INTRODUCTION

The World Health Organization estimates that more than 400 million people worldwide are afflicted by disabling hearing loss (1). Noise-induced hearing loss (NIHL) significantly contributes to this global burden, and its incidence is expected to increase. To date, there are no Food and Drug Administration (FDA)–approved therapeutics to prevent or treat NIHL, although many are currently under investigation, and hearing aids and cochlear implants inadequately address the underlying cause of hearing loss (2).

The pathophysiology of NIHL is multifactorial and involves a host of cellular and molecular aberrations within the numerous cell types of the cochlea (3). In a recently published collaborative study, we describe the cell type–specific transcriptional response to permanent threshold shift (PTS)–inducing noise in the mouse cochlea (4). Of note, in this study, cochlear tissues from male and female mice were pooled and analyzed together. We then sought to identify FDA-approved candidate therapeutics to counteract these noise-induced transcriptional changes. We cross-referenced our data with the drug-target interaction data from the DrugCentral database and generated a list of potential therapeutics. Multiple drugs were identified, including statins and inhaled anesthetics, but the drug with the most potential targets was the antidiabetic drug metformin.

Previous reports in the literature suggest that metformin treatment may reduce hearing loss after exposure to PTS-inducing noise in animal models (5,6). However, to date, the rigorous evaluation of metformin's otoprotective effects against hearing loss in animals of both sexes has not been completed. Furthermore, recent studies demonstrate the importance of hormonal status and biological sex in the susceptibility to NIHL and in the efficacy of potential therapeutics for hearing loss (7,8). In particular, endogenous estrogens are known to have a protective effect on hearing (9). As such, it is critical to perform thorough investigation of candidate therapeutics using subjects from both sexes.

In this study, we treated intact male and female mice with metformin or saline, as a control, in their drinking water, from 7 d before noise exposure until 7 d after. A cohort of ovariectomized female mice was included to simulate menopause and eliminate the effect of ovarian-derived estrogens. We measured the auditory function of all cohorts of mice with auditory brainstem response (ABR) testing 1 week before, as well as 1-day and 1-week after noise exposure. Cochlear tissue was analyzed for outer hair cell (OHC) loss and for loss of inner hair cell (IHC) paired synapses (cochlear synaptopathy). Our results establish metformin as a potential otoprotective agent in male mice.

MATERIALS AND METHODS Animals

All animal testing and procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Male and female B6CBAF1/J mice were purchased at 7 to 8 weeks of age from The Jackson Laboratory and housed in the School of Medicine animal facility. Mice were acclimated for 1 week before any intervention. The facility is temperature controlled and has a 12-hour light/dark cycle. The mice had access to food and water ad libitum.

Ovariectomy

The procedure was performed at 8 weeks of age, after 1 week of acclimation. Anesthesia was achieved with isoflurane gas. The ovariectomy was performed by making a 5-mm, longitudinal, dorsal incision through the skin and muscle wall. The ovary was separated from the uterus lateral to the oviduct using electrocautery, and the uterine horn was repositioned in the abdominal cavity. The muscle wall was closed using a 4-0 poliglecaprone suture (MWI Animal Health, Boise, ID), and the skin was closed using a 4-0 nylon suture (MWI Animal Health). The same procedure was then repeated on the contralateral ovary. Incision sites were treated with a 2.5% lidocaine and 2.5% prilocaine cream (Akorn, Lake Forest, IL) and Bacitracin Zinc antibiotic ointment (Trifecta Pharmaceuticals, Fort Lauderdale, FL). Carprofen (0.05 mg/kg) was administered by subcutaneous injection immediately before the surgical procedure and 24 h postoperatively for analgesia.

Treatments

Control (saline) and metformin treatments were administered via the drinking water. In humans, metformin is dosed at 1000 to 2000 mg/d (10). Based on allometric scaling, the equivalent dose in a mouse is 175 to 300 mg/kg/d (11). We administered a target dose of 200 mg/kg/d, which is consistent with previous studies reported in the literature (10). To achieve this, pharmaceutical-grade metformin hydrochloride was reconstituted in 0.9% normal saline and then injected directly into the water pouch. An equal volume of 0.9% normal saline was injected into the water pouches of the control-treated mice.

