Presbycusis or age-related hearing loss (ARHL) is a complex and multifactorial condition characterized by slow and progressive, auditory sensorineural degeneration, leading to hearing loss, progressing with advancing age (Fetoni et al., 2011; Keithley, 2019). Recent estimates for 2021 indicate that the prevalence of moderate to severe hearing loss in the aging population ranges globally from 15.4% among individuals aged 60–58.2% for those aged 90 or older (World Health Organization, 2021). The aging cochlea undergoes multiple regressive changes at the cellular and subcellular levels. These include loss of sensory cells, a decrease in the number of synaptic ribbons per inner hair cell, degeneration and death of spiral ligament fibrocytes and neurons, strial pathology, vascular dysfunction, and a decline in endocochlear potential, which together contribute to threshold elevation (Gratton et al., 1996; Gordon-Salant, 2005; Kidd and Bao, 2012; Viana et al., 2015; Parthasarathy and Kujawa, 2018; Keithley, 2019; Fischer et al., 2020; Chen et al., 2021). Human studies on the aging ear have extensively documented that damage to the sensory epithelium and the loss of cochlear neurons along with strial degeneration are common age-related features associated with presbycusis (Soucek et al., 1986; Schuknecht and Gacek, 1993; Nelson and Hinojosa, 2006). Indeed, evidence from post-mortem human temporal bones of aged individuals suggests that cochlear synaptopathy and primary cochlear neural degeneration, without significant inner hair cell loss, may also potentially contribute to this age-associated hearing disability (Viana et al., 2015; Wu et al., 2019).

Several animal models that exhibit different patterns of presbycusis-related progressive cochlear degeneration have been developed to investigate the pathogenesis of age-related cochlear damage and predict human disease (Fetoni et al., 2011; Alvarado et al., 2014; Bowl and Dawson, 2014; Escabi et al., 2019). The C57BL/6J (B6) mouse shows mixed sensory-neural-strial ARHL, which is characterized by progressive hearing loss for high frequencies that is already detectable at 6 months of age and extends to lower frequencies by 12 months. Additionally, these features are accompanied by degeneration of the lateral wall, spiral ganglion, and organ of Corti (Li and Borg, 1993; Di Girolamo et al., 2001; Carraro and Harrison, 2016). In contrast, CBA/CaJ mice have near-normal hearing, and exhibit a decline in endocochlear potential with aging, along with lateral wall atrophy and dysregulation of gene and protein expression of NKA subunits by 2 years of age (Sha et al., 2008, 2009; Ohlemiller et al., 2010; Hao et al., 2014; Ding et al., 2018). In rats, the Fischer 344 (F344) albino strain develops a progressive hearing deficit that initiates at 12 months of age, which is accompanied by strial pathology and outer hair cell loss (Buckiova et al., 2007; Bielefeld et al., 2008; Syka, 2010; Balogová et al., 2018). Similarly, Wistar rats also show age-related physiological alterations, including increased auditory thresholds, decreased wave amplitudes, and elongation of wave latencies that initiate at approximately 12–14 months of age and extend to 18–20 months of age; these rats also exhibit reduced excitatory and inhibitory neurotransmission in the aged cochlear nucleus (Alvarado et al., 2014).

Noise exposure may contribute to modify the onset and/or progression of auditory aging (Kujawa and Liberman, 2006; Alvarado et al., 2019; Wang and Puel, 2020). Valuable information has been obtained from reports evaluating the impact of a single noise exposure on aging in young CBA/CaJ mice (Kujawa and Liberman, 2006; Fernandez et al., 2015). However, humans are exposed daily for extended time periods to diverse and complex sources of noise, including urban, recreational, or occupational. Although studies reporting the effects of long-term daily noise exposure on hearing are limited, a previous study (Campo et al., 2011) using a noise exposure paradigm designed to mimic human responses, showed that moderate stimulation [8 kHz, 86.2 dB sound pressure level (SPL)] for 6 h per day, 5 days a week for 1 month in 6-month-old Norway rats accelerates ARHL. Consistent with this, our previous experiments using Wistar rats demonstrated a significant detrimental effect of long-term exposure to short-duration loud sound stimulation on young, 3-month-old animals [1 h of continuous white noise (110 dB SPL), 5 days a week] that were evaluated at 6, 12, and 18 months of age. The data showed that, at 6 months of age, these rats already had impaired auditory function and displayed physiological characteristics similar to those observed in 12-month-old unexposed rats, suggestive of a shortening of presbycusis onset (Alvarado et al., 2019).

