficiency in mice resulted in the formation of abnormal stereocilia in the inner hair cells and hearing loss but not in the loss of these cells. TSK accumulated in nonprosensory regions during early embryonic stages and in both nonprosensory and prosensory regions in late embryonic stages. In adult mice, TSK was localized in the organ of Corti, spiral ganglion cells, and the stria vascularis. Moreover, loss of TSK caused dynamic changes in the expression of key genes that drive the differentiation of the inner hair cells in prosensory regions. Finally, our results revealed that TSK interacted with Sox2 and BMP4 to control stereocilia formation in the inner hair cells. Hence, TSK appears to be an essential component of the molecular pathways that regulate inner ear development.

Oxidative stress is a major contributor to noise-induced hearing loss, the most common cause of hearing loss among military personnel and young adults. HK-2 is a potent, orally-active, multifunctional, redox-modulating drug that has been shown to protect against a wide range of neurological disorders with no observed side effects. HK-2 protected cochlear HEI-OC1 cells against various forms of experimentally-induced oxidative stressors similar to those observed during and after intense noise exposure. The mechanisms by which HK-2 protects cells is twofold, first by its ability to reduce oxidative stress generated by free radicals, and second, by its ability to complex biologically active transition metals such as Fe+2, thus reducing their availability to participate in the Fenton reaction where highly toxic hydroxyl radicals are generated. For the rat in vivo studies, HK-2 provided significant protection against noise-induced hearing loss and hair cell loss. Noise-induced hearing loss was induced by an 8-16 kHz octave band noises presented for 8 h/d for 21 days at an intensity of 95 dB SPL. In the Prevention study, HK-2 was administered orally beginning 5 days before the start of the noise and ending 10 days after the noise. Treatment with HK-2 dose-dependently reduced the amount of noise-induced hearing impairment, reflected in the cochlear compound action potential, and noise-induced hair cell loss. In a subsequent Rescue experiment in which HK-2 was administered for 10 days starting after the noise was turned off, HK-2 also significantly reduced the amount of hearing impairment, but the effect size was substantially less than in the Prevention studies. HK-2 alone did not adversely affect HEI-OC1 cell viability, nor did it cause any adverse changes in rat body weight, behavior, cochlear function or hair cell integrity. Thus, HK-2 is a novel, safe, orally-deliverable and highly effective otoprotective compound with considerable potential for preventing hearing loss from noise and other hearing disorders linked to excessive oxidative stress.

The underlying causes of age-related hearing loss (ARHL) are not well understood, but it is clear from heritability estimates that genetics plays a role in addition to environmental factors. Genome-wide association studies (GWAS) in human populations can point to candidate genes that may be involved in ARHL, but follow-up analysis is needed to assess the role of these genes in the disease process. Some genetic variants may contribute a small amount to a disease, while other variants may have a large effect size, but the genetic architecture of ARHL is not yet well-defined. In this study, we asked if a set of 17 candidate genes highlighted by early GWAS reports of ARHL have detectable effects on hearing by knocking down expression levels of each gene in the mouse and analysing auditory function. We found two of the genes have an impact on hearing. Mutation of Dclk1 led to late-onset progressive increase in ABR thresholds and the A430005L14Rik (C1orf174) mutants showed worse recovery from noise-induced damage than controls. We did not detect any abnormal responses in the remaining 15 mutant lines either in thresholds or from our battery of suprathreshold ABR tests, and we discuss the possible reasons for this.

Acid-base homeostasis is critical for normal growth, development, and hearing function. The sodium–hydrogen exchanger 6 (NHE6), a protein mainly expressed in early and recycling endosomes, plays an important role in regulating organellar pH. Mutations in NHE6 cause complex, slowly progressive neurodegeneration. Little is known about NHE6 function in the mouse cochlea. Here, we found that all NHE isoforms were expressed in wild-type (WT) mouse cochlea. Nhe6 knockout (KO) mice showed significant hearing loss compared to WT littermates. Immunohistochemistry in WT mouse cochlea showed that Nhe6 was localized in the organ of Corti (OC), spiral ganglion (SG), stria vascularis (SV), and afferent nerve fibres. The middle and the inner ears of WT and Nhe6 KO mice were not different morphologically. Given the putative role of NHE6 in early endosomal function, we examined Rab GTPase expression in early and late endosomes. We found no change in Rab5, significantly lower Rab7, and higher Rab11 levels in the Nhe6 KO OC, compared to WT littermates. Because Rabs mediate TrkB endosomal signalling, we evaluated TrkB phosphorylation in the OCs of both strains. Nhe6 KO mice showed significant reductions in TrkB and Akt phosphorylation in the OC. In addition, we examined genes used as markers of SG type I ( Slc17a7 , Calb1 , Pou4f1 , Cal2 ) and type II neurons ( Prph , Plk5 , Cacna1g ). We found that all marker gene expression levels were significantly elevated in the SG of Nhe6 KO mice, compared to WT littermates. Anti-neurofilament factor staining showed axon loss in the cochlear nerves of Nhe6 KO mice compared to WT mice. These findings indicated that BDNF/TrkB signalling was disrupted in the OC of Nhe6 KO mice, probably due to TrkB reduction, caused by over acidification in the absence of NHE6. Thus, our findings demonstrated that NHEs play important roles in normal hearing in the mammalian cochlea.