ABR Testing

ABRs were performed using methods described previously (7,9). Animals were anesthetized using an intraperitoneal injection of ketamine (100 mg/kg; VetOne, Boise, ID) and xylazine (20 mg/kg; Akorn, Gurnee, IL). Using the RZ6 recording system (Tucker-Davis Technologies, Alachua, FL), hearing thresholds were determined at 8, 16, 24, and 32 kHz in an acoustically controlled box (IAC Acoustics, Naperville, IL). Once surgical levels of anesthesia were achieved, animals were positioned so that they directly faced the Fostex FT17H tweeter (Fostex, Tokyo, Japan) with their ears 10 cm from the front of the tweeter. Subcutaneous electrodes were placed at the postauricular area of the left and right ears, and at the vertex of the skull for the reference. The ground electrode was placed at the base of the tail. Output stimuli were calibrated with a measurement microphone (PCB Piezotronics, Depew, NY) placed at the same distance from the tweeter as the mouse's ears (10 cm). Frequency-specific tone bursts 2.5-ms long, with a 0.5-ms sinusoidal on- and off-ramp, were delivered with alternating polarity beginning at 90 dB SPL. Tone bursts were progressively decreased by 5 dB until 10 dB below the measurable hearing threshold for each mouse. Electrophysiologic responses to each tone stimulus, filtered between 300 and 3000 Hz, were recorded for 10 ms starting at the onset of the tone, with a total of 512 sweeps at a rate of 21 sweeps per second, and then averaged at each sound level and frequency tested. The responses from each ear were recorded simultaneously. The hearing threshold was defined as the lowest level at which either ABR waves I and II could be identified. Thresholds were averaged between the two ears and reported as a single value per animal.

Noise Exposure

Noise exposures were performed using the methods previously described (9). All mice underwent calibrated noise exposure at 10 weeks of age at the same time of day (8:00 am). Octave-band noise centered at 11.3 kHz (8–16 kHz) was delivered for 2 h using the Fostex FT17H tweeter (Fostex). Noise was delivered at 102.5 dB sound pressure level (SPL) to induce a PTS in the males and ovariectomized females or at 105 dB SPL to induce a PTS in the female mice. Intact female mice were exposed to a louder noise due to their relative protection from NIHL (7). Calibration was achieved with a measurement microphone (PCB Piezotronics) placed at the same distance from the tweeter as the mouse's ears. The tweeter was placed 20 cm above the mice, and the sound level was measured to be within 0.5 dB of the target level. During the exposure, mice were placed in a custom holder constructed of perforated aluminum sheets (18 × 15 × 5 cm) with eight equal-sized chambers (4.5 × 7.5 × 5 cm) in an acoustically controlled box (IAC Acoustics). Only the four central compartments were used to expose a maximum of four mice simultaneously. The animals were awake and unrestrained throughout the exposure.

Immunostaining

Immunostaining was performed as previously described with minor modifications (7,9). Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, Tewksbury, MA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4°C overnight. After adequate decalcification by incubation in 500 mM EDTA (3–6 d), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories (12). The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technologies, Danvers, MA). Presynaptic ribbons and postsynaptic densities were labeled using a monoclonal mouse anti-CtBP2 antibody (1:200; BD Biosciences, San Jose, CA) and a monoclonal mouse anti-GluR2 antibody (1:2000; Sigma-Aldrich), respectively. After this, the tissue was incubated with the corresponding secondary antibodies, goat antimouse IgG2 Alexa Fluor® 488 and goat antimouse IgG1 Alexa Fluor® 568 (1:1000; ThermoFisher Scientific, Waltham, MA). Nuclei were counterstained with DAPI. The labeled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific).

Cochlear Frequency Mapping

Frequency mapping was performed as previously described (7,9). Tissue was imaged using a Nikon Eclipse E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with an Infinity 3 camera (Lumenera, Ottawa, Canada), and the images were processed with the Measure Line plugin for ImageJ developed by the Eaton-Peabody Laboratories (13).

IHC Synapse Counts

Stained tissues were imaged at the regions around 8, 16, 24, and 32 kHz using a ‌‌Nikon W1 spinning disk confocal on a Nikon Ti2 inverted microscope with a 60× oil objective at 0.2-μm sections. The equipment was provided by the School of Medicine Center for Innovative Biomedical Resources Core Confocal Laboratory. IHC synapses were counted on Z-stacks using the ImageJ Cell Counter plugin. Paired synapses were identified via colocalization of GluR2 and CtBP2.

