A novel model of sensorineural hearing loss induced by repeated exposure to moderate noise in mice: the preventive effect of resveratrol

Abstract

Sensorineural hearing loss (SNHL) induced by noise has increased in recent years due to personal headphone use and noisy urban environments. The study shows a novel model of gradually progressive SNHL induced by repeated exposure to moderate noise (8-kHz octave band noise, 90-dB sound pressure level) for 1 hr exposure per day in BALB/cCr mice. The results showed that the repeated exposure led to gradually progressive SNHL, which was dependent on the number of exposures, and resulted in permanent hearing loss after 5 exposures. Repeated exposure to noise causes a loss of synapses between the inner hair cells and the peripheral terminals of the auditory nerve fibers. Additionally, there is a reduction in the expression levels of c-fos and Arc, both of which are indicators of cochlear nerve responses to noise exposure. Oral administration of resveratrol (RSV, 50 mg/kg/day) during the noise exposure period significantly prevented the noise exposure-induced synapse loss and SNHL. Furthermore, the study found that RSV treatment prevented the noise-induced increase in the gene expression levels of the proinflammatory cytokine interleukin-1β in the cochlea. These results demonstrated the potential usefulness of RSV in preventing noise-induced SNHL in the animal model established as gradually progressive SNHL.

Sensorineural hearing loss (SNHL) reduces the quality of life in humans and has been demonstrated to be a risk factor for cognitive impairment and depression as well as impacting life expectancy [17]. Thus, the prevention of SNHL is important for a healthy human life. In rodents and guinea pigs, a considerable body of evidence suggests that noise-induced SNHL arises due to increased oxidative stress in the cochlea and thus can be prevented by the systemic administration of antioxidant agents [21, 33, 37]. Additional evidence for the involvement of both oxidative stress and inflammation of the cochlea in SNHL comes from reports using animal models of age-related and noise-induced SNHL [13, 20]. The World Health Organization (WHO) is concerned about the potential risk of hearing impairment among 1.1 billion young people (ages 12–35) worldwide, who use portable music players and smartphones [25, 30]. However, the model of hearing loss resulting from the chronic process observed in clinical practice is limited to animals with spontaneous age-related hearing loss. Our previous report has demonstrated that repeated exposure to moderate noise, under conditions where a single exposure produces no effect on hearing ability, leads to hearing impairment [34]. Therefore, the first aim of this study is to establish a model for gradually progressive SNHL induced by moderate noise exposure.

Considerable evidence has demonstrated that the production of excess reactive oxygen species and nitric oxide results in oxidative stress in the cochlea, which is a cause of noise-induced SNHL [8, 22]. The SNHL induced by noise can be prevented by anti-oxidants and curcumin, which has antioxidant and anti-inflammatory effects [34]. These previous findings strongly support the idea that targeting both oxidative stress and inflammation could be useful as therapeutic strategies for noise-induced SNHL. In this study, thus, we focused on resveratrol (RSV), which is a compound derived from grapes and burdock, is a phytochemical that has strong antioxidant and anti-inflammatory effects [2, 3] and has been reported to prevent and treat cancer and cardiovascular diseases [12, 15]. Indeed, it has been shown that RSV suppresses acute hearing loss by promoting activation of silent information regulator-1 (Sirt1) [16]. Because RSV is a lipophilic small molecule, it would be expected to have a protective effect on SNHL as a result of its high permeability of the inner ear and its anti-oxidant/anti-inflammatory effects. This study also aimed to elucidate the impact of RSV on gradually progressive SNHL induced by moderate noise exposure.

MATERIALS AND METHODS

Animals and drug administration

All experiments were conducted in accordance with the guidelines for animal experiments of the Japanese Society of Pharmacology and were approved by the Committee on Animal Experimentation, Faculty of Pharmaceutical Sciences, Setsunan University. (No. 21-8 (2021), No. K22-8 (2022), No. K23-6 (2023)). Five-week-old female BALB/cCr mice (16–20 g) were purchased from Shimizu Experimental Material Co., Ltd. (Kyoto, Japan). Mice were maintained at a room temperature of 25 ± 2°C and humidity of 55% with ad libitum feeding and water intake under a 12 hr light/dark cycle (light period from 7:00 AM to 7:00 PM). To ensure that the mice had normal hearing ability, we checked their hearing ability before use and eliminated mice with natural HL. The inner ear has a blood labyrinth barrier like the blood-retina barrier in the retina and the blood-brain barrier in the brain. In previous studies, doses of 25 mg/kg and 200 mg/kg have been used in the retina [32] and the central nervous system [9], respectively. Based on these studies, RSV was suspended in 50% (w/v) polyethylene glycol, adjusted to a concentration of 1 or 5 mg/mL, and orally administered at 0.1 mL per 10 g body weight.

