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. 2021 Jul 10;12(1):167–181. doi: 10.1016/j.apsb.2021.07.002

Figure 2.

Figure 2

Astaxanthine protection against cisplatin-induced ototoxicity both in cochlear explants and HEI-OC1 cell line modes. (A) A cell pattern for screening in dose–response using HEI-OC1 cell line after 24-h cisplatin exposure. Cisplatin reduced 50% of the cells in the presence of 10 μmol/L that was applied in the following experiments (n = 4–6 for cisplatin groups, details shown in data excel). (B) Astaxanthine responsible for dosage protects against 10 μmol/L cisplatin in cell line (dark blue), accompanied by the medium alone (grey), cisplatin alone (light blue), astaxanthine (orange). Cell viability was measured by the CCK-8 kit. Values were presented as the mean ± SD, ∗∗∗∗P < 0.0001 compared with cisplatin only (black) and medium only (blue) by one-way analysis of variance (ANOVA) with Bonferroni correction (n = 6 for medium group; n = 6 for AST + CDDP group; n = 3 for 80 μmol/L AST group; n = 5–6 for cisplatin group; details shown in data excel). (C) Various regions in cochlear explants were treated with different doses of cisplatin for 24 h. Cytotoxicity was evaluated by HC survival in different regions. The distance from the apex is presented as 20%, 40%, 60%, 80% and 100%. Cisplatin dose- (D) and time-dependent (E) curve in the basal turn of cochlear explants. Cisplatin reduces 50% HCs in the presence of 50 μmol/L within 24 h, which was applied in the following experiments (n = 3–10 for cisplatin group in D; n = 7–15 for cisplatin group in E; details shown in data excel). (F) Dose–response of astaxanthine in postnatal 3-day C57BL/6 mouse cochlear explants damaged with or without cisplatin. Medium only (grey), cisplatin only (light blue), or astaxanthine pretreated for 24 h before cisplatin treatment for 24 h (AST + CDDP, dark blue) in explants. Numbers embedded in each bar represent the number of explants counted per group. HC survival evaluated by the percentage of the remaining numbers of HCs per 150 μm of the basal turn regions that were stained by MYOSIN VIIA, mean ± SD, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared with cisplatin only (black) and medium only (blue) by one-way analysis of variance (ANOVA) with Bonferroni correction. (G) Representative confocal images presenting the protection of astaxanthine (120 μmol/L) against cisplatin (50 μmol/L) injury in the basal turn of the cochlea. (H) and (I) Representative confocal images show cochlear explants treated with either medium only or 50 μmol/L cisplatin for 12 h (G) or 24 h (H) in the basal region. The numbers of HCs were identified using Myosin VIIA staining (green), fluorochrome-labeled Annexin V (red in G), and TUNEL staining (red in H). Scale bar = 20 μm ns, not significant; AST, astaxanthine; CDDP, cisplatin; HC, hair cell; OHC, outer hair cell; IHC, inner hair cell; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.