Hearing disabilities can be the result of exogenous factors such as exposure to noise and treatment with drugs that have ototoxic side effects. Oncologic treatment using cisplatin-based therapy, is a common cause of drug-induced hearing loss, especially when high doses are given. Cisplatin is among the first generation of platinum-based anticancer agents and is widely used to treat different tumors in both children and adults. Cisplatin can enter the cochlea (Laurell et al., 1995; Hellberg et al., 2013), likely via passive membrane diffusion and active uptake to vulnerable cells. The details of this transport are still not fully elucidated, but organic cation transporter 2 (OCT2) (Ciarimboli et al., 2010) and copper transporter 1 (CTR1) (More et al., 2010) have been implicated in cisplatin uptake to the inner ear.

Inside the cochlea, cisplatin can damage the organ of Corti, spiral ganglion, stria vascularis, and spiral ligament (van Ruijven et al., 2005; Rybak, 2007). At the cellular level, cisplatin-induced ototoxicity typically manifests as loss of outer hair cells (OHCs), mainly in the basal turn of the cochlea, resulting in high-frequency hearing loss (Rybak, 2007). Effects on subcellular compartments are less understood. Recent data suggest that cochlear synaptopathy is a key to acquired hearing loss (Liberman and Kujawa, 2017); however, its role in cisplatin-induced ototoxicity is unknown.

Preclinical experiments suggest that underlying mechanisms of cisplatin-induced ototoxicity include the formation of platinum-DNA adducts, inflammation, and oxidative stress (Rybak, 2007). A number of experimental studies have shown that it is possible to reduce cisplatin ototoxicity by co-administrating an otoprotective drug (Rybak, 2007). However, no otoprotective therapy has clearly attenuated cisplatin ototoxicity in clinical practice.

Pharmacological treatment of the inner ear can involve either systemic or local drug administration, and both have advantages and disadvantages. Systemic administration is a well-established method that can be non-invasive (e.g., oral) or invasive (e.g., intravenous). One challenge is that the cochlea possesses at least two barrier systems, the blood-perilymph and intrastrial fluid-blood barriers (Cohen-Salmon et al., 2007; Shi, 2016), that prevent drugs in systemic circulation from accessing the cochlear compartments. Another challenge is that systemic distribution of a drug may lead to unwanted effects in other systems. Unwanted effects of antioxidant treatment to prevent cisplatin-induced ototoxicity include inhibition of the antineoplastic efficacy of cisplatin (Freyer et al., 2017), stimulation of tumor growth (Sayin et al., 2014), and metastasis (Le Gal et al., 2015). These effects are unacceptable in humans with cancer but difficult to study in experimental models. The literature offers limited information in this area, although there are several experimental studies describing otoprotective (Rybak, 2007) and nephroprotective (dos Santos et al., 2012) effects with concomitant use of drugs with antioxidative effects. An alternative to systemic administration is local drug delivery, which makes it possible to achieve a high drug concentration in the target organ, thereby reducing the risk of unwanted effects in other parts of the body. Some pharmacological agents that have been applied systemically have also been tested for intratympanic administration. Local treatment of the inner ear can only be performed with invasive techniques, and its clinical use is currently limited to steroid treatment for sudden sensorineural hearing loss after failure of systemic steroid treatment (Li et al., 2015) and destructive treatment of vertigo with gentamicin in Ménière's disease (Pullens and van Benthem, 2011).