Cytocochleograms

OHC nuclei counterstained with DAPI were imaged throughout the length of the cochlea using a NikonEclipse E600 microscope (Nikon) equipped with an Infinity 3 camera (Lumenera). The counts were expressed as percentage of missing hair cells in 2-kHz intervals and binned within the following frequency regions: 0–4, 4–5.6, 5.6–8, 8–11.3, 11.3–16, 16–22.6, 22.6–32, and 32–45.2 kHz.

Experimental Paradigm

Mice were randomly assigned to the treatment arms. At 9 weeks of age, mice underwent ABR testing to establish baseline auditory thresholds. After the baseline ABR, treatments were administered via the drinking water for the remaining duration of the study. At 10 weeks of age, mice were noise exposed, as described. Twenty-four hours after the noise exposure, the mice underwent ABR testing to quantify the acute hearing loss (also known as the compound threshold shift). One week after the noise exposure, the mice underwent a final round of ABR testing to represent the permanent hearing thresholds. After the 1-week ABR, the mice were euthanized and the temporal bones were harvested for histological analysis of the cochleae.

Statistics

All statistical analyses were performed using Prism 9 software (GraphPad, San Diego, CA). ABR thresholds, OHC counts, and synapse counts were compared between conditions within each cohort using a two-way analysis of variance and the appropriate post-hoc test. An adjusted p value of <0.05 was set as the threshold for statistical significance. Cohen d was calculated for all comparisons that reached statistical significance.

RESULTS Metformin Treatment in Male Mice

The experimental schematic for the treatment of male mice is presented in Figure 1A. Male mice at 8 weeks of age were randomly assigned to treatment with metformin or saline control. A baseline ABR obtained before the initiation of treatment showed no significant difference in hearing thresholds between groups (Fig. 1B). The mice were then subjected to a noise exposure of 102.5 dB SPL. One day after the noise exposure, metformin-treated male mice displayed significantly reduced 24-hour ABR threshold shifts at 16 kHz (mean, 40.2 dB) compared with control-treated mice (mean, 49.8 dB; p < 0.05; d = 1.29; Fig. 1C). One week after noise exposure, metformin-treated male mice displayed significantly reduced 1-week ABR threshold shifts at 16 kHz (mean, 33.9 dB) and 24 kHz (mean, 19.6 dB) compared with control-treated male mice at 16 kHz (mean, 44.8 dB; p < 0.01; d = 1.20) and 24 kHz (mean, 30.4 dB; p < 0.01; d = 1.15; Fig. 1D).

FIG. 1:

Metformin demonstrates otoprotective effects in male mice after a permanent threshold shift–inducing noise exposure. A, Schematic of the experimental protocol for treatment of male mice. B, At baseline, metformin-treated and control-treated male mice display no differences in ABR thresholds. C, Metformin-treated male mice demonstrate a reduced 24-hour post-exposure threshold shift at 16 kHz (mean, 40.2 versus 49.8 dB shift; p < 0.05; d = 1.29). D, Metformin-treated male mice demonstrate a reduced 1-week post-exposure threshold shift at 16 kHz (mean, 33.9 versus 44.8 dB shift; p < 0.01; d = 1.20) and 24 kHz (mean, 19.6 versus 30.4 dB shift; p < 0.01; d = 1.15). E, Compared with metformin-treated male mice, control-treated male mice displayed significantly increased OHC loss between 32 and 45.2 kHz (0.47% versus 4.7% loss; p < 0.0001; d = 2.37). F, Metformin treatment does not protect against inner hair cell–paired synapse loss in male mice. ABR thresholds, percentage of OHC loss, and number of paired synapses were compared using two-way ANOVA. Cohen’s d was calculated for all comparisons that reached statistical significance. Plots: Mean and standard deviation, n = number of mice. *p < 0.05, **p < 0.01, **** p < 0.0001. ABR indicates auditory brainstem response; ANOVA, analysis of variance; NE, noise exposure (102.5 dB SPL, 8–16 kHz, 2 h); OHC, outer hair cell.

