Average ABR threshold shifts at each frequency (0.5–8 kHz) 10 days after noise exposure. Hearing threshold shifts were found in both the noise exposure and the noise/treatment groups at all frequencies with greater shifts in the high frequencies (2–8 kHz). Compared to the noise exposure group, significant reductions in threshold shifts were found in the noise/treatment group at all frequencies. There are significant differences in the threshold shifts between the noise exposure and the noise/treatment groups at all frequencies, especially at high frequencies (***, **, and * indicate P < 0.001, <0.01, and <0.05, resp. Error bars represent standard error of the means. Number of ears = 12 in each group).

CD45⁺ immunostaining and immunostaining density in the stria vascularis. Examples of CD45⁺ immunostaining images obtained from the stria vascularis in the middle turn of cochleae from the normal control (a), noise exposure (b), and noise/treatment (c) groups by light microscopy. Few CD45⁺ cells were found in the stria vascularis of the normal group (arrows in (a)). There was a large increase in the number of CD45⁺ cells in the stria vascularis of the noise exposure group (arrows in (b)). The antioxidant treatment significantly decreased the number of CD45⁺ cells in the stria vascularis (arrows in (c)). CD45⁺ cells were also found in the spiral ligament of the noise and the noise/treatment groups (arrowheads in b and c), but not in the normal control group. The results of the immunostaining density measurement are shown in (d) (number of cochleae = 6 in each group). There were significant differences among the three groups and in pairwise comparisons in the three groups in each turn. The numbers (1, 2, 3) in (d) indicate the normal control, noise exposure, and noise/treatment groups, respectively. Scale bar = 20 μm in (c) for (a)–(c) (error bars represent standard error of the means. *, **, *** indicates P < 0.05, 0.01, 0.001, resp. Number of cochleae = 6 in each group).

NT immunostaining and immunostaining index in the spiral ligament. Examples of NT immunostaining images obtained from the spiral ligament in the middle turn of cochleae in the normal control (a), the noise exposure (B), and the noise/treatment (c) groups by confocal microscopy. No significant NT immunostaining was found in the spiral ligament of the normal control group (A). A large number of NT positive cells were found in the spiral ligament of the noise exposure group (arrows in (b)) while a significantly decreased number of NT positive cells were found in the noise/treatment group (arrows in (c)). The results of the immunostaining index are shown in (d). A high NT immunostaining index was found in the spiral ligament in all three turns of the noise exposure group. There were significant differences between the normal control and the noise exposure groups in the cochlear three turns, and between the noise exposure and the noise/treatment groups in the basal and middle turns. There was no significant difference between the noise exposure and the noise/treatment groups in the apical turn. The numbers (1, 2, 3) in (d) indicate the normal control, noise exposure, and noise/treatment groups, respectively (*** and ** indicate P < 0.001 and <0.01, resp.). Scale bar = 20 μm in (c) for (a)–(c) (error bars represent standard error of the means. Number of cochleae = 6 in each group).

A cytocochleogram representing mean percentage of missing OHCs at the measured percent distance from the cochlear apex in the noise exposure and the noise/treatment groups. Reduced OHC loss was found in the noise/treatment group compared to the noise exposure group, especially at the 55–100% distance from cochlear apex. There is a significant difference between these two groups in average OHC loss in the cochleae (P < 0.001, error bars represent standard error of the means. Number of cochleae = 6 in each group).

4-HNE immunostaining and relative fluorescence intensity in the organ of Corti. Examples of 4-HNE immunostaining images obtained from the organ of Corti in the middle turn of cochleae from the normal control (a), noise exposure (b) and noise/treatment (c) groups by confocal microscopy using the same microscopic and image collecting settings. No significant 4-HNE staining was found in the organ of Corti of the normal control group (a). Strong positive staining was seen in IHC (arrow in (b)), inner and outer pillar cells (open arrowheads in (b)), Deiters cells (arrowheads), and cells of Hensen (starburst) and Boettcher of the noise exposure group. The OHC region also had positive, but relatively weak 4-HNE immunostaining (bracket in B). Significantly lower HNE staining was found in the organ of Corti in the noise/treatment group (c). The results of the relative fluorescence intensity in the organ of Corti measured by LAS AF Lite software are shown in (d). High fluorescence intensity was found only in the organ of Corti of the noise exposure group. There were significant differences among three groups and between the normal control and the noise exposure groups, as well as between the noise exposure and the noise/treatment groups (***indicates P < 0.001). However, there was no significant difference between the normal control and the noise/treatment groups (P > 0.05). There was no significant difference among three turns within each group (P > 0.05). The numbers (1, 2, 3) in (d) indicate the normal control, noise exposure and noise/treatment groups, respectively. Brackets indicate the OHC region in (a)–(c). Scale bar = 20 μm in (c) for (a)–(c) (Error bars represent standard error of the means. Number of cochleae = 6 in each group).