Noise exposure

Mice were once daily exposed to an octave-band of noise (5.4–11.2 kHz), centered at 8 kHz, at a sound pressure level (SPL) of 90-dB within a sound chamber for 1 hr. The sound chamber was equipped with a speaker (300HT, Fostex, Tokyo, Japan) driven by a noise generator (SF-06, Rion, Tokyo, Japan) and a power amplifier (DAD-M100proHT, Flying Mole, Hamamatsu, Japan). The SPL of the noise exposure was measured before and after the exposure using a sound level meter (NL −26; Rion) placed just under the speaker, confirming that there was uniform sound exposure. These operations for 5 days under the same experimental conditions are expressed as “repeated noise exposure” in this paper.

Auditory brainstem response (ABR) recording

Animals were anesthetized following the inhalation of isoflurane delivered at a flow rate of 1 L/min. Following this, stainless steel needle electrodes were subcutaneously inserted at the ventrolateral aspect and vertex of the left and right ears. Sound stimuli were produced using a condenser speaker (ESl; BioResearch Center, Nagoya, Japan) and ABR waveforms were recorded using a programmable attenuator (PA5 and System 3 Quick Start; Tucker-Davis Technologies, Alachua, FL, USA). As a hearing threshold, we defined the lowest stimulus intensity that produced wave I of the ABR waveforms. ABR was recorded from 100 dB to 5-dB SPL intervals until wave I was no longer visible.

Detection of synapses between the inner hair cells (IHCs) and peripheral terminals of the auditory nerve fibers (IHC synapses)

To detect IHC synapses, the organ of Corti was triple immunostained with specific antibodies against C-terminal binding protein-2 (CtBP2, a presynaptic marker in IHCs), the GluA2 subunit of the glutamate receptor (a maker of peripheral terminals of auditory nerve fibers) and myosin VIIa (a marker for IHCs) [5]. Briefly, the cochlea was perfused with 4% paraformaldehyde through an oval window and kept at room temperature for 2 hr to fix the organ of Corti. After fixation, decalcification was carried out in a 10% (w/v) EDTA solution at 4°C for 2 days. The organ of Corti was dissected in phosphate-buffered saline (PBS, pH 7.4) and then permeabilized by the addition of 1% Triton X-100 in PBS (TPBS) at room temperature for 1 hr. Using the mouse cochleogram as a guide [31] the organ of Corti was divided into six sections (Fig. 1a). Each section was incubated in a blocking solution consisting of 5% horse serum and 1% BSA in TPBS for 1 hr at room temperature. The sections were then incubated with the appropriate primary antibodies in 1% horse serum and TPBS at 37°C overnight. The antibodies used were mouse monoclonal antibodies against CtBP2 (1:200, BD Biosciences, Billerica, MA, USA) and GluA2 (1:200, Millipore, Burlington, MA, USA), and a rabbit polyclonal antibody against myosin VIIa (1:100, Proteus Biosciences Inc., Ramona, CA, USA). After washing with TPBS, the sections were then incubated with either an Alexa Fluor 568 goat anti-mouse IgG1 antibody (1:200), an Alexa Fluor 488 goat anti-mouse IgG2a antibody (1:200), or an Alexa Fluor 350 donkey anti-rabbit IgG (H+L) antibody (1:200) at 37°C for 2 hr. Sections were then analyzed using confocal laser microscopy. Adjacent CtBP2-positive patches and GluA2-positive patches stained within the Myosin-VIIa positive area were counted as IHC synapses.

Fig. 1.

Mice were once a day exposed to noise (90-dB sound pressure level, SPL, for 1 hr). The number of synapses per inner hair cell (IHC) was determined one day after noise exposure at each time point. (a) To eliminate area bias in the number of IHC synapses, the organ of Corti in the cochlea was divided into six sections (A–F). (b) To determine the number of IHC synapses, each section was stained with antibodies against myosin VIIa (blue), CtBP2 (red), and GluA2 (green). The number of IHC synapses was estimated by counting the number of patch structures with adjacent staining of CtBP2 and GluA2. The short-dashed lines denote the IHC results. Scale bar=10 μm (c) Change in the number of IHC synapses in each section following noise exposure. The graphs denote the percentage of the control value (number of noise exposures=0) prior to noise exposure. Values are presented as mean ± SEM from to 5–11 separate animals. *P<0.05, **P<0.01, significantly different from each control value obtained for animals immediately before the first noise exposure (number of noise exposures=0).