Histological analysis of cochlear tissue 1 week after noise exposure revealed significantly reduced OHC loss in the 32- to 45.2-kHz frequency region of the cochlea in metformin-treated male mice compared with control-treated male mice (0.47% loss in the metformin cohort versus 4.7% in the control cohort; p < 0.0001; d = 2.37; Fig. 1E). However, there were no differences in OHC loss in the regions corresponding to 16 and 24 kHz. Quantification of the number of paired IHC synapses revealed a robust loss, consistent with the 1-week threshold shifts, with no difference between the two conditions at any frequency examined (Fig. 1F). No IHC loss was observed in either condition.

Metformin Treatment in Female Mice

The experimental schematic for the treatment of intact female mice is presented in Figure 2A. Female mice at 8 weeks of age were randomly assigned to treatment with metformin or saline control. A baseline ABR obtained before the initiation of treatment showed no significant difference in hearing thresholds between groups (Fig. 2B). The mice were then subjected to a noise exposure of 105 dB SPL. In contrast to the metformin-treated males, metformin-treated female mice showed no reduction in the 24-hour threshold shift compared with the control-treated female mice (Fig. 2C). Similarly, there were no differences in the 1-week hearing threshold shifts (Fig. 2D). Analysis of cochlear tissue 1 week after exposure revealed no differences in either OHC loss or paired IHC synapses between control-treated and metformin-treated female mice (Fig. 2, E and F). No IHC loss was observed in either condition.

FIG. 2:

Metformin does not demonstrate an otoprotective effect in intact female mice after a permanent threshold shift–inducing noise exposure. A, Schematic of the experimental protocol for treatment of intact female mice. B, At baseline, metformin-treated and control-treated female mice display no differences in ABR thresholds. C, There are no significant differences in 24-hour post-exposure threshold shifts between metformin-treated and control-treated female mice. D, There are no significant differences in 1-week post-exposure threshold shifts between metformin-treated and control-treated female mice. E, Compared with metformin-treated female mice, control-treated female mice display the same amount of OHC loss. F, Metformin treatment does not affect the number of inner hair cell–paired synapses in female mice. ABR thresholds, percentage of OHC loss, and number of paired synapses were compared using two-way ANOVA. Plots: Mean and SD, n = number of mice. ABR indicates auditory brainstem response; ANOVA, analysis of variance; NE, noise exposure (105 dB SPL, 8–16 kHz, 2 h); OHC, outer hair cell.

Metformin Treatment in Ovariectomized Female Mice

To determine whether ovarian-derived estrogens influence the outcome of metformin treatment in female mice, we included a model of menopause induced via surgical ovariectomy. The experimental schematic for the treatment of ovariectomized female mice is presented in Figure 3A. Female mice underwent bilateral ovariectomy at 8 weeks of age and then were randomly assigned to treatment with metformin or saline control. A baseline ABR obtained before the initiation of treatment showed no significant difference in hearing thresholds between groups (Fig. 3B). The mice were then subjected to a noise exposure of 102.5 dB SPL. Similar to the intact females, there were no changes in 24-hour or 1-week ABR threshold shifts between control-treated and metformin-treated ovariectomized female mice (Fig. 3, C and D). Analysis of cochlear tissue revealed that metformin-treatment in ovariectomized female mice did not affect OHC loss or the number of paired IHC synapses 1 week after exposure (Fig. 3, E and F). No IHC loss was observed in either condition.

FIG. 3:

Metformin does not demonstrate an otoprotective effect in ovariectomized female mice after a permanent threshold shift–inducing noise exposure. A, Schematic of the experimental protocol for treatment of ovariectomized female mice. B, At baseline, metformin-treated and control-treated ovariectomized female mice display no differences in ABR thresholds. C, There are no significant differences in 24-hour post-exposure threshold shifts between metformin-treated and control-treated ovariectomized female mice. D, There are no significant differences in 1-week post-exposure threshold shifts between metformin-treated and control-treated ovariectomized female mice. E, Compared with metformin-treated ovariectomized female mice, control-treated ovariectomized female mice display the same amount of OHC loss. F, Metformin treatment does not affect the number of inner hair cell–paired synapses in ovariectomized female mice. ABR thresholds, percentage of OHC loss, and number of paired synapses were compared using two-way ANOVA. Plots: Mean and SD, n = number of mice. ABR indicates auditory brainstem response; ANOVA, analysis of variance; NE, noise exposure (102.5 dB SPL, 8–16 kHz, 2 h); OHC, outer hair cell; OVX = ovariectomy.