Multiple candidate substances, mostly antioxidants, have been tested to prevent cisplatin-induced ototoxicity, but little is known about the importance of different physicochemical properties for otoprotective efficacy. One interesting candidate is molecular hydrogen (H₂) as it is expected to easily penetrate the different cochlear barriers and reach the organ of Corti. H₂ can be given as a gas or an aqueous solution, and it has been shown to have antioxidant (Fukuda et al., 2007; Ohsawa et al., 2007; Xie et al., 2010) and anti-inflammatory (Gharib et al., 2001; Xie et al., 2010) properties. Gaseous H₂ is not explosive at a concentration <4% in air or pure oxygen (Huang et al., 2010). Inhaled H₂ reduced organ damage in different animal models of ischemia (Fukuda et al., 2007; Ohsawa et al., 2007; Huang et al., 2011; Hugyecz et al., 2011). Importantly, cisplatin-induced nephrotoxicity was attenuated by aqueous H₂ in rats (Kitamura et al., 2010; Matsushita et al., 2011) and by aqueous and gaseous H₂ in mice (Nakashima-Kamimura et al., 2009), likely by reducing oxidative stress (Nakashima-Kamimura et al., 2009). With regard to effects related to hearing, aqueous H₂ reduced antimycin A-induced production of reactive oxygen species (ROS) and increased the survival of inner hair cells (IHCs) and OHCs in a cochlear explant study (Kikkawa et al., 2009). In a gerbil model of auditory neuropathy, gaseous H₂ reduced ouabain-induced hearing threshold shifts and spiral ganglia damage (Qu et al., 2012a). In guinea pig models of noise-induced hearing loss, aqueous H₂ was shown to reduce hearing threshold shifts (Lin et al., 2011; Zhou et al., 2012; Chen et al., 2014) and hair cell loss (Zhou et al., 2012; Chen et al., 2014), likely by alleviating oxidative stress (Chen et al., 2014, 2017) and/or inflammation (Chen et al., 2017). Gaseous H₂ also reduced noise-induced hearing threshold shifts, OHC loss, and oxidative stress in a guinea pig study (Kurioka et al., 2014). An important advantage of H₂ is that it is considered safe for human use (Huang et al., 2010).

Based on existing findings, we presumed that H₂ therapy exerts different protective effects at subcellular levels in the inner ear when given in conjunction with systemic cisplatin. The present study aimed to study the otoprotective effects of gaseous H₂ in guinea pigs treated with cisplatin by evaluating auditory brainstem response (ABR) threshold shifts, hair cell loss, and immunohistochemical findings in subcellular structures damaged by cisplatin.

The median weight change from the day of cisplatin injection to 96 h after cisplatin injection was −9 g (range: −37–9 g) in the Cispt group and −18 g (−49–10 g) in the Cispt+H₂ group (p > 0.05). The values in the H₂ group and two animals in the Control group used for ABR recording were 14 g (2–26 g) and 29 g (26 and 32 g), respectively. Thus, H₂ inhalation did not prevent cisplatin-induced weight loss.

High-dose cisplatin treatment is associated with several toxic insults to the cochlea. Antioxidants were previously shown to protect cochlear structures under experimental conditions. Both systemic and local administration have been employed and demonstrated benefits in experimental animals. Here, we investigated the otoprotective potential of gaseous H₂ given for 60 min starting immediately after albino guinea pigs received a single cisplatin injection. Cisplatin administration caused electrophysiological hearing threshold shifts that were largest at high frequencies. The shifts were partly prevented by H₂ inhalation. The hair cell counts corresponded with the hearing threshold shifts and showed substantial hair cell loss that declined from the base to apex. As previously found (Berglin et al., 2011), OHC loss was greatest in the first row, less in the second row, and least in the third row. There was also some IHC loss in the basal turn of the cochlea, which is less common in cisplatin-treated animals. H₂ inhalation had an important protective effect on OHCs and completely prevented IHC loss. These results agree with those of a previous study, where inhalation of H₂ reduced ABR-assessed threshold shifts and hair cell loss in cisplatin-treated rats (Qu et al., 2012b).

Assessment of threshold shifts and IHC/OHC loss are two rough methods to measure the value of an otoprotective treatment. New, more refined measures are needed to better understand as to the effects of ototoxicologic treatments on cellular and subcellular levels. New insights from animal experiments suggest that synaptopathy plays a key role in hearing damage due to noise, aging, and aminoglycoside treatment, and that it can occur even when hearing thresholds still are normal and hair cells are preserved (Liberman and Kujawa, 2017). These observations indicate that synaptopathy could be an endpoint measure to assess ototoxicological risk and protective treatments. We explored whether synaptopathy may be involved in cisplatin-induced ototoxicity and if it could be prevented by gaseous H₂. We assessed the effect of cisplatin administration without and with concomitant H₂ inhalation on synaptophysin immunoreactivity at the cochlear nerve terminals around the IHCs and OHCs. Measurements were performed 7 and 12 mm from the RW, approximately correlating tonotopically to 12.5 and 3.15 kHz frequencies, respectively (von Békésy, 1960). Although most synapses link IHCs and type I afferent neurons, synapses at the OHCs were also assessed as the OHCs may undergo apoptosis after cisplatin administration. Densitometry analysis was performed to provide a semi-quantitative measure of synaptophysin staining. In many cases, the large destructive effects of cisplatin treatment resulted in collapse of the organ of Corti in the basal turn of the cochlea, which severely hampered the immunohistochemical evaluation at 7 mm. The ROI of synaptophysin was the synapse area of the hair cells. At 12 mm, where threshold changes and OHC loss were less noticeable than at 7 mm and where there was no IHC loss, cisplatin administration still reduced synaptophysin labeling. Notably, this was less pronounced in H₂-treated animals. Evaluation at 7 mm was only possible in the Cispt+H₂ group; there were too few evaluable samples in the Cispt group due to cisplatin cochleotoxicity. Thus, our study shows that synaptopathy might play a role in cisplatin-induced hearing damage. Reduced synaptophysin levels in the inner ear were previously associated with amikacin-induced ototoxicity (Ernfors et al., 1996) and noise trauma (Canlon et al., 1999) in guinea pigs and with aging in mice (Bartolome et al., 2009). Although H₂ inhalation ameliorated the cisplatin-induced change in synaptophysin staining, its protective effect using a post-treatment mode was incomplete. A possible explanation for why partial protection was observed is suboptimal timing. Cisplatin was detected in the scala tympani perilymph just 10 min after i.v. administration in guinea pigs (Laurell et al., 1995; Hellberg et al., 2013). Considering cisplatin's high reactivity, it is possible that a protective agent must already be present in the cochlea to prevent ototoxicity. A second possible explanation is suboptimal dosing. Recognizing the limitations of the methodology and difficulties of knowing the exact pharmacokinetics and pharmacodynamics of H₂ and cisplatin in the inner ear, the results still indicate that H₂ is a promising otoprotective candidate.