Quantitative RT-PCR

Total RNA was extracted from the cochlea using the NucleoSpin RNA/Protein Kit (Machery-Nagel). The total RNA was reverse transcribed using 1 μL of Rever Tra Ase, 4 μL of 5 × RT Buffer, 1 μL of ribonuclease inhibitor, 2 μL of dNTP mixture (10 mM), and 0.1 μL of Oligo (dT) 12–18 Primer to obtain cDNA. Quantitative RT-PCR was performed using SYBR Premix Ex Taq II (Takara Bio Inc., Kusatsu, Japan). The SYBR Green fluorescence intensity was analyzed using the Thermal Cycler Dice Real Time System (Takara Bio). The following primers were used to detect each gene. Mouse ribosomal protein, large, P0 gene (Rplp0; forward, 5′-CACTGGTCTAGGACCCGAGAAG-3′; reverse, 5′-GGTGCCTCTGGAGATTTTCG-3′), mouse c-Fos gene (c-fos; forward, 5′-ATCCTTGGAGCCAGTCAAGA-3′; reverse, 5′-ATGATGCCGGAAACAAGAAG-3′), mouse activity regulated cytoskeleton associated protein gene (Arc; forward, 5′-GTGAAGACAAGCCAGCATGA-3′; reverse, 5′-CCAAGAGGACCAAGGGTACA-3′), mouse sirtuin 1 gene (Sirt1; forward, 5′-CCTGACTTCAGATCAAGAGACGGT-3′; reverse, 5′-CTGATTAAAAATGTCTCCAGAACAG-3′), mouse interleukin-1β (IL-1β) gene (Il1b; forward, 5′-GAGTGT GGATCCCAAGCAAT-3′; reverse, 5′-TACCAGTTGGGGAACTCTGC-3′), mouse tumor necrotic factor-α (TNF-α) gene (Tnf; forward, 5′-CCGATGGGTTGTACCTTGTC-3′; reverse, 5′-AGATAGCAAATCGGCTGACG-3′). The values were normalized to the level of ribosomal protein, large, P0 gene under the same experimental conditions.

Statistical analysis

All values are presented as the mean ± SEM. An unpaired Student’s t-test was used to compare between two groups. A one-way ANOVA, followed by the Tukey-Kramer test was used to compare more than two groups. All statistical analyses were performed using JMP Pro software (SAS Institute Inc., Cary, NC, USA). The significance level was set at P<0.05.

RESULTS

Effects of repeated exposure to noise on neural activity in the cochlea

IHCs are capable of transmitting acoustic information to the brain by generating action potentials in auditory nerves through the release of neurotransmitters. The neural activity of auditory nerves regulating via IHC synapses is important for perception of sounds.

To elucidate if exposure of mice to noise affects the number of IHC synapses, we counted the number of IHC synapses in the organ of Corti. To determine the number of IHC synapses, the organ of Corti was divided into 6 sections (Fig. 1a), each of which was then triple immunostained with antibodies to CtBP2, GluA2, and myosin VIIa to allow for identification of IHC synapses (Fig. 1b). The number of IHC synapses per IHC was 12.1 ± 0.85, 12.7 ± 0.44, 14.1 ± 1.15, 16.5 ± 0.86, 13.3 ± 0.83, and 10.5 ± 1.5 in sections A to F, respectively. In sections D and E, the number of IHC synapses was significantly reduced by noise exposure (Fig. 1c). However, even with repeated noise exposure there were no changes in the number of IHC synapses in sections A, B, C, and F.

During neuronal activation of auditory nerves induced by acoustic stimulation, immediate-early genes such as c-fos and Arc are known to be expressed in the modiolus including auditory nerves [7, 35]. To estimate the cochlear neural activity caused by acoustic stimulation, we measured the expression levels of c-fos and Arc mRNAs after additional noise exposure to activate auditory nerves as acoustic stimulation (90-dB for 1 hr) in the modiolus of mice that were either not exposed or exposed to noise 5 times (Fig. 2). In the animals with not exposure to repeated noise, the expression levels of c-fos and Arc mRNAs were significantly higher than those in naïve animals. However, there was no significant change observed in the level of these mRNAs in animals with repeated exposure to noise. These results suggest that neural activity in auditory nerves is less responsive to noise in animals with SNHL induced by repeated noise exposure.

