Rolando Rolesi

Noise-Induced Hearing Loss (NIHL) as a Target of Oxidative Stress-Mediated Damage: Cochlear and Cortical Responses after an Increase in Antioxidant Defense

This study addresses the relationship between cochlear oxidative damage and auditory cortical injury in a rat model of repeated noise exposure. To test the effect of increased antioxidant defenses, a water-soluble coenzyme Q10 analog (Qter) was used. We analyzed auditory function, cochlear oxidative stress, morphological alterations in auditory cortices and cochlear structures, and levels of coenzymes Q9 and Q10 (CoQ9 and CoQ10, respectively) as indicators of endogenous antioxidant capability. We report three main results. First, hearing loss and damage in hair cells and spiral ganglion was determined by noise-induced oxidative stress. Second, the acoustic trauma altered dendritic morphology and decreased spine number of II–III and V–VI layer pyramidal neurons of auditory cortices. Third, the systemic administration of the water-soluble CoQ10 analog reduced oxidative-induced cochlear damage, hearing loss, and cortical dendritic injury. Furthermore, cochlear levels of CoQ9 and CoQ10 content increased. These findings indicate that antioxidant treatment restores auditory cortical neuronal morphology and hearing function by reducing the noise-induced redox imbalance in the cochlea and the deafferentation effects upstream the acoustic pathway.

Rosmarinic acid up-regulates the noise-activated Nrf2/HO-1 pathway and protects against noise-induced injury in rat cochlea.

Noise-induced hearing loss depends on progressive increase of reactive oxygen species and lipoperoxidative damage in conjunction with the imbalance of antioxidant defenses. The redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a critical role in the regulation of cellular defenses against oxidative stress, including heme oxygenase-1 (HO-1) activation. In this work we describe a link between cochlear oxidative stress damage, induced by noise exposure, and the activation of the Nrf2/HO-1 pathway. In our model, noise induces superoxide production and overexpression of the lipid peroxidation marker 4-hydroxy-nonenals (4-HNE). To face the oxidative stress, the endogenous defense system is activated as well, as shown by the slight activation of superoxide dismutases (SODs). In addition, we observed the activation of the Nrf2/HO-1 pathway after noise exposure. Nrf2 appears to promote the maintenance of cellular homeostasis under stress conditions. However, in this model the endogenous antioxidant system fails to counteract noise-induced cell damage and its activation is not effective enough in preventing cochlear damage. The herb-derived phenol rosmarinic acid (RA) attenuates noise-induced hearing loss, reducing threshold shift, and promotes hair cell survival. In fact, RA enhances the endogenous antioxidant defenses, as shown by decreased superoxide production, reduced expression of 4-HNE, and up-regulation of SODs. Interestingly, RA potentiates the Nrf2/HO-1 signaling pathway, as shown by immunohistochemical and Western blot analyses. Thus, protective effects of RA are associated with the induction/activation of the Nrf2-ARE signaling pathway in addition to RA direct scavenging capability.

Molecular targets for anticancer redox chemotherapy and cisplatin-induced ototoxicity: the role of curcumin on pSTAT3 and Nrf-2 signalling.

Background:In oncology, an emerging paradigm emphasises molecularly targeted approaches for cancer prevention and therapy and the use of adjuvant chemotherapeutics to overcome cisplatin limitations. Owing to their safe use, some polyphenols, such as curcumin, modulate important pathways or molecular targets in cancers. This paper focuses on curcumin as an adjuvant molecule to cisplatin by analysing its potential implications on the molecular targets, signal transducer and activator of transcription 3 (STAT3) and NF-E2 p45-related factor 2 (Nrf-2), in tumour progression and cisplatin resistance in vitro and the adverse effect ototoxicity in vivo.Methods:The effects of curcumin and/or cisplatin treatment have been evaluated in head and neck squamous cell carcinoma as well as in a rat model of cisplatin-induced ototoxicity by using immunofluorescence, western blot, and functional and morphological analysis.Results:This study demonstrates that curcumin attenuates all stages of tumour progression (survival, proliferation) and, by targeting pSTAT3 and Nrf-2 signalling pathways, provides chemosensitisation to cisplatin in vitro and protection from its ototoxic adverse effects in vivo.Conclusions:These results indicate that curcumin can be used as an efficient adjuvant to cisplatin cancer therapy. This treatment strategy in head and neck cancer could mediate cisplatin chemoresistance by modulating therapeutic targets (STAT3 and Nrf2) and, at the same time, reduce cisplatin-related ototoxic adverse effects.