Cisplatin is rapidly distributed to the cochlea after systemic administration (Laurell et al., 1995; Hellberg et al., 2013). The details of this transport remain to be elucidated. A study in mice suggested the involvement of the transport protein OCT2 for cochlear uptake of cisplatin (Ciarimboli et al., 2010). Further support for a role of OCT2 was provided by two recent clinical studies that showed an association between a specific gene variant of the OCT2 gene SLC22A2 and reduced ototoxicity in cisplatin-treated patients (Lanvers-Kaminsky et al., 2015; Spracklen et al., 2016). We therefore wanted to explore the effect of cisplatin administration on OCT2 in the cochlea and whether it was altered by concomitant H₂ inhalation. The ROI of OCT2 included the supporting cells of the organ of Corti (mainly the inner and outer pillar cells) and the stria vascularis. Cisplatin administration reduced OCT2 immunolabeling at 12 mm in both regions, effects that were not prevented by H₂ inhalation. Evaluation at 7 mm was only possible in the Cispt+H₂ group due to extensive cochleotoxicity in the Cispt group. We can only speculate about the causes of reduced OCT2 staining. Possible explanations are that cisplatin alters protein synthesis of OCT2 or promotes the apoptosis of OCT2-expressing cells. Another possibility is that cisplatin binds to OCT2 and thereby impairs binding of the OCT2 antibody. The consequences of reduced OCT2 immunolabeling in the cochlea also warrants further investigation. The influence of OCT2 expression on cisplatin-induced nephrotoxicity was first suggested about a decade ago (Yonezawa et al., 2005) and, as recently reviewed, there are many shared feature between cisplatin's ototoxic and nephrotoxic effects (Karasawa and Steyger, 2015). OCT2 is also a determinant of the efficacy of cisplatin-based cancer treatment (Liu et al., 2012). With regard to OCT2 cochlear localization, others have shown that OCT2 can be found in the mouse stria vascularis (Ciarimboli et al., 2010; More et al., 2010) and in IHCs and OHCs (Ciarimboli et al., 2010). Our previous study of guinea pigs revealed OCT2 in the supporting cells of the organ of Corti when using paraffin-sectioning (Hellberg et al., 2015) in contrast to cryosectioning, which was used in the present investigation and in mouse studies (Ciarimboli et al., 2010; More et al., 2010). The tunnel of Corti bordered by the inner and outer pillar cells communicates with the perilymphatic space, and OCT2 expression in these cells indicates that active uptake of cisplatin through this pathway to the endolymphatic space may contribute to OHC toxicity.