Fig. 2.

In mice that were either not exposure (0) or 5-times exposure to noise (90-dB sound pressure level, SPL, for 1 hr) (5), the expression levels of c-fos and Arc were measured in the modiolus 3 hr after additional exposure to noise (90-dB SPL for 1 hr) to activate auditory nerves. The graphs denote the percentage relative to the values obtained for naïve animals that were untreated. Values are presented as mean ± SEM from three separate animals. *P<0.05, significant difference from naïve animals. ^#P<0.05, significantly different between the values for animals with (5) or without (0) repeated noise exposure.

Effect of repeated exposure to noise on the expression of proinflammatory cytokines in the whole cochlea

Accumulating evidence suggests that inflammatory processes in the cochlea involve noise-induced SNHL in mice. In C57BL/6 mice, acute exposure to noise (100-dB SPL, 8–16 kHz, 24 hr) induced early expression of proinflammatory cytokines (TNF-α, IL-1β), chemokines, and cell adhesion molecules in the cochlea [29]. In rats, acute exposure to noise (124-dB SPL, 4 kHz, 2 hr) has been shown to cause a significant increase in IL-1β and a slight but not significant increase in TNF-α in the cochlea [4]. Changes in the expression of Il1b and Tnf genes, which are associated with inflammation, were analyzed following noise exposure under the experimental conditions using in the present study (Fig. 3). Il1b expression was enhanced 2 hr after final exposure to noise in both animals with once and 5-times noise exposure. However, no significant change in Tnf expression was seen in both animals, suggesting that TNF-α did not affect in the cochlea during developing SNHL under the current experimental conditions.

Fig. 3.

Mice were exposed to noise (90-dB sound pressure level, SPL, for 1 hr) once or once a day for 5 days. Two hours after the final exposure to noise, RNA was prepared from the whole cochlea and used to perform quantitative RT-PCR to determine the gene expression levels of Il1b and Tnf in the whole cochlea. The graphs denote the percentage relative to control naïve animals. Values are presented as mean ± SEM from 4 separate animals. *P<0.05, significantly different from naïve animals.

Effects of RSV on SNHL and IHC synapse loss induced by repeated exposure to noise

Although a single exposure to noise elicited a temporary loss of hearing, a repeated noise exposure led to a permanent loss of hearing at the frequencies of 12 and 20 kHz. The present study shows that a repeated once a day exposure to noise for a total of 5 days elicited a progressive elevation of the auditory threshold at frequencies of 12 and 20 kHz (Fig. 4). To evaluate if 5-time exposure to noise at 90 dB SPL resulted in permanent or temporary hearing loss, we continued to measure the ABR threshold until 6 days after the final exposure. In 5-times noise exposed animals, the elevated auditory threshold at 12 and 20 kHz was maintained for at least 6 days after the final noise exposure (threshold shift after the final noise exposure: 12 kHz: immediately, 10.3 ± 1.7; Day 6, 6.6 ± 1.3: 20 kHz: immediately, 34.2 ± 4.8; Day 6, 31.4 ± 5.3) [34].

Fig. 4.

Mice were once a day exposed to noise (90-dB sound pressure level, SPL, for 1 hr) for 5 days and orally administered vehicle or resveratrol (RSV, 10, 50 mg/kg, once a day) for 8 days before, as well as during, the noise exposure period. The hearing thresholds at frequencies of 12 and 20 kHz were measured 24 hr after noise exposure. Values are presented as mean ± SEM from 7–9 separate animals. *P<0.05, **P<0.01, significantly different from each control value obtained for animals treated with vehicle alone.

To evaluate the effect of RSV on the 5-times repeated noise exposure-induced elevation of the auditory threshold at frequencies of 12 and 20 kHz, RSV at a dose of 10 or 50 mg/kg/day was orally administered to animals exposed to noise (Fig. 4). Although RSV at 10 mg/kg/day was ineffective at altering the auditory threshold following noise exposure, RSV at 50 mg/kg/day was able to significantly reduce noise-induced auditory threshold at both frequencies.