Curcuma longa (curcumin) decreases in vivo cisplatin-induced ototoxicity through heme oxygenase-1 induction.

Hypothesis To investigate whether curcumin may have in vivo protective effects against cisplatin ototoxicity by its direct scavenger activity and/or by curcumin-mediated upregulation of HO-1. Background Cisplatin-induced ototoxicity is a major dose-limiting side effect in anticancer chemotherapy. A protective approach to decrease cisplatin ototoxicity without compromising its therapeutic efficacy remains a critical goal for anticancer therapy. Recent evidences indicate that curcumin exhibits antioxidant, anti-inflammatory, and chemosensitizer activities. Methods In male adult Wistar rats, a curcumin dose of 200 mg/kg, selected from a dose-response curve, was injected 1 hour before cisplatin administration and once daily for the following 3 days. A single dose of cisplatin (16 mg/kg) was administered intraperitoneally. Rats were divided as follows: 1) control, 2) curcumin control, 3) vehicle control, 4) cisplatin, 5) cisplatin+ vehicle, and 6) curcumin+cisplatin. ABRs were measured before and at Days 3 and 5 after cisplatin administration. Rhodamine-phalloidin staining, 4-hydroxy-2-nonenal and heme-oxigenase-1 immunostainings, and Western blot analyses were performed to assess and quantify OHC loss, lipid peroxidation, and the endogenous response to cisplatin-induced damage and to curcumin protection. Results Curcumin treatment attenuated hearing loss induced by cisplatin, increased OHC survival, decreased 4-HNE expression, and increased HO-1 expression. Conclusion This preclinical study demonstrates that systemic curcumin attenuates ototoxicity and provides molecular evidence for a role of HO-1 as an additional mediator in attenuating cisplatin-induced damage.

Efficacy of different routes of administration for Coenzyme Q10 formulation in noise-induced hearing loss: Systemic versus transtympanic modality

Abstract Conclusion: The effectiveness of a coenzyme Q10 formulation, Q-ter, given via transtympanic injection is interesting for the future application of this minimally invasive procedure in the treatment of reactive oxygen species (ROS)-induced hearing loss. Objective: We focused on antioxidant therapy in noise-induced hearing loss (NIHL). Our study was designed to evaluate the effectiveness of Q-ter for different schedules of drug administration to establish the best modality for treatment. Methods: Rats were exposed to acoustic trauma (10 kHz at 120 dB for 60 min) and received Q-ter according to two modalities: systemic (Q-ter 100 mg/kg for 4 days 1 h before and 3 days post noise exposure) and transtympanic (Q-ter 20 and 40% concentration 1 h before noise exposure). Auditory brainstem response (ABR), immunohistochemical and morphological studies were performed. Results: Q-ter administration significantly decreased NIHL at day 21 from noise exposure. The improvement of auditory function by Q-ter was paralleled by a significant reduction in oxidative stress. The transtympanic and systemic routes of drug administration showed a similar degree of protection.

Grafting and early expression of growth factors from adipose-derived stem cells transplanted into the cochlea, in a Guinea pig model of acoustic trauma.

