In vivo fluorescence imaging of hair cells in zebrafish to assess CDDP-induced ototoxicity and otoprotective effects of cimetidine and lansoprazole. (A,B) Zebrafish were treated with CDDP at indicated doses for 2 h, followed by staining with Yo-Pro-1 of sensory hair cells. The fluorescence signals of three neuromasts including hair cells shown in the white rectangles were calculated and are shown as the percentage of the signal in zebrafish without CDDP treatment. (C–F) Zebrafish were treated with or without cimetidine (CMD, (C,D) or lansoprazole (LPZ, (E,F) to examine their effect on the decrease of fluorescence signals in hair cells in zebrafish exposed to CDDP. Quantitative analyses were performed as described above.

In vivo fluorescence imaging of hair cells in crispant zebrafish to assess the involvement of oct2 in the decrease of fluorescence signals caused by CDDP. Crispant zebrafish with knockout (KO) of zgc:64,076 or zgc:175,176, zebrafish homologs of OCT2, were generated using CRISPR-Cas9 technology. (A,E) Electrophoresis of genomic PCR around the sequences targeted by genome editing of zgc:64,076 (A) or zgc:175,176 (E). (B,F) Bright field imaging of both wild-type (WT) and crispant zebrafish for zgc:64,076 (B) and zgc:175,176 (F). C, D, G, and (H) WT and crispant zebrafish for zgc:64,076 (C,D) and zgc:175,176 (G,H) were treated with or without CDDP, followed by staining with Yo-Pro-1 of sensory hair cells. The fluorescence signals of three neuromasts including hair cells shown in the white rectangles were calculated and are shown as the percentage of the signal of WT zebrafish without CDDP treatment.