In mice, sections D and E in the organ of Corti are known to be sensitive to frequencies of approximately 20 kHz [31]. Thus, we assessed the effect of RSV on the decrease in the number of IHC synapses in sections D and E after 5-times repeated noise exposure (Fig. 5). RSV at 10 mg/kg/day was capable of preventing the noise-induced loss of IHC synapses in section E, but not significantly in section D. Importantly, RSV at 50 mg/kg/day was significantly effective at preventing the loss of ICH synapses in both sections.

Fig. 5.

Mice were once a day exposed to noise (90-dB sound pressure level, SPL, for 1 hr) for 5 days and orally administered vehicle or resveratrol (RSV, 10, 50 mg/kg, once a day) for 8 days before, as well as during, the noise exposure period. One day after the final exposure to noise, the number of IHC synapses in sections D and E of the organ of Corti were determined by triple staining for myosin VIIa, CtBP2, and GluA2. The graphs denote the percentage relative to the control naïve animals. Values are presented as mean ± SEM from 4–13 separate animals. *P<0.05, significant difference from each value obtained for naïve animals. ^#P<0.05, significantly different from the control value obtained for animals treated with vehicle alone.

Generally, systemically administered compounds are thought to reach cochlear tissues at a very low rate [11, 27]. To confirm if RSV is distributed and pharmacologically effective in the cochlea following oral administration under the current experimental conditions, we examined the effect of the oral administration of RSV on the expression of Sirt1, the level of which is well-known to increase in various tissues including the hippocampus following RSV treatment [24]. As a result, RSV at a dose of 50 mg/kg for 3 days markedly elevated Sirt1 level in the whole cochlea (Fig. 6), strongly supporting that the oral administration of RSV is useful for evaluating the pharmacological effect of RSV within the cochlea.

Fig. 6.

Mice were orally administered vehicle or resveratrol (RSV, 50 mg/kg, once a day) for 3 days. One day after the final administration, the whole cochlea was dissected to allow for a determination of the gene expression levels of Sirt1. The graph denotes the percentage relative to control values obtained for animals treated with vehicle alone. Values are presented as mean ± SEM from 3–5 separate animals. *P<0.05, significantly different from the control value obtained for animals treated with vehicle alone.

Effect of RSV on noise-induced changes in the expression of proinflammatory cytokines in the whole cochlea

Resveratrol is known to inhibit TNF-α-induce inflammation through activation of sirtuin 1 and suppression of nuclear factor-κB transcription activity in fibroblasts, lymphoid, and epithelial cells [19, 38]. In addition to these findings, the previous findings that resveratrol abolished the elevation of TNF-α and IL-1β in a lipopolysaccharide-induced preterm mouse model [6] led us to evaluate whether RSV abolishes the noise-induced changes in these cytokines.

Figure 7 shows the effect of RSV on noise-induced changes in the levels of Il1b and Tnf in the whole cochlea. Repeated noise exposure significantly elevated the gene expression levels of Il1b. Importantly, RSV (50 mg/kg/day) treatment completely abolished the noise-induced elevation of Il1b levels in the cochlea. However, no significant changes in Tnf levels were seen in the cochlea of RSV-treated animals exposed to repeated noise.

Fig. 7.

Mice were exposed to noise once a day (90-dB sound pressure level, SPL, for 1 hr) for 5 days and orally administered vehicle or resveratrol (RSV, 50 mg/kg, once a day) for 8 days before, as well as during, the noise exposure period. Two hours after the final exposure to noise, RNA was prepared from the whole cochlea and used to perform quantitative RT-PCR in order to determine the gene expression levels of Il1b and Tnf in the whole cochlea. The graphs denote the percentage relative to control naïve animals. Values are presented as mean ± SEM from 3–9 separate animals. **P<0.01, significant difference from each value obtained for naïve animals. ^#P<0.05, significantly different from the control value obtained for animals treated with vehicle alone.

DISCUSSION

SNHL is increasing concomitantly with the increase in the global population, particularly in the elderly population. SNHL progressively exacerbates with age and compromises their quality of life. Recently, noise-induced sensorineural hearing loss (SNHL) has become more prevalent due to personal headphone use and noisy urban environments, including road, rail, and air traffic, industrial facilities, civil construction, and social activities such as parties, fairs, open-air markets, and residential noise. To address these issues, it is crucial to establish animal models that mimic SNHL induced by moderate noise exposure in urban environments and to develop prevention materials for noise-induced SNHL. The aim of this study was to develop an animal model and a prevention material. The results demonstrate that repeated exposure to moderate noise under the experimental conditions produced a useful model of gradually progressive SNHL. The topic of the present study is that daily oral administration of RSV at a dose of 50 mg/kg/day had a preventive effect on a novel model of SNHL, which is gradually developed during repeated exposure to moderate noise.