Noise exposure causes damage of multiple cochlear cell types producing permanent hearing loss with important social consequences. In mammals, no regeneration of either damaged hair cells or auditory neurons has been observed and no successful treatment is available to achieve a functional recovery. Loads of evidence indicate adipose-derived stem cells (ASCs) as promising tools in diversified regenerative medicine applications, due to the high degree of plasticity and trophic features. This study was aimed at identifying the path of in vivo cell migration and expression of trophic growth factors, upon ASCs transplantation into the cochlea, following noise-induced injury. ASCs were isolated in primary culture from the adipose tissue of a guinea pig, transduced using a viral vector to express the green fluorescent protein, and implanted into the scala tympani of deafened animals. Auditory function was assessed 3 and 7 days after surgery. The expression of trophic growth factors was comparatively analyzed using real-time PCR in control and noise-injured cochlear tissues. Immunofluorescence was used to assess the in vivo localization and expression of trophic growth factors in ASCs and cochleae, 3 and 7 days following homologous implantation. ASC implantation did not modify auditory function. ASCs migrated from the perilymphatic to the endolymphatic compartment, during the analyzed time course. Upon noise exposure, the expression of chemokine ligands and receptors related to the PDGF, VEGF, and TGFbeta pathways, increased in the cochlear tissues, possibly guiding in vivo cell migration. Immunofluorescence confirmed the increased expression, which appeared to be further strengthened by ASCs’ implantation. These results indicated that ASCs are able to migrate at the site of tissue damage and express trophic factors, upon intracochlear implantation, providing an original proof of principle, which could pave the way for further developments of ASC-based treatments of deafness.

The redox protein p66(shc) mediates cochlear vascular dysfunction and transient noise-induced hearing loss.

p66shc, a member of the ShcA protein family, is essential for cellular response to oxidative stress, and elicits the formation of mitochondrial Reactive Oxygen Species (ROS), thus promoting vasomotor dysfunction and inflammation. Accordingly, mice lacking the p66 isoform display increased resistance to oxidative tissue damage and to cardiovascular disorders. Oxidative stress also contributes to noise-induced hearing loss (NIHL); we found that p66shc expression and serine phosphorylation were induced following noise exposure in the rat cochlea, together with markers of oxidative stress, inflammation and ischemia as indicated by the levels of the hypoxic inducible factor (HIF) and the vascular endothelial growth factor (VEGF) in the highly vascularised cochlear lateral region and spiral ganglion. Importantly, p66shc knock-out (p66 KO) 126 SvEv adult mice were less vulnerable to acoustic trauma with respect to wild type controls, as shown by preserved auditory function and by remarkably lower levels of oxidative stress and ischemia markers. Of note, decline of auditory function observed in 12 month old WT controls was markedly attenuated in p66KO mice consistent with delayed inner ear senescence. Collectively, we have identified a pivotal role for p66shc -induced vascular dysfunction in a common pathogenic cascade shared by noise-induced and age-related hearing loss.

Pioglitazone Represents an Effective Therapeutic Target in Preventing Oxidative/Inflammatory Cochlear Damage Induced by Noise Exposure

Recent progress in hearing loss research has provided strong evidence for the imbalance of cellular redox status and inflammation as common predominant mechanisms of damage affecting the organ of Corti including noise induced hearing loss. The discovery of a protective molecule acting on both mechanisms is challenging. The thiazolidinediones, a class of antidiabetic drugs including pioglitazone and rosiglitazone, have demonstrated diverse pleiotrophic effects in many tissues where they exhibit anti-inflammatory, anti-proliferative, tissue protective effects and regulators of redox balance acting as agonist of peroxisome proliferator-activated receptors (PPARs). They are members of the family of ligand regulated nuclear hormone receptors that are also expressed in several cochlear cell types, including the outer hair cells. In this study, we investigated the protective capacity of pioglitazone in a model of noise-induced hearing loss in Wistar rats and the molecular mechanisms underlying this protective effects. Specifically, we employed a formulation of pioglitazone in a biocompatible thermogel providing rapid, uniform and sustained inner ear drug delivery via transtympanic injection. Following noise exposure (120 dB, 10 kHz, 1 h), different time schedules of treatment were employed: we explored the efficacy of pioglitazone given immediately (1 h) or at delayed time points (24 and 48 h) after noise exposure and the time course and extent of hearing recovery were assessed. We found that pioglitazone was able to protect auditory function at the mid-high frequencies and to limit cell death in the cochlear basal/middle turn, damaged by noise exposure. Immunofluorescence and western blot analysis provided evidence that pioglitazone mediates both anti-inflammatory and anti-oxidant effects by decreasing NF-κB and IL-1β expression in the cochlea and opposing the oxidative damage induced by noise insult. These results suggest that intratympanic pioglitazone can be considered a valid therapeutic strategy for attenuating noise-induced hearing loss and cochlear damage, reducing inflammatory signaling and restoring cochlear redox balance.

