Macrophage depletion protects against cisplatin-induced ototoxicity and nephrotoxicity.

Cisplatin is a widely used anticancer drug with notable side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced toxicities, we used PLX3397, a U.S. Food and Drug Administration-approved inhibitor of the colony-stimulating factor 1 receptor, to eliminate tissue-resident macrophages. Mice treated with cisplatin alone had considerable hearing loss (ototoxicity) and kidney injury (nephrotoxicity). Macrophage ablation resulted in significantly reduced hearing loss and had greater outer hair cell survival. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together, our data indicate that ablation of tissue-resident macrophages represents an important strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

ERK1/2 Inhibition via the Oral Administration of Tizaterkib Alleviates Noise-Induced Hearing Loss While Tempering down the Immune Response.

Noise-induced hearing loss (NIHL) is a major cause of hearing impairment and is linked to dementia and mental health conditions, yet no FDA-approved drugs exist to prevent it. Downregulating the mitogen-activated protein kinase (MAPK) cellular pathway has emerged as a promising approach to attenuate NIHL, but the molecular targets and the mechanism of protection are not fully understood. Here, we tested specifically the role of the kinases ERK1/2 in noise otoprotection using a newly developed, highly specific ERK1/2 inhibitor, tizaterkib, in preclinical animal models. Tizaterkib is currently being tested in phase 1 clinical trials for cancer treatment and has high oral bioavailability and low predicted systemic toxicity in mice and humans. In this study, we performed dose-response measurements of tizaterkib's efficacy against permanent NIHL in adult FVB/NJ mice, and its minimum effective dose (0.5 mg/kg/bw), therapeutic index (>50), and window of opportunity (<48 h) were determined. The drug, administered orally twice daily for 3 days, 24 h after 2 h of 100 dB or 106 dB SPL noise exposure, at a dose equivalent to what is prescribed currently for humans in clinical trials, conferred an average protection of 20-25 dB SPL in both female and male mice. The drug shielded mice from the noise-induced synaptic damage which occurs following loud noise exposure. Equally interesting, tizaterkib was shown to decrease the number of CD45- and CD68-positive immune cells in the mouse cochlea following noise exposure. This study suggests that repurposing tizaterkib and the ERK1/2 kinases' inhibition could be a promising strategy for the treatment of NIHL.

Effects of Cochlear Implantation and Steroids on the Aging Guinea Pig Cochlea.

OBJECTIVE: The objective was to determine the effects of older age on hearing preservation after cochlear implantation (CI), and whether steroids improve hearing preservation in older animals. We hypothesized greater hearing preservation would be observed in (1) young animals compared to older animals and (2) older animals receiving steroids compared to no steroids. The secondary objective was to assess levels of fibrosis utilizing optical coherence tomography (OCT). STUDY DESIGN: Experimental Animal Study. SETTING: Laboratory. METHODS: Three groups of guinea pigs: young (YCI; 8.5 ± 0.5 weeks; n = 10), old (OCI; 19.1 ± 1.0 months; n = 9) and old + steroids (OCI+S; 19.1 ± 1.0 months; n = 9) underwent CI. The OCI+S group received a steroid taper over 7 days starting 2 days before surgery to 4 days after. Auditory brainstem response (ABR) measurements were performed preoperatively and postoperatively. OCT imaging was performed to assess cochleae for extent of fibrotic tissue growth in the scala tympani. RESULTS: The YCI group had significantly better hearing preservation as measured by smaller increases in ABR thresholds [mean shift: 2.79 ± 0.66] compared to the OCI group [mean shift = 12.44 ± 5.6]. The OCI+S group had significantly better hearing preservation [2.66 ± 1.50] compared to the OCI group. No significant differences was seen in fibrosis across groups. CONCLUSIONS: Young animals and older animals that received steroids had better hearing after CI than older animals not given steroids, but hearing preservation was not correlated with the level of fibrosis assessed using OCT. This work is the first to investigate differences in hearing preservation by age in an animal model, and supports the protective effects of steroids on hearing preservation in older individuals.