In the present study, a single exposure to noise caused temporary elevation of the auditory threshold at frequencies of 12 and 20 kHz, and decreased the number of IHC synapses in sections D and E, which are coincident with frequencies of approximately 20 kHz according to the mouse cochleogram. These findings let us speculate that a decrease in IHC synapses is not essential for permanent SNHL. However, a previous report demonstrated that acoustic overexposure causing temporary hearing impairment resulted in an acute loss of afferent nerve terminals (equivalent to IHC synapses in the current study) and a delayed degeneration of the cochlear nerve [14]. A further important point in the current study is that the decrease in the number of IHC synapses was maintained in animals with repeated noise exposure, causing permanent SNHL. These findings strongly support the idea that prolonged loss of IHC synapses triggers severe degeneration of the cochlear nerve and permanent SNHL. On the other hand, the data presented in Fig. 1 raises the question as to why the noise-induced decrease in synapses was not significantly exacerbated by exposure to noise 5 times, compared to exposure only once, in sections D and E. The interpretation of this phenomenon can be challenging. It could be approached in the following way: the decrease in synapse count is due to excessive acoustic stimulation of the inner ear. This is followed by an enhanced protective mechanism against excessive stimulation and an enhanced regeneration mechanism for lost synapses. If noise exposure is repeated, the protective effect may prevent further synapse reduction. It is speculated that the lack of statistical change after one exposure compared to five exposures may be related to this protective effect. However, further investigation is required to fully understand these phenomena.

The inner ear is known to have a blood-labyrinth barrier, which has a structure similar to the blood-brain barrier, for regulating the transportation of substances from blood to the inner ear tissues. As a result, a low rate of delivery of systemically administered drugs into the inner ear tissues is often observed [23, 36]. In the present study, however, we found that oral administration of RSV significantly increased the gene levels of Sirt1 in the whole cochlea, suggesting that RSV is effectively delivered to the cochlea. This finding is in keeping with the known properties of RSV. It is a highly lipophilic compound with a molecular weight of 500 or less with high tissue permeability. In addition, a kinetic study using rats orally treated with isotopically labeled RSV, found that RSV was delivered into the brain through the blood-brain barrier [1].

In the present study, repeated noise exposure (90-dB SPL, 5.4–11.2 kHz, 1 hr/day, 5 days) of BALB/cCr mice significantly elevated the expression of Il1b, concomitant with no change in Tnf, in the cochlea. The findings that permanent SNHL was observed under the experimental conditions in the present study led us to propose that the proinflammatory cytokine IL-1β functions as a common signaling factor in the cochlea to cause permanent SNHL induced by noise. This proposal is supported by the current finding that continuous administration of RSV prevented the repeated noise-induced elevation in the expression of Il1b in the cochlea. Interestingly, RSV was capable of protecting IHC synapses in the organ of Corti from the effects of exposure to repeated noise. This finding led us to speculate that elevated expression of Il1b contributes to the noise-induced decrease in IHC synapses. Noise exposure can infiltrate immune cells, such as monocytes, into the inner ear [10, 28]. Additionally, it has been reported that the NOD-like receptor protein 3 inflammasome is involved in noise-induced SNHL through an inflammatory cascade [26]. The inflammasome activates caspase-1, which causes the maturation of Il1b [18]. Therefore, RSV may exert its anti-inflammatory effects by suppressing Il1b expression, which subsequently inhibits macrophage infiltration. However, the preventive effect of RSV may be due to various functions rather than anti-inflammatory effects. Further studies are needed to elucidate the involvement of IL-1β in noise-induced SNHL through a decrease in IHC synapses in the organ of Corti.

In conclusion, a new model has been developed for gradually progressive SNHL caused by repeated exposure to moderate noise, which is equivalent to personal headphone use and noisy urban environments. Additionally, it was found that the oral administration of RSV had a preventive effect on gradually progressive SNHL by alleviating the decrease in the number of IHC synapses in the cochlea of BALB/cCr mice. Therefore, the supplemental intake of RSV could be a new strategy for preventing SNHL.

CONFLICT OF INTEREST

We declare that we have no conflicts of interest.