Oxidative stress, with imbalances in the antioxidant defense system and adaptive and/or dysregulated inflammation are also crucial biological responses associated with ARHL and noise-induced hearing loss (NIHL) (Henderson et al., 2006; Tan, 2013; Okano, 2014; Kalinec et al., 2017; Fetoni et al., 2019). Key pathophysiological elements associated with the noise-exposed and aging cochlea include immune system dysregulation, reshaping of cytokine expression profiles, and dysregulation of redox homeostasis, including decreases in antioxidant protein and enzyme concentrations and increases in the levels of glutathione-conjugated proteins and the products of hydroxyl radical and peroxynitrite activity (Hirose et al., 2005; Fujioka et al., 2006; Tornabene et al., 2006; Jiang et al., 2007; Carroll et al., 2017; Hu et al., 2018; He et al., 2020; Lyu et al., 2020). As demonstrated in numerous studies on the aging and noise-exposed cochlea, the above-mentioned cellular processes are closely interlinked and are associated with detrimental effects on cochlear tissue and hearing (Nagashima et al., 2010; Fetoni et al., 2013, 2015b; Jayakody et al., 2018; Bermúdez-Muñoz et al., 2020). Despite the many studies that have addressed the link between age and noise, few have evaluated the chronic effect of noise exposure on the aging cochlea. Therefore, using a noise-accelerated presbycusis (NAP) protocol (Alvarado et al., 2019) and functional, histological, and molecular techniques, the aim of this study was to further evaluate the effects of long-term exposure to a short, loud noise (1 h of continuous white noise at 110 dB SPL) for 5 days beginning at 3 months and lasting until 16 months of age and assess which pathological and/or adaptive cellular processes might contribute to exacerbating the progression of presbycusis induced by chronic noise exposure.

The present results demonstrate that exposure to repetitive long-term short-duration noise overstimulation in young Wistar rats, starting at the age of 3 months, aggravates age-dependent hearing deterioration, as seen in significantly higher thresholds and larger threshold shifts in aged, 16-month-old, noise-exposed animals (E16), compared with age-matched, non-exposed animals (NE16). In response to NAP protocol, significant increases in auditory threshold and decreases in waves amplitudes, relative to unexposed 3-month-old, were already observed in E6 rats. In correlation with such threshold shifts, rats in the E6 group exhibited early SV alterations and loss of spiral limbus fibrocytes relative to unexposed rats. On the other hand, NE16 rats showed age-related loss of type IV fibrocytes in the spiral ligament and loss of spiral ganglion cells that, when combined with noise exposure (E16), resulted in increased vulnerability of lateral wall fibrocytes, and also degeneration of SGN. Additionally, the effect of NAP on adult (E6) and aged (E16) animals resulted in increased NKA-immunostaining in the SV, decreased levels of CTGF-stained type IV fibrocytes, increased expression of 4-HNE, and prolonged cochlear inflammatory response.