Antioxidative Stress-Induced Destruction to Cochlear Cells Caused by Blind Antioxidant Therapy.

OBJECTIVE: Verification that blind and excessive use of antioxidants leads to antioxidant stress which exacerbates cochlear cell damage. STUDY DESIGN: Basic research. SETTING: The Third Affiliated Hospital of Sun Yat-Sen University. METHODS: We compared and quantified hair cell-like house ear institute-organ of corti 1 (HEI-OC1) cell density, cell viability, and apoptosis caused by different concentrations of N-acetylcysteine (NAC) via Hoechst staining, Cell Counting Kit 8, Hoechst with propidium iodide staining, and Annexin V with propidium iodide (PI) staining. Apoptosis induced by high concentrations of M40403 and coenzyme Q10 in cochlear explants was analyzed and compared by cochlear dissection and activated caspase 3 labeling. RESULTS: With the increase of NAC concentration (0-1000 µmol/L), cell density decreased consequently and reached the lowest at 1000 µmol/L (****P ≤ .0001). Cell viability is also declining (**P < .01). The number of Annexin V-fluorescein isothiocyanate-labeled cells and PI-labeled cells increased with increasing NAC concentration after treatment of HEI-OC1 cells for 48 hours. The proportion of apoptotic cells also rose (*P < .05, **P < .01). Cochlear hair cells (HCs) treated with low concentrations of M40403 and coenzyme Q10 for 48 hours showed no damage. When the concentrations of M40403 and coenzyme Q10 were increased (concentrations＞30 µmol/L), HC damage began, followed by a dose-dependent increase in HC loss (*P < .001, **P < .0001). Activated caspase-3 was clearly apparent in cochlear explants treated with 50 µmol/L M40403 and coenzyme Q10 compared with cochlear explants without added M40403 and coenzyme Q10. CONCLUSION: These experimental results suggest that inappropriate application of antioxidants can cause severe damage to normal cochlear HCs.

Protective effect of berberine chloride against cisplatin-induced ototoxicity.

BACKGROUND: Cisplatin (CP) is an effective anticancer drug broadly used for various types of cancers, but it has shown ototoxicity that results from oxidative stress. Berberine has been reported for its anti-oxidative stress suggesting its therapeutic potential for many diseases such as colitis, diabetes, and vascular dementia. OBJECTIVE: Organ of Corti of postnatal day 3 mouse cochlear explants were used to compare hair cells after the treatment with cisplatin alone or with berberine chloride (BC) followed by CP. METHODS: We investigated the potential of the anti-oxidative effect of BC against the cisplatin-induced ototoxicity. We observed a reduced aberrant bundle of stereocilia in hair cells in CP with BC pre-treated group. Caspase-3 immunofluorescence and TUNEL assay supported the hypothesis that BC attenuates the apoptotic signals induced by CP. Reactive oxygen species level in the mitochondria were investigated by MitoSOX Red staining and the mitochondrial membrane potentials were compared by JC-1 assay. RESULTS: BC decreased ROS generation with preserved mitochondrial membrane potentials in mitochondria as well as reduced DNA fragmentation in hair cells. In summary, our data indicate that BC might act as antioxidant against CP by reducing the stress in mitochondria resulting in cell survival. CONCLUSION: Our result suggests the therapeutic potential of BC for prevention of the detrimental effect of CP-induced ototoxicity.

The disruption and hyperpermeability of blood-labyrinth barrier mediates cisplatin-induced ototoxicity.

