Hydrogen Inhalation Protects against Ototoxicity Induced by Intravenous Cisplatin in the Guinea Pig

Abstract

Introduction: Permanent hearing loss and tinnitus as side-effects from treatment with the anticancer drug cisplatin is a clinical problem. Ototoxicity may be reduced by co-administration of an otoprotective agent, but the results in humans have so far been modest. Aim: The present preclinical in vivo study aimed to explore the protective efficacy of hydrogen (H₂) inhalation on ototoxicity induced by intravenous cisplatin. Materials and Methods: Albino guinea pigs were divided into four groups. The Cispt (n = 11) and Cispt+H₂ (n = 11) groups were given intravenous cisplatin (8 mg/kg b.w., injection rate 0.2 ml/min). Immediately after, the Cispt+H₂ group also received gaseous H₂ (2% in air, 60 min). The H₂ group (n = 5) received only H₂ and the Control group (n = 7) received neither cisplatin nor H₂. Ototoxicity was assessed by measuring frequency specific ABR thresholds before and 96 h after treatment, loss of inner (IHCs) and outer (OHCs) hair cells, and by performing densitometry-based immunohistochemistry analysis of cochlear synaptophysin, organic transporter 2 (OCT2), and copper transporter 1 (CTR1) at 12 and 7 mm from the round window. By utilizing metabolomics analysis of perilymph the change of metabolites in the perilymph was assessed. Results: Cisplatin induced electrophysiological threshold shifts, hair cell loss, and reduced synaptophysin immunoreactivity in the synapse area around the IHCs and OHCs. H₂ inhalation mitigated all these effects. Cisplatin also reduced the OCT2 intensity in the inner and outer pillar cells and in the stria vascularis as well as the CTR1 intensity in the synapse area around the IHCs, the Deiters' cells, and the stria vascularis. H₂ prevented the majority of these effects. Conclusion: H₂ inhalation can reduce cisplatin-induced ototoxicity on functional, cellular, and subcellular levels. It is proposed that synaptopathy may serve as a marker for cisplatin ototoxicity. The effect of H₂ on the antineoplastic activity of cisplatin needs to be further explored.

Keywords: ABR; copper transporter 1; in vivo; inner hair cells; organic cation transporter 2; outer hair cells; perilymph metabolomics; synaptophysin.

Figure 1

Micrograph showing a mid-modiolar cross-section of a normal guinea pig cochlea. The circles indicate the two points on the basilar membrane, 12 mm (middle turn) and 7 mm (basal turn) from the round window (RW), where the immunoreactivity of synaptophysin, organic cation transporter 2 (OCT2), and copper transporter 1 (CTR1) were analyzed.

Figure 2

(A) A fluorescent image (40 X) of the organ of Corti from a normal guinea pig. The orange and green labeling indicates immunoreactivity of synaptophysin and OCT2. In order to perform a quantification using densitometry measuring, the software converts the colors to a gray scale, (B) synaptophysin and (C) OCT2, (CTR1 image not shown). Scale bar: 25 μm.

Figure 3

Guinea pigs were subjected to frequency-specific ABR assessment in the left ear before and 96 h after cisplatin injection (8 mg/kg b.w., i.v.). The graph shows the marginal means of the threshold elevations obtained in the Cispt group (n = 11; empty circles), which received cisplatin, and in the Cispt+H₂ group (n = 11; filled circles), which received cisplatin immediately followed by inhalation of H₂ (2% in air) during 60 min. The horizontal lines with an asterisk indicate a significant difference (p < 0.05) between two frequencies, which was obtained only in the Cispt group. The vertical lines with an asterisk indicate a frequency-specific significant difference (p < 0.05) between the Cispt group and the Cispt+H₂ group.

Figure 4

OPLS-DA scores plot (R2X 0.219, R2Y 0.375, Q2 0.222 in the predictive component) of sample Cispt group (triangles) and sample Cispt+H₂ group (circles).

Figure 5

Representative cochleograms from animals in (A–C) the Cispt group and (D–F) the Cispt+H₂ group. IHC, inner hair cells; OHC 1, outer hair cells of the first row; OHC 2, outer hair cells of the second row; OHC 3, outer hair cells of the third row.

Figure 6

The percentage of lost IHCs and OHCs in the Cispt and Cispt+H₂ groups was summarized in three different regions, Apex (18-14.1 mm from RW), Middle (14-9.1 mm from RW), and Base (9-2.1 mm from RW). ^*p < 0.05 and ^**p < 0.01.

Figure 7

Micrograph (40 X) from a normal guinea pig cochlea (Control group) showing immunoreactivity of (A) synaptophysin, (B) OCT2, and (C) CTR1.

Figure 8

Quantification of the immunoreactivity of synaptophysin, using densitometry, in the synapse area of (A) IHCs and (B) OHCs at a distance of 12 mm from the RW in the Control group (blue), Cispt group (red), and Cisp+H₂ group (green). The bars illustrate the mean intensity and the error bars represent SD. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 9

Quantification of the immunoreactivity of OCT2, using densitometry, in (A) inner and outer pillar cells and (B) stria vascularis at a distance of 12 mm from the RW in the Control (blue), Cispt (red), and Cispt+H₂ (green) groups. The bars illustrate the mean intensity obtained in the three groups and the error bars represent SD. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 10

Quantification of immunoreactivity of CTR1, using densitometry. Upper row show the results from the 12 mm region from the RW and the lower row show the results from the 7 mm region from the RW. (A) The synapse area of IHCs, (B) Dieters' cells, and (C) stria vascularis of the cochlea in the control (blue), Cispt (red), and Cispt+H₂ (green) groups. The bars illustrate the mean intensity obtained in the three groups and the error bars represent SD. ^*p < 0.05 and ^**p < 0.01.