Although noise is an important risk factor for the onset and/or progression of presbycusis, the exact nature of their interrelation, and its impact on vulnerable cochlear structures and auditory processing are not clearly understood (Bielefeld et al., 2010; Fetoni et al., 2011). Previous research on animal models of NIHL and ARHL have shown that these degenerative diseases are driven by similar pathogenic events involved in decline of cochlear function, including inflammation, increased oxidative stress, and degenerative changes in cochlear vasculature (Fetoni et al., 2011, 2013; Alvarado et al., 2015, 2018, 2020; Tavanai and Mohammadkhani, 2017). Therefore, the coexistence of these common etiopathogenic mechanisms underlying hearing loss would have a significant impact on cochlear tissue damage, inducing premature presbycusis. Physiological studies leading to accelerated long-term decline of auditory function in response to noise exposure have demonstrated that either single or repeated overstimulation with subtraumatic or traumatic sound levels (higher than 85dB SPL) has a deleterious impact on hearing during aging, accelerating and/or aggravating presbycusis (Gates and Mills, 2005; Kujawa and Liberman, 2006; Campo et al., 2011; Fernandez et al., 2015; Alvarado et al., 2019). Consistent with these observations, our findings demonstrate that repeated daily exposure for 1 h to 110 dB SPL noise induces statistically significant increases in auditory threshold and threshold shifts in young adult rats. These include reduction in amplitude and alterations in morphology of ABR waves similar to that observed in presbycusic aged rats. However, it should be highlighted that there is a critical age period (Kujawa and Liberman, 2006; Alvarado et al., 2015), that for Wistar rats is between 3 and 6 months of age, during which the effects of the interaction between noise and age are stronger, and prolonged noise exposure each day for even a short period of time is sufficient to induce NAP.

Studies in animal models of ARHL mimicking aspects of the human clinical condition have shown that this sensory disability, which is characterized by metabolic, sensory, and neural phenotypes, results from pathological changes in the function and structure of vulnerable cochlear regions that collectively impact hearing loss progression (Kalinec et al., 2017; Watson et al., 2017). Previous studies in CBA/J and CBA/CaJ mice have demonstrated that different inbred mouse strains exhibit different aspects of presbycusis. For instance, whereas CBA/J mice show sensorineural presbycusis characterized by delayed high-frequency hearing loss along with loss of outer hair cells and degeneration of the spiral ganglion, CBA/CaJ mice show aspects of strial presbycusis including accelerated hearing loss, strial degeneration, declined endocochlear potential, and loss of outer sulcus/root cells (Sha et al., 2008; Ohlemiller, 2009; Ohlemiller et al., 2010). C57BL/6J (B6) mice show early progressive high-frequency hearing loss and degeneration of the lateral wall, sensory receptors, and SGN (Li and Borg, 1991; Di Girolamo et al., 2001; Ohlemiller, 2009; Carraro and Harrison, 2016). Fisher 344 albino rats show strial pathology and outer hair cell loss correlated with progressive hearing loss by 12 months of age (Buckiova et al., 2007; Bielefeld et al., 2008; Syka, 2010; Balogová et al., 2018). Consistent with these studies, our results demonstrate that aging Wistar rats (NE16) display SV dystrophy, loss of type IV fibrocytes in the lateral wall, and loss of fibrocytes in the spiral limbus, suggesting that fibrocyte degeneration contributes to metabolic/strial presbycusis in this animal model. Similar degenerative cellular patterns in cochlear fibrocytes coupled with hearing dysfunction have been reported in aged 129S6/SvEv and C57BL/6 mice (Zheng et al., 1999; Yoshida et al., 2000; Hequembourg and Liberman, 2001; Ohlemiller and Gagnon, 2004). Because the accelerated presbycusic 6-month-old rats presented physiological characteristics similar to those observed in unexposed old rats (NE16), it would be logical to expect that they also present early pathogenic features like those observed in the unexposed group. Consistent with previous studies in noise-exposed mice (Wang et al., 2002; Shin et al., 2019), our data confirm that E6 rats shared the alterations observed in older unexposed animals, including initial strial pathology and fibrocytes loss in the spiral limbus. Our results also show that when ARHL and NIHL coexist, as in E16 animals, further structural damage occurs including loss of type IV fibrocytes and SGN, suggesting accelerated onset and/or progression of hearing loss.