In all commercial cochlear implant (CI) devices, the electric stimulation is performed with a rectangular pulse that generally has two phases of opposite polarity. To date, developing new stimulation strategies has relied on the efficacy of this shape. Here, we investigate the potential of a novel stimulation paradigm that uses biophysically-inspired electrical ramped pulses. Using electrically-evoked auditory brainstem response (eABR) recordings in mice, we found that less charge, but higher current level amplitude, is needed to evoke responses with ramped shapes that are similar in amplitude to responses obtained with rectangular shapes. The most charge-efficient pulse shape had a rising ramp over both phases, supporting findings from previous in vitro studies. This was also true for longer phase durations. Our study presents the first physiological data on CI-stimulation with ramped pulse shapes. By reducing charge consumption ramped pulses have the potential to produce more battery-efficient CIs and may open new perspectives for designing other efficient neural implants in the future.

The experience of variation in maternal licking and grooming (LG) is considered a critical influence in neurodevelopment related to stress and cognition, but little is known about its relationship to early sensory development. In this study, we used a maternal selection approach to test the hypothesis that differences in LG during the first week of life influence the timing of hearing onset in Wistar rat pups. We performed a range of tests, including auditory brainstem responses (ABR), tracking of eye opening (EO), micro-CT X-ray tomography, and qRT-PCR to monitor neurodevelopmental changes in the female and male progeny of low-LG and high-LG dams. Our results show that variation in maternal LG is not overtly associated with different timing of ABR onset and EO in the progeny. However, the data provide insight on the delay between hearing onset and EO, on key functional and structural properties that define hearing onset at the auditory periphery, and on changes in brain gene expression that include the first evidence that: a) the hypoxia-sensitive pathway is regulated in subcortical and cortical auditory brain regions before hearing onset, and b) implicates maternal LG in regulation of Bdnf signaling in auditory cortex after hearing onset. Altogether, these findings provide a baseline to evaluate how factors that severely disrupt the early maternal environment may affect the expression of robust developmental sensory programs. SIGNIFICANCE STATEMENT Early life experience during sensitive developmental periods can induce long-term effects on the neurobiological development of the offspring. In the present work we tested the hypothesis that variation in maternal licking and grooming (LG) affects the timing of hearing onset in Wistar rat pups. To our surprise the results did not support the hypothesis. Instead, we found a robust range of early and late auditory development that was independent of maternal LG. Nevertheless, the study provides new findings on the delay between hearing onset and eye opening, on key functional and structural properties that define hearing onset at the auditory periphery, and the first evidence that a) the hypoxia-sensitive pathway is regulated in the central auditory system during the sensitive period before hearing onset, and b) maternal LG is implicated in regulation of Bdnf signaling during the sensitive period after hearing onset. These findings provide a baseline to evaluate how factors that severely disrupt the early maternal environment may affect the expression of robust developmental sensory programs.

The non-conventional neurotrophic factor mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-resident protein that promotes ER homeostasis. MANF has a cytoprotective function, shown in the central nervous system neurons and pancreatic beta cells. Here, we report that MANF is expressed in the hair cells and neurons and in selected non-sensory cells of the cochlea and that Manf inactivation triggers upregulation of the ER chaperones in these cells. However, Manf inactivation resulted in the death of only outer hair cells (OHCs), the cells responsible for sound amplification in the cochlea. All OHCs were formed in Manf -inactivated mice, but progressive OHC death started soon after the onset of hearing function. The robust OHC loss was accompanied by strongly elevated hearing thresholds. Conditional Manf inactivation demonstrated that MANF has a local function in the cochlea. Immunostainings revealed the upregulation of CHOP, the pro-apoptotic component of the unfolded protein response (UPR), in Manf -inactivated OHCs, linking the UPR to the loss of these cells. The phenotype of Manf -inactivated OHCs was distinctly dependent on the mouse strain, such that the strains characterized by early-onset age-related hearing loss (C57BL/6J and CD-1) were affected. These results suggest that Manf deficiency becomes detrimental when accompanied by gene mutations that predispose to hearing loss, by intensifying ER dyshomeostasis. Together, MANF is the first growth factor shown to antagonize ER stress-mediated OHC death. MANF might serve as a therapeutic candidate for protection against hearing loss induced by the ER-machinery-targeting stressors.

