Antioxidative stress-induced damage in cochlear explants

Abstract

The imbalance of reactive oxygen species and antioxidants is considered to be an important factor in the cellular injury of the inner ear. At present, great attention has been placed on oxidative stress. However, little is known about fighting oxidative stress. In the current study, we evaluated antioxidant-induced cochlear damage by applying several different additional antioxidants. To determine whether excessive antioxidants can cause damage to cochlear cells, we treated cochlear explants with 50 μM M40403, a superoxide dismutase mimetic, 50 μM coenzyme Q-10, a vitamin-like antioxidant, or 50 μM d-methionine, an essential amino acid and the important antioxidant glutathione for 48 h. Control cochlear explants without the antioxidant treatment maintained their normal structures after incubation in the standard serum-free medium for 48 h, indicating the maintenance of the inherent oxidative and antioxidant balance in these cochlear explants. In contrast, M40403 and coenzyme Q-10-treated cochlear explants displayed significant hair cell damage together with slight damage to the auditory nerve fibers. Moreover, d-methiodine-treated explants exhibited severe damage to the surface structure of hair cells and the complete loss of the spiral ganglion neurons and their peripheral fibers. These results indicate that excessive antioxidants are detrimental to cochlear cells, suggesting that inappropriate dosages of antioxidant treatments can interrupt the balance of the inherent oxidative and antioxidant capacity in the cell.

Keywords: Antioxidative stress; Cochlea; Coenzyme Q-10; D-methiodine; M40403.

Fig. 1

Photomicrographs and cochleograms showing the surface preparation of cochlear basilar membrane and percentage of hair cell missing 48 h after cultures in standard serum-free medium without antioxidant (A. B), or 48 h after 50 μM M40402 treatment (C. D), or 48 h post-50 μM coenzyme Q-10 cultures (E. F), or 48 h after 50 μM d-methionine treatment (G. H). Specimens were triple stained with Alexa Fluor 555 conjugated phalloidin (red, arrow) to label the stereocilia bundles and cuticular plate of sensory hair cells, with Alexa Fluor 488-conjugated beta-tubulin antibody (green) to stain the auditory nerve fibers, and with ToPro-3 (blue) to label the nuclei. (A) The sensory hair cells and nerve endings present normal 48 h after culture in control condition. (B) The averaged cochleogram showed that most of the hair cells were intact except for the partial hair cell missing at the hook region that might be induced by dissection-induced mechanical damage in control group. (C) 48 h after 50 μM M40402 treatment, massive hair cell missing and mild damage in auditory nerve fibers were detected. (D) In the mean cochleogram from M40403 treated group, mor than 50% hair cell missing throughout entire length of the cochlear basilar membrane. (E) 48 h treatment with 50 μM coenzyme Q-10 caused a large number of hair cell loss and mild damage in auditory nerve fibers. (F) More than 65% hair cell missing were found as shown in the averaged cochleogram in the experimental condition of 50 μM coenzyme Q-10 treatment for 48 h. (G) Although the cochlear hair cells were visible in cochlear explants 48 h after 50 μM d-methionine treatment. However, the damaged and disarrayed stereocilia were detected on all sensory hair cells. (H) The mean cochleogram of 50 μM d-methionine treated group displayed no obvious hair cell missing.

Fig. 2

Photomicrographs and histogram showing the spiral ganglion neuron damage and percentage of degenerating ganglion neurons 48 h after cultures in standard serum-free medium without antioxidant (A), 48 h after 50 μM M40402 treatment (B), 48 h post-50 μM coenzyme Q-10 cultures (C), or 48 h after 50 μM d-methionine treatment (D). Specimens were double stained with Alexa Fluor 488 conjugated beta-tubulin antibody (green) to stain the soma of spiral ganglion neurons, and with ToPro-3 (blue) to label the nuclei. (A) The somas and nuclei of spiral ganglion neurons in normal control cochleae 48 h after culture have a large, oval-shaped soma with a large, round, homogeneously labeled nucleus. (B) 48 h after 50 μM M40402 cultures, the nuclear condensation were detected (white arrows) in some spiral ganglion neurons. (C) Nuclear shrinkage were detected in few spiral ganglion neurons (white arrows) 48 h after 50 μM coenzyme Q-10 treatment. (D) 48 h after d-methionine treatment, all spiral ganglion neurons were greatly shrinken with nuclei fragmentation (white arrows). (E) The histogram showing the percentage of cell shrinkage with nuclear fragmentation. *Significantly different from control (P < 0.05).