Aging and noise-exposure also impacted CTGF levels. In this regard, unexposed aged rats (NE16) and NAP-exposed rats (E6 and E16) showed decreased CTGF staining and increased cytotoxic levels of 4-HNE in type IV fibrocytes, relative to the NE6 group. These results are supported by previous studies showing that CTGF-stained type IV fibrocytes in CBA/CaJ mice are extremely vulnerable to noise trauma (6 h post-exposure at 8–16 kHz and to 95 dB) (Wang et al., 2002; Adams, 2009), implying that deteriorated and dysfunctional fibrocytes contribute to gradual hearing loss. However, despite that the progression of age-related functional disability differs both across and within species, the prevailing view is that metabolic presbycusis is predominant in ARHL, with the consequent alteration of endocochlear potential indispensable for proper auditory receptor function (Fetoni et al., 2011; Keithley, 2019; Ohlemiller, 2019).

The ubiquitous enzyme complex NKA, located in cell plasma membranes in the SV and the spiral ligament, is critical for maintenance of the electrochemical composition of the endolymph in the inner ear (Schulte and Adams, 1989; Cate et al., 1994; Nakazawa et al., 1995; Erichsen, 1996; Gratton et al., 1996; Delprat et al., 2007; Patuzzi, 2011; Liu et al., 2017). Accumulating evidence suggests that dysfunction of lateral wall structures (spiral ligament and SV) leading to disruption of the endocochlear potential affects hearing ability. Aged gerbils, mice, and rats display atrophy of the cochlear lateral wall, which correlates with decreased NKA activity and immunostaining (Schulte and Schmiedt, 1992; Gratton et al., 1995, 1997; Spicer and Schulte, 2002; Buckiova et al., 2007; Ding et al., 2018). Our findings in 16-month-old unexposed cochleae show age-related decreases in immunostaining of NKA-expressing type II fibrocytes, which are essential for regulating potassium homeostasis. This provides evidence of lateral wall pathology and ATPase-dysregulation, leading to declined endocochlear potential and contributing to ARHL. In the present study, adult (E6) and aged (E16) rats exposed to NAP showed decreased NKA-immunostaining in the spiral ligament and the spiral ganglion relative to unexposed rats, however, staining in the SV was significantly increased relative to that in the NE6 group. Noise-exposure associated perturbations in NKA levels in the cochlear spiral ligament have been described previously (Hsu et al., 2000; Yamaguchi et al., 2014). In mice, in vivo acoustic noise stimulation (1 h–8 kHz octave band noise at 110 dB SPL) results in decreased mRNA levels of Na +, K + -ATPase in spiral ligament fibrocytes 2 h post-exposure which remains until day seven (Yamaguchi et al., 2014). However, white noise exposure (10 min or 40 h at 105 dB) in adult guinea pigs leads to a significant decline in NKA activity 7 days post-exposure in lateral wall structures and followed by a return to basal levels on days 10 and 28 (Hsu et al., 2000, 2002). In these studies, Na +, K + -ATPase activity was evaluated using different noise parameters (duration, intensity, and noise paradigm) and at shorter survival times. The noise-induced increases in NKA in the SV of E6 and E16 cochleae observed in the present study may reflect an ion homeostatic response to chronic daily and short-duration noise stimulation as an attempt to preserving cochlear function.