The ototoxic mechanisms of cisplatin on the organ of Corti and spiral ganglion neurons have been extensively studied, while few studies have been focused on the stria vascularis (SV). Herein, we verified the functional and morphological impairment in SV induced by a single injection of cisplatin (12 mg/kg, I.P.), represented by a reduction in Endocochlear Potentials (EP) and strial atrophy, and explored underlying mechanisms. Our results revealed increased extravasation of chromatic tracers (Evans blue dye and FITC-dextran) around microvessels after cisplatin exposure. The increased vascular permeability could be attributed to changes of pericytes (PCs) and perivascular-resident macrophage-like melanocytes (PVM/Ms) in number or morphology, as well as the enhanced level of HIF-1α and downstream VEGF. This capillary leakage led to a high accumulation of cisplatin in the perivascular space in SV, and disrupted the integrity of blood-labyrinth barrier (BLB). Also, tight junction (ZO-1) loosening and Na+, K+-ATPase damage was considered to be other critical contributors of BLB breakdown, which resulted in EP drop and consequent hearing loss. This study explored the role of stria vascularis in cisplatin-induced ototoxicity in terms of BLB hyperpermeability and pointed to a novel therapeutic target for the prevention of cisplatin-related hearing loss.

Telmisartan Attenuates Kanamycin-Induced Ototoxicity in Rats.

Telmisartan (TM) has been proposed to relieve inflammatory responses by modulating peroxisome proliferator activator receptor-γ (PPARγ) signaling. This study aimed to investigate the protective effects of TM on kanamycin(KM)-induced ototoxicity in rats. Forty-eight, 8-week-old female Sprague Dawley rats were divided into four groups: (1) control group, (2) TM group, (3) KM group, and (4) TM + KM group. Auditory brainstem response was measured. The histology of the cochlea was examined. The protein expression levels of angiotensin-converting enzyme 2 (ACE2), HO1, and PPARγ were measured by Western blotting. The auditory threshold shifts at 4, 8, 16, and 32 kHz were lower in the TM + KM group than in the KM group (all p < 0.05). The loss of cochlear outer hair cells and spiral ganglial cells was lower in the TM + KM group than in the KM group. The protein expression levels of ACE2, PPARγ, and HO1 were higher in the KM group than in the control group (all p < 0.05). The TM + KM group showed lower expression levels of PPARγ and HO1 than the KM group.TM protected the cochlea from KM-induced injuries in rats. TM preserved hearing levels and attenuated the increase in PPARγ and HO1 expression levels in KM-exposed rat cochleae.

Effect of Phlorofucofuroeckol A and Dieckol Extracted from Ecklonia cava on Noise-induced Hearing Loss in a Mouse Model.

One of the well-known causes of hearing loss is noise. Approximately 31.1% of Americans between the ages of 20 and 69 years (61.1 million people) have high-frequency hearing loss associated with noise exposure. In addition, recurrent noise exposure can accelerate age-related hearing loss. Phlorofucofuroeckol A (PFF-A) and dieckol, polyphenols extracted from the brown alga Ecklonia cava, are potent antioxidant agents. In this study, we investigated the effect of PFF-A and dieckol on the consequences of noise exposure in mice. In 1,1-diphenyl-2-picrylhydrazyl assay, dieckol and PFF-A both showed significant radical-scavenging activity. The mice were exposed to 115 dB SPL of noise one single time for 2 h. Auditory brainstem response(ABR) threshold shifts 4 h after 4 kHz noise exposure in mice that received dieckol were significantly lower than those in the saline with noise group. The high-PFF-A group showed a lower threshold shift at click and 16 kHz 1 day after noise exposure than the control group. The high-PFF-A group also showed higher hair cell survival than in the control at 3 days after exposure in the apical turn. These results suggest that noise-induced hair cell damage in cochlear and the ABR threshold shift can be alleviated by dieckol and PFF-A in the mouse. Derivatives of these compounds may be applied to individuals who are inevitably exposed to noise, contributing to the prevention of noise-induced hearing loss with a low probability of adverse effects.

Ultrasound Microbubbles Enhance the Efficacy of Insulin-Like Growth Factor-1 Therapy for the Treatment of Noise-Induced Hearing Loss.

