Antioxidant and anti-apoptotic effect of melatonin on the vestibular hair cells of rat utricles

Abstract

Objectives: Aminoglycosides are commonly used antibiotic agents, and they are known to generate free oxygen radicals within the inner ear and to cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Melatonin, a pineal secretory product, has the properties of being a powerful direct and indirect antioxidant. The aim of the present study was to prove the antioxidant effect of melatonin against gentamicin-induced ototoxicty.

Methods: The utricular maculae of Sprague-Dawley rats were prepared from postnatal day 2-4, and these maculae were were divided into 6 groups as follows: 1) control, 2) melatonin only, 3) gentamicin only, and 4), 5), and 6) gentamicin plus melatonin (10, 50, and 100 microM, respectively). To count the number of hair cells, 5 utricles from each group were stained with phalloidin-FITC on the 1st, 4th, and 7th days after drug administration. Reactive oxygen species (ROS) was assessed by using the fluorescent probe hydrofluorescent diacetate acetyl ester. The caspase-3 activity was also examined with using the fluorescent caspase-3 substrate and performing Western blotting.

Results: The result of this study showed that gentamicin induced the loss of utricular hair cells, and this loss of hair cells was significantly attenuated by co-administration of melatonin. Melatonin reduced ROS production and caspase-3 activation in the gentamicin treated utricular hair cells.

Conclusion: Our findings conclusively reveal that melatonin has protective effects against gentamicin-induced hair cell loss in the utricles of rat by inhibiting both ROS production and caspase-3 activity.

Keywords: Antioxidants; Melatonin; Ototoxicity; Utricle.

Fig. 1

Photomicrographs showing utricular organotypic cultures labeled phalloidin-FITC in control group. Left column shows rat's utricle at the 1st day after utricle culture (A). Median (B) and right (C) column are the 4th and 7th days'utricle findings. The number of live hair cells were 292.8±21.8 (n=5), 292.0±10.1 (n=5), and 288.4±8.0 (n=5) in group C at the 1st, 4th, and 7th day after culture stated. Scale bar shown in each panels.

Fig. 2

Photomicrographs showing utricular organotypic cultures labeled phalloidin-FITC in gentamicin (G) group. The number of live hair cells in group G were 48.2±15.9 (n=5), 38.4±8.1 (n=5), and 19.8±11.1 (n=5) at 1st, 4th, and 7th day after culture stated. The utricles cultured for 48 hr in the presence of 1 mM gentamicin show extensive loss of hair cells.

Fig. 3

Mean number of utricular hair cells per 20,000 µm² in each the groups. Melatonin-treated group was not significantly different from control group. GM2 & GM3 groups had more hair cells than G group significantly.

Fig. 4

Photomicrographs showing utricular organotypic cultures labeled phalloidin-FITC in melatonin (M) group. The number of live hair cells in group M were 281.0±11.2 (n=5), 276.0±22.2 (n=5), and 277.6±15.2 (n=5) at the 1st, 4th, and 7th day after culture stated. Utiricular hair cells were observed as many as control group.

Fig. 5

Photomicrographs showing utricular organotypic cultures labeled phalloidin-FITC in GM1 group. The number of live hair cells in group GM1 were 58.2±10.1 (n=5), 54.2±5.5 (n=5), and 33.0±6.4 (n=5) at the 1st, 4th, and 7th day after culture stated. The utricles show extensive loss of hair cells.

Fig. 6

Photomicrographs showing utricular organotypic cultures labeled phalloidin-FITC in GM2 group. The number of live hair cells in group GM2 stained phalloidin-FITC by CLSM were 130.6±13.4 (n=5), 118.4±13.4 (n=5), and 103.2±19.0 (n=5) at the 1st, 4th, and 7th day after culture stated. The utricular hair cells were observed more than GM1 group.

Fig. 7

Photomicrographs showing utricular organotypic cultures labeled phalloidin-FITC in GM3 group. The number of live hair cells in groups GM3 were 168.6±19.5 (n=5), 161.0±22.0 (n=5), and 154.4±14.3 (n=5) at the 1st, 4th, and 7th day after culture stated. The utricular hair cells were observed more than GM1 & GM2 group, significantly.

Fig. 8

Effects of melatonin on gentamicin-induced production of reactive oxygen species (ROS). Photomicrographs showing ROS activation in control (A), 1 mM gentamicin only (B), 50 µM melatonin only (C), and 1 mM gentamicin plus 50 µM melatonin (D). Scale bar shown in panels.

Fig. 9

Photomicrographs showing red caspase-3 substrate activation in normal utricular culture (A column), utricular cultures treated with 1 mM gentamicin (B column), utricular cultures treated with 50 µM melatonin (C column), and utricular cutures treated with gentamicin 1 mM plus 50 µM melatonin (D column). Caspase-3 expression of utricle presented in following order of intensity, group G (B), group GM2 (D), group M (C), and group C (A). Scale bar shown in panels.

Fig. 10

The protein of caspase-3 on western blotting were detected in rat vestibular hair cells. 1: control, 2: group M, 3: group GM2, 4: group G. Group C was weakly expressed caspase-3 band. Group G was increased caspase-3 activation compared to group GM and M.