DISCUSSION

Metformin is a biguanide antihyperglycemic agent, currently used as the first-line oral treatment for type 2 diabetes. It is an inexpensive, well-tolerated, FDA-approved medication with a potential for impact in the treatment of many disease states (14,15). There are several ongoing studies to investigate a role for metformin in cardiac protection, neural protection, anti-neoplastic effects, and hearing loss (5,6,10,16). For example, the use of metformin has demonstrated potential otoprotective effects, such as preventing ototoxicity from gentamicin treatment in murine cochlear explants (17) and protecting against NIHL in a female rat model (5). Metformin also has a potential role for hearing protection in humans; in a retrospective review, the incidence of sudden sensorineural hearing loss was lower in diabetic patients treated with metformin than those without metformin treatment (18). Of note, the hazard ratio was even lower in male patients on metformin (18). In addition, we identified metformin as the top-ranking candidate therapeutic predicted to reverse transcriptomic changes that are acutely observed in mice with NIHL (4).

This study validates our molecular analyses suggesting a role for metformin in protection from noise exposure (4) and establishes that metformin does have otoprotective effects in vivo. The exact mechanism of how metformin protects against NIHL remains to be identified. Metformin has a complex and pleiotropic mechanism of action. In addition to glucose control, metformin plays key roles in decreasing oxidative stress and fatty acid synthesis. Metformin activates AMP kinase via phosphorylation, leading to downstream inhibition of HMG-CoA reductase, which results in decreased mevalonic acid production (Fig. 4) (10). A study investigating the effects of AMP kinase found that AMP kinase–deficient mice are more susceptible to acoustic trauma, indicating that this pathway is likely involved in the otoprotective effects of metformin (19). Interestingly, in addition to metformin, statin drugs and bisphosphonates were also identified as highly ranking therapeutic candidates to target transcriptomic changes observed in mice with NIHL (3). Statins are cholesterol-lowering drugs that directly inhibit HMG-CoA reductase and have been shown to reduce noise-induced hearing loss in animal models (20) and protect from cisplatin-induced ototoxicity in humans (21). Bisphosphonates have also been shown to reduce NIHL in animal models, potentially mediated by the mevalonic acid pathway (22), and to prevent progressive sensorineural hearing loss in patients with otosclerosis (23). Our previous cochlear transcriptomic analysis of mice exhibiting NIHL showed significantly increased expression of the HMG-CoA reductase encoding gene, Hmcgr, after noise exposure in both OHCs and supporting cells (4). Therefore, inhibition of the mevalonic acid pathway serves as a potential mechanism of action for metformin to protect against NIHL.

FIG. 4: Metformin's role in the mevalonic acid pathway. Metformin activates AMP kinase, which leads to downstream inhibition of HMG-CoA reductase, ultimately resulting in a decrease in mevalonic acid production. Of note, the statins and bisphosphonate classes of medication also inhibit this pathway. Statins directly inhibit HMG-CoA reductase, and bisphosphonates inhibit a downstream target, farnesyl diphosphate synthase. Our previous transcriptomic analysis of mice with NIHL identified metformin as the top candidate therapeutic with the most potential molecular targets of action (3). This analysis also identified statins and bisphosphonates as potential therapeutics. The mevalonic acid pathway serves as a potential unifying mechanism of action for these medications to provide otoprotective effects. NIHL indicates noise-induced hearing loss.

Our calibrated sound exposure is an 8- to 16-kHz exposure resulting in a maximal acoustic trauma at the 16- to 24-kHz range, and our results demonstrate that metformin can protect against hearing loss in this range. Although a potential molecular pathway has been identified, the cellular mechanism of action remains unclear. Metformin's otoprotection may not be mediated by prevention of OHC loss or IHC synapse loss in the 16- to 24-kHz range; it may still prevent OHC dysfunction or synaptopathy. Our noise exposure for the male mice also results in milder trauma at the higher frequencies. Interestingly, the metformin-treated male mice displayed significantly reduced OHC loss in the 32- to 45.2-kHz range. The protection at the higher frequencies may indicate a role for metformin in age-related hearing loss, which predominantly affects this frequency range. It would be clinically significant if metformin is found to be effective in preventing both noise-induced and age-related hearing loss. Although some studies have identified potential mechanisms for metformin to prevent aging (24) and age-related hearing loss (25), there are no existing studies of age-related hearing loss in humans, to our knowledge. Unfortunately, retrospective analysis of existing large human cohorts is limited, as currently, metformin is not routinely prescribed to patients without diabetes. To fully understand the effects of metformin on hearing loss, further investigation into the cellular and molecular mechanisms of action in a variety of hearing loss models is required. Although this study demonstrates promising results for the use of metformin as an otoprotective agent in mice, additional studies need to be performed in human subjects.