The application of insulin-like growth factor 1 (IGF-1) to the round window membrane (RWM) is an emerging treatment for inner ear diseases. RWM permeability is the key factor for efficient IGF-1 delivery. Ultrasound microbubbles (USMBs) can increase drug permeation through the RWM. In the present study, the enhancing effect of USMBs on the efficacy of IGF-1 application and the treatment effect of USMB-mediated IGF-1 delivery for noise-induced hearing loss (NIHL) were investigated. Forty-seven guinea pigs were assigned to three groups: the USM group, which received local application of recombinant human IGF-1 (rhIGF-1, 10 µg/µL) following application of USMBs to the RWM; the RWS group, which received IGF-1 application alone; and the saline-treated group. The perilymphatic concentration of rhIGF-1 in the USM group was 1.95- and 1.67- fold of that in the RWS group, 2 and 24 h after treatment, respectively. After 5 h of 118 dB SPL noise exposure, the USM group had the lowest threshold shift in auditory brainstem response, least loss of cochlear outer hair cells, and least reduction in the number of synaptic ribbons on postexposure day 28 among the three groups. The combination of USMB and IGF-1 led to a better therapeutic response to NIHL. Two hours after treatment, the USM group had significantly higher levels of Akt1 and Mapk3 gene expression than the other two groups. The most intense immunostaining for phosphor-AKT and phospho-ERK1/2 was detected in the cochlea in the USM group. These results suggested that USMB can be applied to enhance the efficacy of IGF-1 therapy in the treatment of inner ear diseases.

Effects of Androgen Receptor Inhibition on Kanamycin-Induced Hearing Loss in Rats.

Megalin has been proposed as an endocytic receptor for aminoglycosides as well as estrogen and androgen. We aimed to investigate the otoprotective effects of antiandrogens (flutamide, FM) on kanamycin (KM)-induced hearing loss in rats. Rats were divided into four groups. The KM group was administered KM (20 mg/kg/day) for 5 days, while the FM group received FM (15 mg/kg/day) for 10 days. In the KM + FM group, KM and FM (15 mg/kg/day) were simultaneously injected for 5 days and then FM was injected for 5 days. Auditory brainstem responses were measured. Western blotting and/or quantitative reverse transcriptase-polymerase chain reaction were performed for megalin, cytochrome P450 1A1 (Cyp1a1), Cyp1b1, metallothionein 1A (MT1A), MT2A, tumor necrosis factor (TNF)-α, caspase 3, and cleaved caspase 3. The FM + KM group showed attenuated auditory thresholds when compared with the KM group at 4, 8, 16, and 32 kHz (all p < 0.05). The KM + FM group showed lower megalin and Cyp1b1 levels than the KM group (all p < 0.05). The KM + FM group revealed lower MT1A, TNFα, and caspase 3 protein levels, compared with those in the KM group (all p < 0.05). Androgen receptor inhibition protects against cochlear injuries in KM-induced hearing loss rats by attenuating megalin expression, revealing anti-inflammatory and anti-apoptotic effects.

An in vitro model to assess the peripheral vestibulotoxicity of aromatic solvents.

Epidemiological and experimental studies indicate that a number of aromatic solvents widely used in the industry can affect hearing and balance following chronic exposure. Animal studies demonstrated that long-term exposure to aromatic solvents directly damages the auditory receptor within the inner ear: the cochlea. However, no information is available on their effect on the vestibular receptor, which shares many structural features with the cochlea and is also localized in inner ear. The aim of this study was to use an in vitro approach to assess and compare the vestibular toxicity of different aromatic solvents (toluene, ethylbenzene, styrene and ortho-, meta-, para-xylene), all of which have well known cochleotoxic properties. We used a three-dimensional culture model of rat utricles ("cysts") with preserved functional sensory and secretory epithelia, and containing a potassium-rich (K+) endolymph-like fluid for this study. Variations in K+ concentrations in this model were considered as biomarkers of toxicity of the substances tested. After 72 h exposure, o-xylene, ethylbenzene and styrene decreased the K+ concentration by 78 %, 37 % and 28 %, respectively. O- xylene and styrene both caused histopathological alterations in secretory and sensory epithelial areas after 72 h exposure, whereas no anomalies were observed in ethylbenzene-exposed samples. These in vitro results suggest that some widely used aromatic solvents might have vestibulotoxic properties (o-xylene, styrene and ethylbenzene), whereas others may not (p-xylene, m-xylene, toluene). Our results also indicate that variations in endolymphatic K+ concentration may be a more sensitive marker of vestibular toxicity than histopathological events. Finally, this study suggests that cochleotoxic solvents might not be necessarily vestibulotoxic, and vice versa.

