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H₂O₂, capsaicin, protease inhibitor, and phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma-Aldrich (St. Louis, MO, United States). TNF-α cytokine was purchased from Sigma-Aldrich (St. Louis, MO, United States). Etanercept or Enbrel was purchased from Walgreens pharmacy for animal use. Various primary antibodies used were as follows: p-ERK1/2, ERK1/2, NOX3, β-actin from Santa Cruz Biotechnology (Dallas, TX, United States); TNF-α, Cox2 and iNOS from Cell Signaling Technology (Danvers, MA, United States); Alexa Fluor 488 Phalloidin from Thermo Fisher Scientific (Waltham, MA, United States). Secondary antibodies used were as follows: donkey anti-rabbit IRDye 680RD, goat anti-mouse IRDye 800RD (no. 926-32214) from LI-COR Biosciences (Lincoln, NE, United States); Alexa Fluor 488 goat anti-rabbit (no. A11008) and RNA Later were purchased from Thermo Fisher Scientific (Berkeley, MO, United States); Alexa Fluor 647 goat anti-mouse IgG1 (no. A-21240) from Molecular Probes (Eugene, OR, United States). Apoptosis was measured using ApopTag^® Red In Situ Apoptosis Detection Kit (Millipore Sigma, United States). Prolong gold anti-fade (Invitrogen) was used to mount immunohistochemistry samples for imaging.

Statistical significance differences among groups were performed using either student’s t-Test or ANOVA, followed by Tukey’s post hoc test. p < 0.05 was considered to be statistically significant.

Noise exposure induces ABR threshold shifts of 25–50 ± 8 dB in rats at 8, 16, and 32 kHz tested (Figure 1A), and is associated with increased cochlear TRPV1 and TNF-α expression. Scanning electron microscopic (SEM) images of the organ of Corti show ∼40–50% damage/loss of OHCs (Figures 1B,C) in the noise-exposed animals. The expression of cochlear stress and inflammatory genes measured by qPCR at 48 h and 21 days following NE were elevated in a time-dependent manner. Interestingly, the increases for TRPV1 and TNF-α were significantly greater at 21 days than at 48 h. TRPV1 expression was increased to 2.0 ± 0.2 fold at 48 h and further increased to 3 ± 0.2 fold by 21 days while TNF-α expression increased from 2.1 ± 0.2 to 3.5 ± 0.2 fold over the same time period. NOX3, iNOS, and COX2 expression increased significantly at 48 h to 2.2 ± 0.4, 1.5 ± 0.2, and 2.3 ± 0.2 fold, respectively, with no significant changes at 21 days post-NE (Figure 1D). These findings demonstrate the induction of TRPV1, TNF-α, and NOX3 as early and persistent markers of NIHL.

To establish the role of TRPV1 channel in NIHL in, in vitro studies and examine whether TNF-α potentiates TRPV1-induced Ca²⁺ influx, H₂O₂ was used to mimic ROS produced following NE. HEKVR1 and immortalized organ of Corti (UB/OC-1) cells were used. The cells were loaded with Ca²⁺ dye - Fluo-4AM washed and imaged every 3 s for 300 s. Basal fluorescence was captured for 10 scans and 100μM H₂O₂ was added at 30 s and the resulting fluorescence captured. H₂O₂ treatment elicited a rapid robust Ca²⁺ response within 10 s which returned to baseline by 60 s. Similar robust Ca²⁺ response was seen in UB/OC-1 cells starting at 30 s that returned to baseline by 3 min. The differences in temporal profile are due to fewer TRPV1 channels in UB/OC-1 cells (data not shown) compared to HEKVR1 cells. Pretreatment with TNF-α (0.1 μg/ml) for 10 min prior to treatment with H₂O₂ resulted in a prolonged and sustained Ca²⁺ fluorescence in both HEKVR1 and UB/OC-1 cells which is observed until 5 min (Figures 2A,B). Pictorial representation of baseline, maximal and fluorescence after 5 min is shown in HEKVR1 cells (Figure 2C) and in UB/OC-1 cells (Figure 2D). Since extracellular signal-regulated kinase (ERK) activation contributes to the pathology of NIHL (Meltser et al., 2010; Maeda et al., 2013), we tested the involvement of this kinase in TRPV1-mediated Ca²⁺ influx by pre-treating the ERK inhibitor, PD98059. Our data indicate that TRPV1-mediated Ca²⁺ entry is ERK1/2 dependent, as pretreatment of the cells with 10 μM PD98059 blunted the Ca²⁺ release in both HEKVR1 as well as UB/OC-1 cells (Figures 2A,B).