Another cellular hallmark in the pathogenesis of ARHL and NIHL is the imbalance of redox homeostasis in the injured cochlea. Studies in chinchillas, mice, and rats have shown that aging and noise trauma in vivo increase oxidative stress biomarkers such as 4-HNE in lateral cochlear wall structures, organs of Corti, and spiral ganglion, and this correlates with hearing loss (Henderson et al., 2006; Jiang et al., 2007; Du et al., 2011; Yamaguchi et al., 2014; Fetoni et al., 2015a,2019; Wang et al., 2019). Increased antioxidant defenses to minimize oxidative stress damage have been reported in the injured cochlea (Fetoni et al., 2019). Upregulation of Cat expression by 10 days and Gpx1 expression between 1 and 10 days after exposure to broadband noise (118 dB SPL for 4 h a day for 4 consecutive days) suggests that Gpx1 plays a key role in early cochlear enzyme-mediated antioxidation responses to NIHL (Alvarado et al., 2020). In addition, our present findings show that NAP significantly increases Cat, Gpx1, Sod1, and Sod2 in E6 rats relative to unexposed rats (NE6) and unexposed and noise-exposed aging animals (NE16 and E16), suggesting that early noise exposure increases cochlear susceptibility to aging. We also report that the transcription levels of all four antioxidant enzymes evaluated decreased significantly in both unexposed (NE16) and exposed (E16) aged animals, suggesting that antioxidant capacity is depleted in these animals. Accumulation of free radical damage and decline in the expression of major antioxidant enzymes in cochlear tissue accelerates age-related oxidative damage (Staecker and Zheng, 2001; Jiang et al., 2007; Menardo et al., 2012).

When cultures from murine SL fibrocytes are stimulated by proinflammatory cytokines IL-1β or TNFα, they secrete cellular mediators involved in prolonging inflammation (Yoshida et al., 1999; Ichimiya et al., 2000). Given that these cells may regulate inflammatory-associated events in response to cochlear aging or noise injury, dysfunctional fibrocytes may have a profound impact on hearing function (Fujioka et al., 2014; Okano, 2014; Watson et al., 2017; Peeleman et al., 2020). In the present study, we further demonstrate chronic inflammation characterized by increased IL-1β protein levels primarily in type IV fibrocytes in the spiral ligaments and spiral ganglia of adult NAP-exposed, aged unexposed, and aged NAP-exposed rats. However, our results provide evidence of increased IL-1β and TNFα transcription in noise-exposed cochleae (E6) but not in aged animals (NE16 and E16). Previous studies have also shown noise-induced upregulation of IL-1β and TNFα in cochlear fibrocytes and reactive microglia (Fujioka et al., 2006; Tornabene et al., 2006; Fuentes-Santamaría et al., 2017). Decreased IL-1β and TNFα expression has also been reported in the perilymph of 2-year-old mice relative to younger mice, suggesting that aging and dysfunctional cochlear fibrocytes are not capable of cytokine biosynthesis and/or secretion (Landegger et al., 2019). Our results also showed that macrophages in the spiral ligament were unchanged in E6, NE16, and E16 groups, suggesting that their contribution to the inflamed spiral ligament is primarily regulated by fibrocytes. Macrophages in the spiral ganglia in the same groups switched to an active phenotype, suggesting that microglia also actively contribute to aging and/or noise-induced cochlear inflammation.

In summary, the present findings support the notion that a common pathogenic pathway is involved in NIHL and ARHL genesis. They demonstrate that noise and aging target similar cochlear cell types through similar etiopathogenic mechanisms including inflammation and oxidative stress, and this results in early presbycusis. This implies that the comorbidity between noise and aging, acting in a synergistic and/or redundant manner, leads to premature long-term progressive deterioration of auditory function that accelerates/aggravates presbycusis.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

All procedures involving the use and care of the animals were approved by the Ethics Committee on Animal Experimentation at the University of Castilla-La Mancha (Permit Number: PR-2019-02-05) and conformed to European Union (Directive 2010/63/EU) and Spanish (R.D. 53/2013; Law 32/2007) regulations for the care and use of animals in research.

VF-S and JA: study concept, design, and drafting of the manuscript. VF-S, JA, SM, PM-R, MG-U, and JC-S: acquisition of data. JA, VF-S, and SM: statistical analysis and interpretation of data. JA, VF-S, PM-R, SM, and JJ: critical revision of the manuscript for important intellectual content. JJ, VF-S, and JA: obtaining funding. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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