Cisplatin-induced ototoxicity: Updates on molecular mechanisms and otoprotective strategies.

Cisplatin is a highly effective antitumor drug generally used in the treatment of solid malignant tumors. However, cisplatin causes severe side effects such as bone marrow depression, nephrotoxicity, and ototoxicity, thus limiting its clinical application. The incidence of ototoxicity induced by cisplatin ranges from 20% to 70%, and it usually manifests as a progressive, bilateral and irreversible hearing loss. Although the etiology of cisplatin-induced ototoxicity remains unclear, an increasing body of evidence suggests that the ototoxicity of cisplatin is mainly related to the production of reactive oxygen species and activation of apoptotic pathway in cochlear tissues. Many drugs have been well proved to protect cisplatin-induced hearing loss in vitro and in vivo. However, the anti-tumor effect of cisplatin is also weakened by systemic administration of those drugs for hearing protection, especially antioxidants. Therefore, establishing a local administration strategy contributes to the otoprotection without affecting the effect of cisplatin. This review introduces the pathology of ototoxicity caused by cisplatin, and focuses on recent developments in the mechanisms and protective strategies of cisplatin-induced ototoxicity.

Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

Investigation of Astaxanthin Effect on Cisplatin Ototoxicity in Rats by Using Otoacoustic Emission, Total Antioxidant Capacity, and Histopathological Methods.

BACKGROUND: Cisplatin-induced ototoxicity is related to oxidative stress. Astaxanthin is one of the most powerful antioxidants in nature. AIMS/OBJECTIVES: To investigate the protective effect of astaxanthin on cisplatin-induced ototoxicity. MATERIALS AND METHODS: Thirty-five Sprague Dawley female rats were divided into 5 groups: control, cisplatin, and cisplatin with 10, 20, and 40 mg/kg astaxanthin groups. Cisplatin group received a single intraperitoneal injection of 14 mg/kg cisplatin. While saline was administered in the control group, in the other 3 groups, 10, 20, and 40 mg/kg daily doses of astaxanthin were administered through orogastric cannula before administration of cisplatin. Baseline and 10th day otoacoustic emission tests were administered. An intracardiac blood sample was taken to measure total antioxidant capacity (TAC), and the cochleas of the animals were investigated histopathologically. RESULTS: Hearing level of astaxanthin 40 mg/kg + cisplatin group was higher at 24 kHz and 32 kHz frequencies compared to the cisplatin group. The TAC value of the cisplatin group was lower than both the control and astaxanthin + cisplatin groups (P < .05). On histopathological examination, the other groups were deformed compared to the control group, but no statistically significant difference was observed between the astaxanthin + cisplatin and cisplatin groups. CONCLUSIONS AND SIGNIFICANCE: Astaxanthin showed protective effect at high frequencies when it was administered at high dose. Thus, astaxanthin may have protective effect against cisplatin-induced ototoxicity.

Dose-Dependent Effects of Resveratrol on Cisplatin-Induced Hearing Loss.