Anodal transcranial direct current stimulation affects auditory cortex plasticity in normal-hearing and noise-exposed rats

BACKGROUND Transcranial direct current stimulation (tDCS) is a non-invasive tool capable to modulate cortical functions by affecting neuronal excitability and synaptic plasticity. OBJECTIVE Here we investigated the effects of anodal tDCS on auditory cortex (ACx) in normal-hearing rats and following a paradigm of noise-induced hearing loss (NIHL), that causes morphological alterations in ACx pyramidal neurons. METHODS Male rats exposed to intense pure tone (10 kHz) were subsequently subjected to unilateral anodal tDCS of ACx and changes in dendritic morphology and spines were assessed by Golgi-Cox staining 30 days after the onset of the acoustic trauma. Molecular and functional changes were investigated by Western immunoblotting, immunofluorescence experiments and electrophysiological recordings in brain slices. RESULTS We found that NIHL altered dendritic morphology by decreasing spine density, mostly in layer 2/3 pyramidal neurons. Interestingly, tDCS increased ACx spine density, targeting apical dendrites of layer 2/3 and 5/6 pyramidal neurons in rats with normal auditory function and both apical and basal arborizations in layer 2/3 of NIHL rats. Twenty-four hours after tDCS, Bdnf and synaptophysin levels in ACx increased both in normal-hearing and noise-exposed rats. Field recordings showed that basal synaptic transmission at layer 2/3 horizontal connections was significantly reduced in noise-exposed rats compared to normal-hearing animals and, notably, input-output curves of noise-exposed animals subjected to tDCS were similar to those of normal-hearing rats. CONCLUSIONS Our findings provide novel evidence that anodal tDCS affects structural plasticity in the ACx suggesting that it might be beneficial in treating cortical alterations due to cochlear damage.

Role of antioxidant supplementation in preventing noise induced hearing loss

Objective: Oxidative stress plays a significant role in noise induced hearing loss (NIHL) as it largely participates in the mechanisms that underlie cell death after noise exposure and leads to sensorineural hearing loss. Many antioxidant drugs have been tested to prevent NIHL. Study design: We compared the protective effects of five molecules having antioxidant properties (vitamin E, ferulic acid, active CoQ10, its synthetic analogue – idebenone, and the soluble formulation CoQter) tested in our laboratory. This study has been conducted in a model of acoustic trauma in rats in which the molecules were given intraperitoneally 1 h before and once daily for three days after pure tone noise exposure (10 kHz for 1 h at 100dB SPL).We evaluated their hearing function via electrophysiological measurements at 2, 7 and 21 days and performed morphological studies with scanning electron microscopy and TUNEL assay as a parameter of apoptotic activation. Results: All molecules decreased threshold shift, reaching almost a complete recovery starting from day 7 with a further light recovery at day 21. At this time-point all treatments reached almost 80–90% of protection; however, CoQter and vitamin E were the most effective treatments. A decreasing number of TUNEL-positive nuclei for each treatment were observed two days after noise exposure. The cochleogram and morphological observations were consistent with the protective effects measured by ABR, 21 days after trauma. Conclusion: These results indicate that the best protection can be achieved by using antioxidant molecules acting against mitochondrial induced oxidative stress, such as vitamin E and CoQter and the degree of protection depends on the pharmacological properties of the molecules.