Hypothesis: Microneedles can create microperforations in the round window membrane (RWM) without causing anatomic or physiologic damage. Background: Reliable delivery of agents into the inner ear for therapeutic and diagnostic purposes remains a challenge. Our novel approach employs microneedles to facilitate intracochlear access via the RWM. This study investigates the anatomical and functional consequences of microneedle perforations in guinea pig RWMs in vivo. Methods: Single three-dimensional-printed, 100 μm diameter microneedles were used to perforate the guinea pig RWM via the postauricular sulcus. Hearing was assessed both before and after microneedle perforation using compound action potential and distortion product otoacoustic emissions. Confocal microscopy was used ex vivo to examine harvested RWMs, measuring the size, shape, and location of perforations and documenting healing at 0 hours (n = 7), 24 hours (n = 6), 48 hours (n = 6), and 1 week (n = 6). Results: Microneedles create precise and accurate perforations measuring 93.1 ± 29.0 μm by 34.5 ± 16.8 μm and produce a high-frequency threshold shift that disappears after 24 hours. Examination of perforations over time demonstrates healing progression over 24 to 48 hours and complete perforation closure by 1 week. Conclusion: Microneedles can create a temporary microperforation in the RWM without causing significant anatomic or physiologic dysfunction. Microneedles have the potential to mediate safe and effective intracochlear access for diagnosis and treatment of inner ear disease.

The round window membrane (RWM) is the most common entryway for local drug and gene delivery into the inner ear, but its permeability can change the treatment outcome. We previously demonstrated a feasible and highly efficient approach using ultrasound-aided microbubble (USMB) cavitation to enhance the permeability of the RWM. Here, we investigated the safety of USMB exposure and the association between temporal changes in RWM permeability and ultrastructure. Experimental guinea pigs were divided into two treatment groups: a control group receiving round window soaking (RWS) with MBs and treatment (USM) groups undergoing 3 (USM-3) or 5 (USM-5) consecutive USMB exposures (1 min/exposure) at an acoustic intensity of 3 W/cm2 and 1 MHz frequency. The trans-RWM delivery efficiency of biotin-fluorescein isothiocyanate conjugates, used as permeability tracers, revealed a greater than 7-fold higher delivery efficiency for the USM groups immediately after 3 or 5 exposures than for the RWS group. After 24 h, the delivery efficiency was 2.4-fold higher for the USM-3 group but was 6.6-fold higher for the USM-5 group (and 3.7-fold higher after 48 h), when compared to the RWS group. Scanning electron microscopy images of the RWM ultrastructure revealed USMB-induced sonoporation effects that could include the formation of heterogeneous pore-like openings with perforation diameters from 100 nm to several micrometers, disruption of the continuity of the outer epithelial surface layer, and loss of microvilli. These ultrastructural features were associated with differential permeability changes that depended on the USMB exposure course. Fourteen days after treatment, the pore-like openings had significantly decreased in number and the epithelial defects were healed either by cell expansion or by repair by newly migrated epithelial cells. The auditory brainstem response recordings of the animals following the 5-exposure USMB treatment indicated no deterioration in the hearing thresholds at a 2-month follow-up and no significant hair cell damage or apoptosis, based on scanning electron microscopy, surface preparations, and TUNEL assays. USMBs therefore appear to be safe and effective for inner ear drug delivery. The mechanism of enhanced permeability may involve a disruption of the continuity of the outer RWM epithelial layer, which controls transmembrane transport of various substances.