Our physiological and histological data demonstrate that metformin treatment protects against NIHL in male mice but not in intact or ovariectomized female mice. Importantly, these data demonstrate the critical importance of evaluating therapeutic efficacy in a biological sex–specific manner. It is well demonstrated in the literature that there are sex differences in the susceptibility to NIHL as well as the response to therapeutics (7,9). For example, in a mouse model of cisplatin-induced ototoxicity, lovastatin provided otoprotection, but the relationship is dose dependent by sex; the female mice were found to have greater protection by a higher dose of lovastatin, whereas the male mice were found to have greater protection by a lower dose (26). Other studies suggest that there may be a relationship between metformin and hormonal signaling. For example, metformin has been found to cause changes in estrogen receptor expression in endometrial and breast cancer (16,27). Mindful of sex differences in hearing based on our previous work (7), multiple cohorts were included in this study. Intact female mice were exposed to a louder noise intensity to generate a consistent PTS. A cohort of ovariectomized female mice was also included and exposed to an identical noise intensity to the male mice because they have lost endogenous protection from noise exposure (9). Although the ovariectomized female mice again demonstrated a similar response to the noise stimulus as the male mice, neither female cohort benefitted from metformin. A lack of otoprotective effects of metformin in female mice may be secondary to insufficient metformin dosing or different metabolism in the female mice (8). Overall, there is a known paucity of clinical trial data regarding the study of disease and effects of medications on women (28). Further work is required to understand sex differences in the metabolism and effects of metformin in animal models and humans (29).

The lack of otoprotective effects in our female mice cohorts contradicts previous data obtained by Gedik et al. (5), which demonstrated a protective effect in female rats at all frequencies. However, the discrepancy may be explained by a few factors. The results obtained by Gedik et al. may be confounded by the method of metformin administration. Treated rats received metformin via oral gavage, although the control mice did not undergo gavage with a control substance. This is significant because other studies have demonstrated that the restraint stress causes a glucocorticoid response that can protect against NIHL (30,31). Furthermore, the differences in our results may be attributed to differences in the species of the rodent model. This highlights the importance of ongoing rigorous and comprehensive testing of metformin to determine its role in otoprotection.

In summary, this study established the otoprotective effect of metformin in male mice and is the first study, to our knowledge, to provide rigorous testing of metformin in both sexes. Although further testing is required to understand the mechanism of action in hearing protection, this study offers a foundation for the future use of metformin to prevent hearing loss. Because there are currently no FDA-approved drugs to prevent NIHL, this line of research has significant clinical potential.

ACKNOWLEDGMENTS

We would like to thank Didier Depireux (Otolith Labs) and Katharine Fernandez (National Institute on Deafness and Other Communication Disorders, National Institutes of Health) for their critical review of this work. We appreciate Joseph Mauban and the University of Maryland School of Medicine Center for Innovative Biomedical Resources Core Confocal Facility for providing equipment for this research.