To further confirm the involvement of TRPV1 in H₂O₂ mediated Ca²⁺ entry, knockdown of TRPV1 was achieved by transfecting the UB/OC-1 cells with siRNA for TRPV1 (Mukherjea et al., 2008). Knockdown of TRPV1 by siTRPV1 abolished the Ca²⁺ response in UB/OC-1 cells by H₂O₂ and pretreatment with TNF-α did not have any effect, suggesting that H₂O₂-stimulated Ca²⁺ entry was mediated via TRPV1 (Figure 3A). Furthermore, inhibition of TRPV1 by transfection of UB/OC-1 cells with siRNA inhibited apoptosis induced by 100 μM H₂O₂ as seen by TUNEL staining (Figure 3B). To further confirm the role of TNF-α in potentiating the Ca²⁺ response via the TRPV1 channel, UB/OC-1, and HEKVR1 cells were loaded with Fluo-4AM dye and pretreated with either PBS or TNF-α (0.1 μg/ml) for 10 min. Addition of capsaicin (2.5 μM for UB/OC-1 cells and 100 nM for HEKVR1 cells) produced a robust increase in Ca²⁺ uptake in both UB/OC-1 and HEKVR1 cells compared to capsaicin alone (Figures 3C,D for UB/OC-1 and Figures 3E,F for HEKVR1). More prolonged exposure of UB/OC-1 cells to the capsaicin + TNF-α combination for 30 min increased baseline fluorescence from 100 ± 2% to 741 ± 22% with capsaicin treatment to 3,146 ± 532% increase with capsaicin + TNF-α (Figures 3G,H). These data indicate that ROS (such as H₂O₂) can activate TRPV1 to increase intracellular Ca²⁺ accumulation and that this action is potentiated by TNF-α via an ERK 1/2 dependent pathway in the cochlea.

To establish the role of ROS activation of ERK phosphorylation, UB/OC-1 cells were treated with H₂O₂ (100 μM). Increase in ERK1/2 phosphorylation in UB/OC-1 cells treated with 100 μM H₂O₂ was observed in a time-dependent manner (Figures 4A,B), with significantly elevated expression seen up to 120 min post-treatment. Additionally, inhibition of ERK activation by PD98059 prior to treatment with 100 μM H₂O₂ showed significantly decreased cell death by TUNEL (Figure 4C). Interestingly, TNF-α (0.1 μg/ml) treatment alone significantly activates ERK 1/2 phosphorylation and elicited a bell-shaped curve with the highest expression seen at 15 min post-treatment (Figures 4D,E).