Lateral olivocochlear (LOC) efferent neurons modulate auditory nerve fiber (ANF) activity using a large repertoire of neurotransmitters, including dopamine (DA) and acetylcholine (ACh). Little is known about how individual neurotransmitter systems are differentially utilized in response to the ever-changing acoustic environment. Here we present quantitative evidence in rodents that the dopaminergic LOC input to ANFs is dynamically regulated according to the animal’s recent acoustic experience. Sound exposure upregulates tyrosine hydroxylase, an enzyme responsible for dopamine synthesis, in cholinergic LOC intrinsic neurons, suggesting that individual LOC neurons might at times co-release ACh and DA. We further demonstrate that dopamine down-regulates ANF firing rates by reducing both the hair cell release rate and the size of synaptic events. Collectively, our results suggest that LOC intrinsic neurons can undergo on-demand neurotransmitter re-specification to re-calibrate ANF activity, adjust the gain at hair cell/ANF synapses, and possibly to protect these synapses from noise damage.

Platinum-based agents, such as cisplatin, form the mainstay of currently used chemotherapeutic regimens for several malignancies; however, the main limitations are chemoresistance and ototoxic side effects. In this study we used two different polyphenols, curcumin and ferulic acid as adjuvant chemotherapeutics evaluating (1) in vivo their antioxidant effects in protecting against cisplatin ototoxicity and (2) in vitro the transcription factors involved in tumor progression and cisplatin resistance. We reported that both polyphenols show antioxidant and oto-protective activity in the cochlea by up-regulating Nrf-2/HO-1 pathway and downregulating p53 phosphorylation. However, only curcumin is able to influence inflammatory pathways counteracting NF-κB activation. In human cancer cells, curcumin converts the anti-oxidant effect into a pro-oxidant and anti-inflammatory one. Curcumin exerts permissive and chemosensitive properties by targeting the cisplatin chemoresistant factors Nrf-2, NF-κB and STAT-3 phosphorylation. Ferulic acid shows a biphasic response: it is pro-oxidant at lower concentrations and anti-oxidant at higher concentrations promoting chemoresistance. Thus, polyphenols, mainly curcumin, targeting ROS-modulated pathways may be a promising tool for cancer therapy. Thanks to their biphasic activity of antioxidant in normal cells undergoing stressful conditions and pro-oxidant in cancer cells, these polyphenols probably engage an interplay among the key factors Nrf-2, NF-κB, STAT-3 and p53.

It has been demonstrated that isolated auditory sensory cells, outer hair cells, can generate distortion products at low frequencies. It remains unknown, however, whether or not motile outer hair cells are able to generate two-tone distortion at high frequencies in living cochleae under the mechanical loads caused by surounding tissues and fluids. By measuring sub-nanometer vibration directly from the apical ends of outer hair cells using a custom-built heterodyne low-coherence interferometer, here we show outer hair cell-generated two-tone distortion in reticular lamina motion in the living cochlea. Reticular-lamina distortion is significantly greater and occurs at a broader frequency range than that of the basilar membrane. Contrary to expectations, our results indicate that motile outer hair cells are capable of generating two-tone distortion in vivo not only at the locations tuned to primary tones but also at a broad region basal to these locations.

Etimicin (ETM), a fourth-generation aminoglycosides (AGs), is now widely clinically used in China due to its high efficacy and low toxicity. However, the mechanisms underlying its low nephrotoxicity and ototoxicity remain unclear. In the present study we compared the antibacterial and toxicity profiles of etimicin, gentamicin (GM, a second-generation AG), and amikacin (AMK, a third-generation AG), and investigated their pharmacokinetic properties in the toxicity target organs (kidney and inner ear) and subcellular compartments. We first demonstrated that ETM exhibited superior antibacterial activities against clinical isolates to GM and AMK, and it exerted minimal nephrotoxicity and ototoxicity in rats following multi-dose administration. Then, we conducted pharmacokinetic studies in rats, showed that the three AGs accumulated in the kidney and inner ear with ETM being distributed to a lesser degree in the two toxicity target organs as compared with GM and AMK high-dose groups. Furthermore, we conducted in vitro experiments in NRK-52E rat renal tubular epithelial cells and HEI-OC1 cochlear hair cells, and revealed that all the three AGs were distributed predominantly in the mitochondria with ETM showing minimal accumulation; they not only directly inhibited the activity of mitochondrial complexes IV and V but also inhibited mitochondrial function and its related PGC-1α-NRF1-TFAM pathway; ETM caused minimal damage to the mitochondrial complex and mitochondrial biogenesis. Our results demonstrate that the minimal otonephrotoxicity of ETM results from its lesser accumulation in mitochondria of target cells and subsequently lesser inhibition of mitochondrial function. These results provide a new strategy for discovering novel AGs with high efficacy and low toxicity.