Previous preclinical studies have demonstrated the otoprotective effects of resveratrol (RV) at low doses. This study aimed to investigate the dose-dependent effects of RV in rats with cisplatin (CXP)-induced hearing loss. Sprague-Dawley rats (8-weeks old) were divided into six treatment groups (n = 12/group) and treated as follows: control, 0.5 mg/kg RV, 50 mg/kg RV, CXP, 0.5 mg/kg RV + CXP), and 50 mg/kg RV + CXP groups. CXP (3 mg/kg) was intraperitoneally injected for 5 days. RV (0.5 or 50 mg/kg) was intraperitoneally injected for 10 days from the first day of CXP administration. Auditory brainstem response (ABR) thresholds were measured before and within 3 days at the end of the drug administration. Cochlear tissues were harvested, and the outer hair cells were examined using cochlear whole mounts. The mRNA expression of NFκB, IL6, IL1ß, and CYP1A1, and protein levels of aryl hydrocarbon receptor (AhR) and cytosolic and nuclear receptor for advanced glycation endproducts (RAGE) were evaluated. The ABR threshold increased in the 50 mg/kg RV and CXP groups at 4, 8, 16, and 32 kHz. The 0.5 mg/kg RV + CXP group demonstrated decreased hearing thresholds at 4 and 32 kHz compared to the CXP group. Cochlear whole-mount analysis revealed loss of outer hair cells in the 50 mg/kg RV and CXP groups and partial prevention of these cells in the 0.5 mg/kg RV + CXP group. The mRNA expressions of NFκB, IL6, and IL1ß were increased in the 50 mg/kg RV and CXP groups compared to the control group. In contrast, these levels were decreased in the 0.5 mg/kg RV + CXP group compared to the CXP group. The mRNA expression of CYP1A1 was increased in the CXP group, while it was decreased in the 0.5 mg/kg RV + CXP group compared to the control group. The protein levels of AhR and cytosolic RAGE decreased in the 0.5 mg/kg RV group. Low-dose RV had partial otoprotective effects on CXP ototoxicity. The otoprotective effects of RV may be mediated through anti-oxidative (CYP1A1 and RAGE) and anti-inflammatory (NFκB, IL6, and IL1ß) responses. High-dose RV exerted an inflammatory response and did not ameliorate CXP-induced ototoxicity.

Regulator of G Protein Signalling 4 (RGS4) as a Novel Target for the Treatment of Sensorineural Hearing Loss.

We and others have previously identified signalling pathways associated with the adenosine A1 receptor (A1R) as important regulators of cellular responses to injury in the cochlea. We have shown that the "post-exposure" treatment with adenosine A1R agonists confers partial protection against acoustic trauma and other forms of sensorineural hearing loss (SNHL). The aim of this study was to determine if increasing A1R responsiveness to endogenous adenosine would have the same otoprotective effect. This was achieved by pharmacological targeting of the Regulator of G protein Signalling 4 (RGS4). RGS proteins inhibit signal transduction pathways initiated by G protein-coupled receptors (GPCR) by enhancing GPCR deactivation and receptor desensitisation. A molecular complex between RGS4 and neurabin, an intracellular scaffolding protein expressed in neural and cochlear tissues, is the key negative regulator of A1R activity in the brain. In this study, Wistar rats (6-8 weeks) were exposed to traumatic noise (110 dBSPL, 8-16 kHz) for 2 h and a small molecule RGS4 inhibitor CCG-4986 was delivered intratympanically in a Poloxamer-407 gel formulation for sustained drug release 24 or 48 h after noise exposure. Intratympanic administration of CCG-4986 48 h after noise exposure attenuated noise-induced permanent auditory threshold shifts by up to 19 dB, whilst the earlier drug administration (24 h) led to even better preservation of auditory thresholds (up to 32 dB). Significant improvement of auditory thresholds and suprathreshold responses was linked to improved survival of sensorineural tissues and afferent synapses in the cochlea. Our studies thus demonstrate that intratympanic administration of CCG-4986 can rescue cochlear injury and hearing loss induced by acoustic overexposure. This research represents a novel paradigm for the treatment of various forms of SNHL based on regulation of GPCR.