REFERENCES 1. World Health Organization. Deafness and Hearing Loss. 2020. Available at: https://www.who.int/news-room/fact-sheets/detail/deafness-and-hearing-loss. Accessed March 27, 2023. 2. Crowson MG, Hertzano R, Tucci DL. Emerging therapies for sensorineural hearing loss. Otol Neurotol 2017;38:792–803. 3. Kurabi A, Keithley EM, Housley GD, Ryan AF, Wong ACY. Cellular mechanisms of noise-induced hearing loss. Hear Res 2017;349:129–37. 4. Milon B, Shulman ED, So KS, et al. A cell-type-specific atlas of the inner ear transcriptional response to acoustic trauma. Cell Rep 2021;36:109758. 5. Gedik Ö, Dogan R, Babademez MA, et al. Therapeutic effects of metformin for noise induced hearing loss. Am J Otolaryngol 2020;41:102328. 6. Kesici GG, Öcal FCA, Gürgen SG, et al. The protective effect of metformin against the noise-induced hearing loss. Eur Arch Otorhinolaryngol 2018;275:2957–66. 7. Milon B, Mitra S, Song Y, et al. The impact of biological sex on the response to noise and otoprotective therapies against acoustic injury in mice. Biol Sex Differ 2018;9:12. 8. Reavis KM, Bisgaard N, Canlon B, et al. Sex-linked biology and gender-related research is essential to advancing hearing health. Ear Hear 2023;44:10–27. 9. Shuster B, Casserly R, Lipford E, et al. Estradiol protects against noise-induced hearing loss and modulates auditory physiology in female mice. Int J Mol Sci 2021;22:12208. 10. Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: From mechanisms of action to therapies. Cell Metab 2014;20:953–66. 11. Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 2016;7:27–31. 12. Mouse Cochlear Dissection. 2015. Available at: https://vimeo.com/144531710. Accessed March 27, 2023. 14. Triggle CR, Mohammed I, Bshesh K, et al. Metformin: Is it a drug for all reasons and diseases? Metabolism 2022;133:155223. 15. Mbara KC, Mofo Mato PE, Driver C, et al. Metformin turns 62 in pharmacotherapy: Emergence of non-glycaemic effects and potential novel therapeutic applications. Eur J Pharmacol 2021;898:173934. 16. Lee TY, Martinez-Outschoorn UE, Schilder RJ, et al. Metformin as a therapeutic target in endometrial cancers. Front Oncol 2018;8:341. 17. Oishi N, Kendall A, Schacht J. Metformin protects against gentamicin-induced hair cell death in vitro but not ototoxicity in vivo. Neurosci Lett 2014;583:65–9. 18. Chen HC, Chung CH, Lu CH, Chien WC. Metformin decreases the risk of sudden sensorineural hearing loss in patients with diabetes mellitus: A 14-year follow-up study. Diab Vasc Dis Res 2019;16:324–7. 19. Föller M, Jaumann M, Dettling J, et al. AMP-activated protein kinase in BK-channel regulation and protection against hearing loss following acoustic overstimulation. FASEB J 2012;26:4243–53. 20. Prayuenyong P, Kasbekar AV, Baguley DM. The efficacy of statins as otoprotective agents: A systematic review. Clin Otolaryngol 2020;45:21–31. 21. Fernandez KA, Allen P, Campbell M, et al. Atorvastatin is associated with reduced cisplatin-induced hearing loss. J Clin Invest 2021;131:e142616. 22. Seist R, Tong M, Landegger LD, et al. Regeneration of cochlear synapses by systemic administration of a bisphosphonate. Front Mol Neurosci 2020;13:87. 23. Quesnel AM, Seton M, Merchant SN, Halpin C, McKenna MJ. Third-generation bisphosphonates for treatment of sensorineural hearing loss in otosclerosis. Otol Neurotol 2013;33:1308–14. 24. Kanigur Sultuybek G, Soydas T, Yenmis G. NF-κB as the mediator of metformin's effect on ageing and ageing-related diseases. Clin Exp Pharmacol Physiol 2019;46:413–22. 25. Cai H, Han B, Hu Y, et al. Metformin attenuates the d-galactose–induced aging process via the UPR through the AMPK/ERK1/2 signaling pathways. Int J Mol Med 2020;45:715–30. 26. Fernandez K, Spielbauer KK, Rusheen A, et al. Lovastatin protects against cisplatin-induced hearing loss in mice. Hear Res 2020;389:107905. 27. Kim J, Lee J, Jang SY, et al. Anticancer effect of metformin on estrogen receptor-positive and tamoxifen-resistant breast cancer cell lines. Oncol Rep 2016;35:2553–60. 28. Steinberg JR, Turner BE, Weeks BT, et al. Analysis of female enrollment and participant sex by burden of disease in US clinical trials between 2000 and 2020. JAMA Netw Open 2021;4:e2113749. 29. Ilias I, Rizzo M, Zabuliene L. Metformin: Sex/gender differences in its uses and effects—Narrative review. Medicina (Kaunas) 2022;58:430. 30. Wang Y, Liberman MC. Restraint stress and protection from acoustic injury in mice. Hear Res 2002;165:96–102. 31. Tahera Y, Meltser I, Johansson P, Canlon B. Restraint stress modulates glucocorticoid receptors and nuclear factor kappa B in the cochlea. Neuroreport 2006;17:879–82.