Another transporter implicated in cisplatin-induced ototoxicity is CTR1. This protein is abundantly expressed in the mouse cochlea, and pretreatment with the CTR1 substrate copper sulfate decreased cisplatin-induced ototoxicity (More et al., 2010). The ROI of CTR1 was the synapse area of the IHC, Deiters' cells, and the stria vascularis. Cisplatin treatment reduced CTR1 immunolabeling at 12 mm in all these regions, an effect that was prevented by H₂ inhalation. The reduction pattern was similar in the 7-mm region, but the difference between the Cispt and Cispt+H₂ groups did not reach statistical significance. As for the reduction of OCT2 immunolabeling, we can only hypothesize about the causes and consequences of our CTR1 findings. Clinical support for a role of CTR1 in cisplatin-induced ototoxicity remains sparse (Xu et al., 2012), but its importance for cisplatin-induced anticancer efficacy is more established (Liu et al., 2012). There are a number of copper influx and efflux transporters, and several have been shown to be involved in the cellular passage of cisplatin. Future studies that simultaneously investigate both types of copper transporters might increase our understanding in this field. With regard to CTR1 localization within the cochlea, CTR1 was abundantly expressed in the stria vascularis, OHCs, IHCs, and supporting cells in a previous mouse study (More et al., 2010).

As the precise trafficking of cisplatin within the cochlea is not clear, we wanted to further explore the effect of gaseous H₂ on scala tympani perilymph. We aspirated scala tympani perilymph for metabolomics analysis. Although preliminary, the data in the OPLS-DA score plot indicate a clear separation of the Cispt and Cispt+H₂ groups. This finding further supports the possibility that H₂ inhalation can reduce the ototoxic effect of cisplatin within the cochlea before the drug affects the OHCs (i.e., a preventive mechanism before the drug reaches the organ of Corti). This is most likely in line with strategies to prevent cisplatin ototoxicity by intratympanic antioxidant administration. We recently showed that injection of an ototoxic dose of cisplatin to guinea pigs induces a wide range of changes in the blood metabolome (Videhult Pierre et al., 2017).

It is reasonable to speculate that there are important differences between ototoxicity and otoprotection in healthy experimental animals and human beings treated for platinum-sensitive cancer. A major limitation of the study is the focus on the cochlear effects of H₂ administration without an oncologic perspective. We did not evaluate any influence of H₂ on cisplatin cytotoxicity in a tumor target. In cancer research, it is important to assess antitumor activity when introducing a novel co-administrated compound to reduce side effects. Moreover, the hearing situation of patients undergoing cisplatin treatment is more complex, with a number of confounding factors influencing the ototoxic effect. Caution must be taken when interpreting our results obtained in guinea pigs. However, the in vivo model reflects a clinically relevant question, although cisplatin administration routes and methods differ from that in clinical practice. For example, we administered a bolus injection of cisplatin, whereas repeated injections are usually given in clinical practice.

The findings in the present investigation raise a number of interesting questions that have to be further studied. Even though our method could be a potentially attractive approach in clinical settings, several issues must be addressed before it can be used as a proof of concept. Antioxidant treatment may have negative effects on cisplatin cytotoxicity in tumors (Freyer et al., 2017) and could even promote tumor growth (Sayin et al., 2014) and metastasis (Le Gal et al., 2015), so more research is needed to develop strategies that circumvent ototoxicity in patients undergoing cisplatin treatment. Substantial effort will be required to assess the effects of H₂ with and without cisplatin on tumor growth and spread. One finding that has not received enough attention is cisplatin's effect on IHC synapses. The observed synaptopathy after a single cisplatin injection could be a target for additional research. In the present study, only H₂ administered after cisplatin was used to prevent ototoxicity. Pre-loading H₂ could be a more effective way to protect hearing. Apart from the discrepancy between the influence of H₂ on OCT2 and CTR1 immunoreactivities, one striking finding in this study was that cisplatin altered immunolabeling of the transport proteins in cochlear tissue. Additional work is needed to clarify the active transport processes involved in cisplatin ototoxicity. Nevertheless, our findings suggest that H₂ administration could prevent cisplatin ototoxicity, but further evaluation is necessary.

Although cisplatin is an old drug, it is still frequently used in oncology despite its side effects. There have been no clinical breakthroughs for otoprotection during cisplatin therapy. The present results demonstrate that gaseous H₂ is a promising agent that can protect against cochlear injury. First, H₂ showed an effect on frequency-specific electrophysiological hearing thresholds. Second, surface preparations of the cochlea revealed that OHC loss was attenuated in H₂-treated animals. Third, at the subcellular level, H₂ produced a protective effect on IHC synapses. Hearing loss after cisplatin treatment is multifactorial, and our results implicate synaptopathy as an additional mechanism. Using a guinea pig model for an otoprotective study has some obvious limitations as they cannot be compared with cancer patients. The advantage is the ability to study the inner ear structures in detail after cisplatin treatment, which is not possible in human subjects.

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.