In these studies, pre-treatment ABRs from naïve rats were recorded and rats were then treated with daily oral PBS or capsaicin starting 24 h prior to noise, on the day of NE, and at 24 h post NE. The final ABRs were recorded 21 days post NE. Schematic representation of the treatment paradigm has been depicted in Figure 5A. The noise produced ABR threshold shifts of 21.6 ± 1.7, 35.5 ± 1.9, and 43.9 ± 2.3 dB at 8, 16, and 32 kHz, respectively. Oral capsaicin reduced threshold shifts to 3.9 ± 1.4, 6.1 ± 1.8, and 8.9 ± 2.1 dB at 8, 16, and 32 kHz respectively. Oral capsaicin administration did not show any significant ABR threshold shift compared to control (1 ± 1.1, 1 ± 1.1, 3 ± 1.6 dB threshold shift at 8, 16, and 32 kHz respectively). Statistical significance was calculated using one way ANOVA with Tukey’s post hoc analyses, p < 0.05, N = 6 (Figure 5B). Gene expression analyses of the various stress response, inflammatory and apoptotic markers by qPCR of the rat cochleae showed that oral capsaicin inhibits the upregulation of all the stress markers by noise (Figure 5C). Fold increases in gene expressions by noise over oral PBS treated control rat cochleae were 4.4 ± 0.7 fold for TRPV1, 4.7 ± 0.6 for TNF-α, 4.6 ± 0.7 for NOX3, 3 ± 0.2 for COX2 and 3 ± 0.1 for Bax, while the cochleae from rats administered oral capsaicin + noise group showed basal levels of gene expression and oral capsaicin treatment alone showed basal levels of all genes tested (Figure 5C). Fluorescent immunolabelling of the mid-modiolar sections of the rat cochleae with TRPV1 and TNF-α antibody shows a robust increase in fluorescence in cochleae from noise-exposed rats but not in cochleae from rats administered oral capsaicin followed by noise (Figure 5D). Additionally, immunofluorescent staining of inflammatory mediators such as NOX3 and COX2 also show a similar decrease in expression in rat cochleae pre-treated with oral capsaicin (Figure 5E). Thus, capsaicin protects against NIHL and associated cochlear inflammatory markers.

To determine whether inhibition of the inflammatory TNF-α signaling could ameliorate NIHL, we administered ETA by either TT or s.c injections. The drug treatment was administered either as a single preventive pretreatment given prior to noise or as a single rescue treatment administered after noise. Schematic representation of the 4 different treatment paradigms have been shown in Figure 6A. We hypothesized that sequestering TNF-α by ETA pretreatment either 3 days or 7 days prior to NE should diminish TNF-α-mediated inflammation in the cochlea. Further, in the event of unanticipated NE, rescue ETA treatment if given within a window period of either 2 or 24 h post-NE, would alleviate the TNF-α induced inflammation and ameliorate NIHL.

Single ETA administration (TT-150 μg/30 μl, or s.c: 3 mg/kg) or sterile PBS (TT-30 μl, s.c:1 ml) was performed either before or after NE according to the experimental design. Post-treatment ABR assessments were performed 21 days following NE. The cochleae were collected and processed for various biochemical and immunohistochemical assays. At least four animals were used per treatment group. Noise produced 36.25 ± 3, 45 ± 3, and 48.75 ± 2.5 dB threshold shifts, recorded at 8, 16, and 32 kHz. Gene expression analyses by real-time RT-PCR showed that noise increased the expression of cochlear TRPV1, NOX3, TNF-α, iNOS and COX2 by ∼2–5 fold. ETA was administered either 7 or 3 days prior to NE by either TT or s.c administration. The 7 days pretreatment paradigm showed significant protection from NIHL with threshold shifts measuring at s.c ETA being 12.5 ± 3.6, 15 ± 5, and 16.25 ± 5.3 dB at 8, 16, and 32 kHz, respectively. The TT ETA produced threshold shifts of 16.3 ± 3.3, 16.3 ± 3.8, and 16.3 ± 4.6 dB at 8, 16, and 32 kHz, respectively (Figures 6B,C). Immunohistochemistry of mid-modiolar sections of treated animals show decreased TNF-α and NOX3 staining by both routes of administration compared to NE group alone (Figure 6D). Similar otoprotection was achieved by ETA administered 3 days prior to NE (Figures 6B,C).

