Anti-apoptotic effect of dexamethasone in an ototoxicity model

Abstract

Background: Dexamethasone (DEX) is used for the treatment of various inner ear diseases. However, the molecular mechanism of DEX on gentamicin induced hair cell damage is not known. Therefore, this study investigated the protective effect of DEX on gentamicin (GM)-induced ototoxicity and the effect of GM on the expression of apoptosis related genes.

Methods: The protective effects of DEX were measured by phalloidin staining of explant cultures of organ of Corti from postnatal day 2-3 mice with GM-induced hair cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to detect apoptosis and immunofluorescence was done to analyze the effect of DEX on the expression of apoptosis related genes.

Results: Cochlear explant cultures of postnatal day-4-old mice were exposed to 0, 1, 5, 10, 30, 50, and 100 μg/ml DEX and GM during culture. DEX protected from GM-induced hair cell loss in the inner ear of postnatal day 4 mice. To understand the molecular mechanisms by which DEX pre-treatment decreased hair cell loss, the testes of cochlear explant cultures of postnatal day 4 mice were examined for changes in expression of cochlear apoptosis mediators. The pro-apoptotic protein Bax was significantly down-regulated and numbers of apoptotic hair cells were decreased.

Conclusions: DEX has a protective effect on GM-induced hair cell loss in neonatal cochlea cultures and the protective mechanism may involve inhibition of the mitochondrial apoptosis pathway. The combination with scaffold technique can improve delivery of DEX into the inner ear to protect GM-induced ototoxicity.

Keywords: Dexamethasone; Explant culture; Gentamicin; Ototoxicity.

Fig. 1

Protective effect of DEX against GM-induced hair cell loss in organotypic cochlear cultures from postnatal mice. GM induced hair cell loss in the control (Con) group and DEX had a significant effect at concentrations higher than 5 μg/mL. p < 0.005 as compared with the GM-alone group; p < 0.05*, p < 0.005**, p < 0.0005***)

Fig. 2

Cochlea histology in mice exposed to 0.3 mM GM and dose-dependence of DEX treatment. Phalloidin stained cochlear explant cultures of postnatal day-4-old mice pretreated with PBS (a), 0.3 mM GM only (b), 1 μg/mL DEX (c), 5 μg/mL DEX (d), 10 μg/mL DEX (e), 30 μg/mL DEX (f), 50 μg/mL DEX (g), and 300 μg/mL DEX (h) (400x magnification)

Fig. 3

Decreased apoptosis in organotypic cochlear cultures of P4 mice exposed to GM and DEX. A. TUNEL assay was conducted on explant cultures of P4 mice cochlea prepared from groups treated with PBS only (a), 0.3 mM GM only (b), and following pretreatment with DEX concentrations of 5 μg/mL (c), 100 μg/mL (d) prior to GM exposure. B. Apoptotic hair cells (green fluorescence) were counted from the cochlear explant cultures of P4 mice. Data are mean ± SEM from three independent tests in each dose of pre-treated DEX with 0.3 mM GM. p < 0.05 as compared with the GM-alone group. (p < 0.05* and p < 0.005**, 400x magnification.)

Fig. 4

Down-regulated Bax expression (red fluorescence following counterstaining with 4',6-diamidino-2-phenylindole) in P4 mice cochlea treated with low dose and high dose of DEX. Cochlear explant cultures of P4 mice were used for IHC examination for Bax; PBS only (a), 0.3 mM GM only (b), 5 μg/mL (c), and 100 μg/mL (d) DEX with 0.3 mM GM (400x magnification)