N-acetylcysteine and N-nitroarginine methyl ester attenuate Carboplatin-induced ototoxicity in dissociated spiral ganglion neuron cultures

Abstract

Objectives: Carboplatin, a platinum-containing anti-cancer drug used to treat a variety of cancers, induces ototoxicity. Since, reactive oxygen species (ROS) and nitric oxide (NO) seem to be responsible for this toxicity, the antioxidant, N-acetyl-L-cysteine (L-NAC), and NO synthetase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) were predicted to have protective effects against carboplatin ototoxicity. The aim of this study was to test for the protective effects of L-NAC and L-NAME on cochlear hair cells and spiral ganglion neurons (SGNs).

Methods: Cochlear organotypic cultures and dissociated spiral ganglion neuron cultures, from mice postnatal day 5 cultures were used in this study. The cultures were treated with carboplatin alone or in combination with L-NAC or L-NAME, and carboplatin-induced damage was monitored.

Results: Treatment with carboplatin induced a significant loss of outer hair cells, while inner hair cells were preserved in the cochlear organotypic cultures. Addition of L-NAC or L-NAME reduced the amount of carboplatin-induced hair cell damage; the differences did not reach statistical significance. However, carboplatin significantly decreased the number of surviving SGNs in dissociated cultures. The toxic effects were significantly reduced by addition of L-NAC or L-NAME. In addition, carboplatin induced the loss of neurites from the SGN somata, and this was not blocked with L-NAC or L-NAME.

Conclusion: The results of this study suggest that ROS and NO are involved in carboplatin-induced damage to hair cells and SGNs, and administration of L-NAC/L-NAME can be used to attenuate the toxicity.

Keywords: Carboplatin; Inner hair cell; Mouse; Nitric oxide; Ototoxicity; Outer hair cell; Spiral ganglion neuron.

Fig. 1

Confocal microscopic photomicrographs of cochlear organotypic cultures stained with FITC-conjugated phalloidin (green) and NF-200 (red). (A) Control showing a typical arrangement of three rows of outer hair cells, and single row of inner hair cells. (B) Cochlear cultures treated for 48 hours with carboplatin at a concentration of 100 mg/mL. (C) Cochlear cultures treated for 48 hours with 100 µg/mL carboplatin plus 100 µM N-nitro-L-arginine methyl ester. (D) Cochlear cultures treated for 48 hours with 100 µg/mL carboplatin plus 10 mM N-acetyl-L-cysteine. Scale bar represents 10 µm.

Fig. 2

Histogram shows mean number of outer hair cells (OHCs), per (A) and inner hair cells (IHCs) (B) per 0.2 mm length of the cochlea. Asterisk indicates that the carboplatin-treated group shows a statistically significant decrease in the OHC number compared to the control group. L-NAME: N-nitro-L-arginine methyl ester; L-NAC: N-acetyl-L-cysteine.

Fig. 3

Fluorescence microscopic photographs of dissociated spiral ganglion neuron cultures stained with NF-200 (green) and Hoechst (blue) after 72 hours of culture. (A) Untreated control, (B) 50 mg/mL carboplatin only, (C) 100 mM N-nitro-L-arginine methyl ester (L-NAME) only, (D) 100 mM N-acetyl-L-cysteine (L-NAC) only, (E) 50 mg/mL carboplatin+100 mM L-NAME, (F) 50 mg/mL carboplatin+100 mM L-NAC (×400).

Fig. 4

Histogram shows relative percentages of NF-200 positive cells (survived spiral ganglion neurons) in the dissociated spiral ganglion neuron (SGN) cultures for 72 hours. The survival of SGNs in each condition is shown as a relative percentage of survival to control (100%). The Wilcoxon rank sum test was used to compare the surviving SGNs in the conditioned culture groups with the control group. L-NAME: N-nitro-L-arginine methyl ester; L-NAC: N-acetyl-L-cysteine.