To determine whether ETA treatment could be used to alleviate or lessen the extent of NIHL due to unanticipated NE and provide a window of opportunity for treatment, we administered a single dose of ETA 2 h or 24 h after noise by either TT or s.c routes. The treatment paradigms are depicted schematically in Figure 6A. We show that ETA administered within 2 h of noise by the s.c route provides significantly better protection from NIHL compared to the TT route (s.c. ETA: 9.16 ± 2.6, 15.8 ± 2.8 and 20.8 ± 4.0 dB threshold shifts) compared to TT route of administration (20 ± 2.6, 30 ± 2.7 and 33.75 ± 2.7 dB threshold shifts) at 8, 16, and 32 kHz respectively (Figures 6B,C). Immunolabelling of the mid-modiolar sections of the rat cochleae for TNF-α and NOX3 indicate that ETA treatment decreases the expression of these proteins in the cochlea. Gene expression analyses of the stress response genes also show decreased expression of TNF-α and NOX3 compared to noise. Interestingly, TRPV1 gene expression though lower than NE are still high. Initiation of ETA 24 h post NE by the s.c route provided significantly greater protection from NIHL with threshold shifts of (s.c. ETA: 21.25 ± 3.9, 22.5 ± 4.9, and 26.25 ± 5.6 dB) compared to trans-tympanic administration (TT ETA: 22.5 ± 2.5, 31.25 ± 2.9, and 37.5 ± 3.6 dB) at 8, 16, and 32 kHz respectively (Figures 6B,C). Immunolabelling of the cochlear sections for TNF-α and NOX3 indicate decreased expression of these two proteins (Figure 7A). Gene expression studies also show decreased cochlear TNF-α and NOX3, however, the levels of TRPV1 and COX2 are not significantly different than NE (Figure 7B). We believe that the increased TRPV1 and COX2 gene expression levels signify that the cochlea is severely compromised and TNF-α sequestration at 24 h (especially for the TT administration) is too late to reverse/significantly decrease the chronic inflammatory cascade.

Cochlear injury induced by noise trauma activates and increases NOX3 and TRPV1 expression which contributes to increases in TNF-α production, ERK activation, and inflammation. Unresolved chronic inflammation results in damage or death of cells in the cochlea and hearing loss. Blocking any one of these moieties of the pathway will prevent and treat NIHL (Figure 8).

Chronic TRPV1 channel activation leads to chronic increases in intracellular Ca²⁺ accumulation. Thus, induction of TRPV1 by noise could lead to Ca²⁺ overload in cochlear structures expressing these channels. Interestingly, chronic increases in Ca²⁺ and ROS have been shown to increase inflammation and apoptosis in various tissues (Nicotera and Orrenius, 1992; Orrenius et al., 1992a, b; Vass et al., 1995; Sinha et al., 2013). Our studies of cisplatin-induced hearing loss have implicated TRPV1 activation in the generation of ROS via NOX3, inflammation, and apoptosis in the cochlea (Mukherjea et al., 2008; Mukherjea et al., 2011). In the present study, we have shown that noise-induced ROS mimicked by H₂O₂ in vitro, can activate TRPV1 channel and increase Ca²⁺ influx in an immortalized organ of Corti cell line UB/OC-1 as well as in HEKVR1 cells. However, when these cell lines were pretreated with TNF-α for 10 min, the addition of H₂O₂ elicited a strong persistent opening of the TRPV1 channel. Thus, we show that NE not only dysregulates the TRPV1 channel expression but that the accompanying inflammation as mediated by TNF-α (and possibly other cytokines) will further potentiate this action, eventually leading to cell death. The novelty of this observation is that this is the first time TNF-α mediated potentiation of TRPV1 mediated Ca²⁺ entry has been implicated in hearing loss. Similar phenomenon of TNF-α induced potentiation of Ca²⁺ current evoked by TRPV1 has been reported in rat pulmonary sensory neurons in asthma (Hu et al., 2010), in cultured trigeminal ganglions isolated from neonatal rats (Meng et al., 2016), in human synoviocytes (Kochukov et al., 2009) and in sensitization of the spinal cord in the pain model (Spicarova and Palecek, 2010).

Direct TRPV1 agonist capsaicin can activate and subsequently desensitize the TRPV1 receptor rapidly. Interestingly, our data indicate that rats pre-treated with oral capsaicin prior to noise, show complete protection from ABR threshold shifts. Protein, as well as gene expression studies of the cochleae of these animals, indicate that pretreatment with capsaicin not only abolishes the noise-induced increases in TRPV1 but also inhibits NOX3, TNF-α, COX2, and iNOS. It was interesting to note that capsaicin administered prior to noise suppresses ROS generation as well as inflammation.