Ferrostatin-1 protects auditory hair cells from cisplatin-induced ototoxicity in vitro and in vivo.

Cisplatin is used in a wide variety of malignancies, but cisplatin-induced ototoxicity remains a major issue in clinical practice. Experimental evidence indicates that ferroptosis plays a key role in mediating the unwanted cytotoxicity effect caused by cisplatin. However, the role of ferroptosis in cisplatin-induced ototoxicity requires elucidation. Ferrostatin-1 (Fer-1) was identified as a potent inhibitor of ferroptosis and radical-trapping antioxidant with its ability to reduce the accumulation of lipid peroxides and chain-carrying peroxyl radicals. In the current study, we investigated the effects of Fer-1 in cisplatin-induced ototoxicity in in vitro, ex vivo, and in vivo models. We found, for the first time that Fer-1 efficiently alleviated cisplatin-induced cytotoxicity in HEI-OC1 cells via a concentration-dependent manner. Furthermore, Fer-1 mitigated cisplatin cytotoxicity in transgenic zebrafish sensory hair cells. In HEI-OC1 cells, Fer-1 pretreatment not only drastically reduced the generation of intracellular reactive oxygen species but also remarkably decreased lipid peroxidation levels induced by cisplatin. This was not only ascribed to the inhibition of 4-hydroxynonenal, the final product of lipid peroxides, but also to the promotion of glutathione peroxidase 4, the protein marker of ferroptosis. MitoTracker staining and transmission electron microscopy of mitochondrial morphology suggested that in HEI-OC1 cells, Fer-1 can effectively abrogate mitochondrial damage resulting from the interaction with cisplatin. In addition, Fer-1 pretreatment of cochlear explants substantially protected hair cells from cisplatin-induced damage. Therefore, our results demonstrated that ferroptosis might be involved in cisplatin ototoxicity. Fer-1 administration mitigated cisplatin-induced hair cell damage, further investigations are required to elucidate the molecular mechanisms of its otoprotective effect.

Circadian vulnerability of cisplatin-induced ototoxicity in the cochlea.

The chemotherapeutic agent cisplatin is renowned for its ototoxic effects. While hair cells in the cochlea are established targets of cisplatin, less is known regarding the afferent synapse, which is an essential component in the faithful temporal transmission of sound. The glutamate aspartate transporter (GLAST) shields the auditory synapse from excessive glutamate release, and its loss of function increases the vulnerability to noise, salicylate, and aminoglycosides. Until now, the involvement of GLAST in cisplatin-mediated ototoxicity remains unknown. Here, we test in mice lacking GLAST the effects of a low-dose cisplatin known not to cause any detectable change in hearing thresholds. When administered at nighttime, a mild hearing loss in GLAST KO mice was found but not at daytime, revealing a potential circadian regulation of the vulnerability to cisplatin-mediated ototoxicity. We show that the auditory synapse of GLAST KO mice is more vulnerable to cisplatin administration during the active phase (nighttime) when compared to WT mice and treatment during the inactive phase (daytime). This effect was not related to the abundance of platinum compounds in the cochlea, rather cisplatin had a dose-dependent impact on cochlear clock rhythms only after treatment at nighttime suggesting that cisplatin can modulate the molecular clock. Our findings suggest that the current protocols of cisplatin administration in humans during daytime may cause a yet undetectable damage to the auditory synapse, more so in already damaged ears, and severely impact auditory sensitivity in cancer survivors.

2-Hydroxypropyl-ß-cyclodextrin Ototoxicity in Adult Rats: Rapid Onset and Massive Destruction of Both Inner and Outer Hair Cells Above a Critical Dose.

