Clin Exp Otorhinolaryngol.  2013 Dec;6(4):219-225.

Protective Role of Trimetazidine Against Neomycin-induced Hair Cell Damage in Zebrafish

• Chang J
• Im GJ
• Chae SW
• Lee SH
• Kwon SY
• Jung HH
• Chung AY
• Park HC
• Choi J

Abstract

OBJECTIVES
Trimetazidine (TMZ) is known to reduce the generation of oxygen-derived free radicals. The objective of the present study was to evaluate the effects of TMZ on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP).
METHODS
Five-day, postfertilization zebrafish larvae were exposed to 125 microM neomycin and one of the following TMZ concentrations for 1 hour: 10 microM, 100 microM, 500 microM, 1,000 microM, 1,500 microM, or 2,000 microM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of hair cells in the control group that were not exposed to neomycin. Ultrastructural changes were evaluated using scanning electron microscopy.
RESULTS
TMZ protected against neomycin-induced hair cell loss in the neuromasts (TMZ 1,000 microM, 11.2+/-0.4 cells; 125 microM neomycin only, 4.2+/-0.5 cells; n=10; P<0.05) and decreased the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. In the ultrastructural analysis, structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125 microM neomycin and 1,000 microM TMZ.
CONCLUSION
TMZ attenuated neomycin-induced hair cell loss in zebrafish. The results of this study suggest that neomycin induces apoptosis, and that apoptotic cell death can be prevented by treatment with tremetazidine.

Keyword

Trimetazidine; Neomycin; Ototoxicity; Zebrafish

MeSH Terms

Apoptosis
Cell Death
Cell Survival
DNA Nucleotidylexotransferase
Free Radicals
Hair*
Larva
Microscopy, Confocal
Microscopy, Fluorescence
Mitochondria
Neomycin
Trimetazidine*
Zebrafish*
DNA Nucleotidylexotransferase
Free Radicals
Neomycin
Trimetazidine

Figure

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  Fig. 1 Quantitative assay of neuromasts in zebrafish. Hair cells from four neuromasts (supraorbital 1, supraorbital 2, otic 1, and occipital 1 were counted. Treatment of zebrafish with 125 µM Neo for 1 hour significantly decreased the number of hair cells in the neuromasts. TMZ protected against neomycin-induced hair cell loss in neuromasts (NC, 13.1±0.9 cells; 125 µM Neo only 4.2±0.5 cells; TMZ 10 µM, 4.2±0.7 cells; TMZ 100 µM, 4.1±0.6 cells; TMZ 500 µM, 9.4±1.1 cells; TMZ 1,000 µM, 11.2±0.4 cells; TMZ 1,500 µM, 10.0±0.7 cells; TMZ 2,000 µM, 8.7±1.7 cells; n=10; P<0.05). NC, negative control; Neo, neomycin; TMZ, trimetazidine.

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  Fig. 2 Confocal microscopy (×40). The 5-dpf transgenic zebrafish were treated simultaneously with 125 µM Neo and 1,000 µM TMZ for 1 hour. Treatment with 125 µM Neo resulted in a significant decrease in the number of hair cells in the neuromasts, and TMZ protected against this neomycin-induced hair cell damage. TMZ, trimetazidine; SO1, supraorbital 1; SO2, supraorbital 2; O1, otic 1; OC1, occipital 1; NC, negative control; Neo, neomycin.

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  Fig. 3 Evaluation of mitochondrial damage with DASPEI (fluorescent microscopy, O1, ×40). The 5-dpf wild-type zebrafish were treated with 125 µM Neo and 1,000 µM TMZ for 1 hour. Treatment with 125 µM Neo resulted in a significant decrease in mitochondrial staining with DASPEI, and this mitochondrial damage was prevented by TMZ. NC, negative control; Neo, neomycin; TMZ, trimetazidine; O1, otic.

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  Fig. 4 Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay (fluorescent microscope, ×10). Neo-induced apoptotic cells were confirmed by the TUNEL assay. Apoptotic cells are marked as light red dots in red-colored fish after the TUNEL reaction under a fluorescent microscope (arrow indicates TUNEL-positive cells). No light-red dots were found in the NC group. A comparison of the color intensity between the group treated with 125 µM Neo and the group treated with 1,000 µM TMZ for 1 hour showed that TMZ significantly decreased the number of TUNEL-positive cells and protected against Neo-induced apoptotic cell death of the neuromast hair cells (green-colored fish of right column, control for TUNEL reaction; red-colored fish of middle column, TUNEL reaction; green- and red-colored fish of left column, combination of control and TUNEL reaction). NC, negative control; Neo, neomycin; TMZ, trimetazidine.

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  Fig. 5 Scanning electron microscopy (SEM; O1, ×3,000). Compared to control neuromasts, neuromasts in zebrafish treated with 125 µM Neo for 1 hour showed severe morphological damage to stereocilia and kinocilium under SEM. However, this damage was prevented by TMZ. NC, negative control; Neo, neomycin; TMZ, trimetazidine, O1, otic.

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