Our next hypothesis was that if inflammation due to TNF-α were to be inhibited prior to NE, the degree of NIHL will be ameliorated. We further hypothesized that in the event of unanticipated NE, TNF-α sequestration within a limited time window will afford some degree of protection from NIHL. We used ETA, a fusion protein consisting of two p75 TNF receptors bound to human IgG1receptor (Mohler et al., 1993) and currently used to treat autoimmune diseases like plaque psoriasis, psoriatic arthritis, ankylosing spondylitis among others. Prophylactic treatment with ETA performed either 3 or 7 days prior to noise, significantly protected from the ABR threshold shift measured at 21 days post NE. It appears that either systemic administration by subcutaneous injections or local delivery by TT injection were equally effective at protecting from NIHL when given 7 days prior to noise. Similarly, both routes of administration are equally effective in preventing NIHL when given 3 days prior to NE. The 3 days ETA pre-treatment regimen shows stronger protection than the 7 days pre-treatment regimen. This is a very interesting observation that inhibition of inflammation either systemically or locally prior to noise would ameliorate noise-induced ABR threshold shift. This implies that when used prophylactically in an uninjured body with basal levels of inflammation, the availability of a TNF-α antagonist can reduce the local levels of TNF-α and this prevents subsequent up regulation of TRPV1, thus lessening the impact of traumatic noise on the cochlea. It also indicates that lower levels of TNF-α in systemic circulation prevent recruitment of immune-competent cells by the cochlea. Lower levels of circulating TNF-α would also modulate the expression of adhesion molecules such as ICAM-1 in the cochlea (Kim H. et al., 2008) which have been shown to be upregulated by noise (Tan et al., 2016). Additionally, local inhibition of TNF-α is equally relevant as this lowers the cochlear ability to ramp up inflammation in response to traumatic noise. Most importantly, the effectiveness of trans-tympanic ETA indicates that in an uninjured cochlea, this large molecule (∼150 kD) can cross the round window and protect against permanent NIHL. Furthermore, the trans-tympanic administration of ETA would avoid the global suppression of immunity associated with the systemic route.

In unanticipated NEs, we show that systemic ETA can decrease hearing loss when administered within a 2–24 h time period following NE. An interesting observation is that while post noise administration of ETA reduces the cochlear expression of TNF-α and NOX3, the high levels of TRPV1 persists and could contribute to the residual hearing loss observed in these animals. This finding could also indicate a slower turnover of TRPV1 mRNA or to differential regulation of TRPV1 and TNF-α by noise. ETA administered by the localized TT injection show a significant lowering of ABR threshold shifts when administered within 2 h of noise but not at 24 h of noise. This could reflect a slower onset of action of ETA via the TT route post injury (probably due to slower/decreased entry into the endolymph following TT injections) to prevent irreversible changes in the cochlea which are initiated within 24 h following noise. We hypothesize that systemic (peripheral) administration of the drug could enable rapid delivery to the SV via the blood stream while dampening the systemic inflammation and possibly decreasing the recruitment of immune-competent cells from systemic circulation. This would enable the cochlea to mount the resolution phase of the inflammatory cascade, which is reflected as decreased threshold shifts or amelioration of hearing loss. Thus, as a rescue treatment, the subcutaneous route provides a wider window of opportunity than the TT route.

ETA has been shown to inhibit cochlear inflammation caused by immunologic stimulation (Satoh et al., 2003; Wang et al., 2003) and cochlear electrode insertion trauma (Ihler et al., 2014). ETA has also been studied in the treatment of immune-mediated cochlea-vestibular disorders as well as autoimmune inner ear diseases in humans with varying successes (Cohen et al., 2005; Matteson et al., 2005; Van Wijk et al., 2006). Subcutaneous administration of ETA in noise-exposed guinea pigs showed hearing preservation at 8 kHz, when determined at 3 h post noise, due to the preservation of cochlear blood flow (Arpornchayanon et al., 2013). This study complements our data which indicates a window of opportunity for subcutaneous treatment from 7 days prior to NE to within 24 h post NE.