2-Hydroxypropyl-ß-cyclodextrin (HPßCD), a cholesterol chelator, is being used to treat diseases associated with abnormal cholesterol metabolism such as Niemann-Pick C1 (NPC1). However, the high doses of HPßCD needed to slow disease progression may cause hearing loss. Previous studies in mice have suggested that HPßCD ototoxicity results from selective outer hair cell (OHC) damage. However, it is unclear if HPßCD causes the same type of damage or is more or less toxic to other species such as rats, which are widely used in toxicity research. To address these issues, rats were given a subcutaneous injection of HPßCD between 500 and 4000 mg/kg. Distortion product otoacoustic emissions (DPOAE), the cochlear summating potential (SP), and compound action potential (CAP) were used to assess cochlear function followed by quantitative analysis of OHC and inner hair cell (IHC) loss. The 3000- and 4000-mg/kg doses abolished DPOAE and greatly reduced SP and CAP amplitudes. These functional deficits were associated with nearly complete loss of OHC as well as ~ 80% IHC loss over the basal two thirds of the cochlea. The 2000-mg/kg dose abolished DPOAE and significantly reduced SP and CAP amplitudes at the high frequencies. These deficits were linked to OHC and IHC losses in the high-frequency region of the cochlea. Little or no damage occurred with 500 or 1000 mg/kg of HPßCD. The HPßCD-induced functional and structural deficits in rats occurred suddenly, involved damage to both IHC and OHC, and were more severe than those reported in mice.

Pterostilbene protects cochlea from ototoxicity in streptozotocin-induced diabetic rats by inhibiting apoptosis.

Diabetes mellitus (DM) causes ototoxicity by inducing oxidative stress, microangiopathy, and apoptosis in the cochlear sensory hair cells. The natural anti-oxidant pterostilbene (PTS) (trans-3,5-dimethoxy-4-hydroxystylbene) has been reported to relieve oxidative stress and apoptosis in DM, but its role in diabetic-induced ototoxicity is unclear. This study aimed to investigate the effects of dose-dependent PTS on the cochlear cells of streptozotocin (STZ)-induced diabetic rats. The study included 30 albino male Wistar rats that were randomized into five groups: non-diabetic control (Control), diabetic control (DM), and diabetic rats treated with intraperitoneal PTS at 10, 20, or 40 mg/kg/day during the four-week experimental period (DM + PTS10, DM + PTS20, and DM + PTS40). Distortion product otoacoustic emission (DPOAE) tests were performed at the beginning and end of the study. At the end of the experimental period, apoptosis in the rat cochlea was investigated using caspase-8, cytochrome-c, and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin end labeling (TUNEL). Quantitative real-time polymerase chain reaction was used to assess the mRNA expression levels of the following genes: CASP-3, BCL-associated X protein (BAX), and BCL-2. Body weight, blood glucose, serum insulin, and malondialdehyde (MDA) levels in the rat groups were evaluated. The mean DPOAE amplitude in the DM group was significantly lower than the means of the other groups (0.9-8 kHz; P < 0.001 for all). A dose-dependent increase of the mean DPOAE amplitudes was observed with PTS treatment (P < 0.05 for all). The Caspase-8 and Cytochrome-c protein expressions and the number of TUNEL-positive cells in the hair cells of the Corti organs of the DM rat group were significantly higher than those of the PTS treatment and control groups (DM > DM + PTS10 > DM + PTS20 > DM + PTS40 > Control; P < 0.05 for all). PTS treatment also reduced cell apoptosis in a dose-dependent manner by increasing the mRNA expression of the anti-apoptosis BCL2 gene and by decreasing the mRNA expressions of both the pro-apoptosis BAX gene and its effector CASP-3 and the ratio of BAX/BCL-2 in a dose-dependent manner (P < 0.05 compared to DM for all). PTS treatment significantly improved the metabolic parameters of the diabetic rats, such as body weight, blood glucose, serum insulin, and MDA levels, consistent with our other findings (P < 0.05 compared to DM for all). PTS decreased the cochlear damage caused by diabetes, as confirmed by DPOAE, biochemical, histopathological, immunohistochemical, and molecular findings. This study reports the first in vivo findings to suggest that PTS may be a protective therapeutic agent against diabetes-induced ototoxicity.