In the current study, HET mice did not show acceleration in ABR threshold shifts with aging when compared to WT mice. This physiological finding was confirmed by histological analysis, which revealed no significant differences in HC survival rates, SGC density, and SV thickness between HET and WT mice at either 4 or 16 months of age. The HET mice had reduced Mn-SOD activity (~50%) in the SGC and SV at either age when compared to WT mice. The expression of 8-OHdG, a marker of DNA oxidation, was increased in the SGC with aging in both WT and HET mice, and the expression of 8-OHdG in the SGC and SV at 16 months was significantly greater in HET mice compared to WT mice. The expression of 4-HNE, a marker of lipid peroxidation, was increased in the SGC with aging and tended to increase in the SV in both HET and WT, although it was not significantly different between HET and WT mice. These findings suggest that half reduction of Mn-SOD may accelerate oxidative stress, predominantly to DNA, to some extent, but may not be sufficient to increase the damage to the cochlear tissues under normal aging process. Since both WT and HET mice carry a specific mutation in the cadherin 23 gene, it is possible that the effect of Mn-SOD might be masked by C57 genetic pathology. However, we enrolled wild-type C57BL/6 littermates as a control, which we consider made the influence of the C57 background minimal.
It has been reported that mice lacking Cu/Zn-SOD exhibited 30% decrease in life expectancy [42
], whereas overexpression of Cu/Zn-SOD and catalase extended lifespan in Drosophila
]. Further, small synthetic mimetics of SOD/catalase increased lifespan in C. elegans
]. Collectively, these results imply that interplay between ROS and protective responses by antioxidant enzymes is an important factor in determining aging and lifespan. Nevertheless, the role of these antioxidant enzymes in aging is still controversial. Sod2+/−
mice have been reported to have reduced Mn-SOD activity (~50%) in all tissues throughout life, increased oxidative damage as demonstrated by elevation of 8-OHdG in all tissues (significantly higher compared with WT mice), and increase in tumor incidence. However, the lifespans of Sod2+/−
mice were identical to those of WT mice and biomarkers of aging, such as cataract formation, immune response, and formation of glycoxidation products carboxymethyl lysine and pentosidine in skin collagen changed with age to the same extent in both WT and Sod2+/−
], indicating that life-long reduction of Mn-SOD activity leads to increased levels of oxidative damage to DNA and increased cancer incidence but does not appear to affect aging. Overexpression of antioxidant enzymes in mice, such as Cu/Zn-SOD or catalase, did not extend lifespan [31
]. The median lifespan of mice heterozygous of glutathione peroxidase 4 was significantly longer than that of wild-type mice, even though they showed increased sensitivity to oxidative stress-induced apoptosis [32
]. These results pose a question in terms of the importance of antioxidant enzymes in preventing aging process.
As of age-related degeneration in the cochlea, it has been reported that overexpression of Cu/Zn-SOD did not prevent or slow AHL, whereas Cu/Zn-SOD KO mice exhibited acceleration of AHL due to massive loss of HCs and auditory neurons in an earlier onset [34
]. Interestingly, half-expressed Cu/Zn-SOD did not accelerate AHL in mice [33
]. Similarly, Le and Keithley [36
] reported that Mn-SOD heterozygous transgenic mice showed no deterioration in the extent of ABR threshold shifts compared to the background strain but did not address the histological findings. Although it is ideal to investigate Mn-SOD homozygous knockout mice to assess the importance of Mn-SOD in AHL, systemic Mn-SOD-deficient mice are known to die at an early stage after birth. Because of this reason, we are forced to use Mn-SOD heterozygous knockout mice, but we examined not only their auditory function but also cochlear histology and immunohistology. The present study demonstrated no significant difference in ABR thresholds between HET and WT mice at either 4 or 16 months of age, which is consistent with the report by Le and Keithley [36
]. We also found no significant differences in SGC densities or SV thickness between HET and WT mice at either 4 or 16 months, supporting that reduction of Mn-SOD by half did not accelerate age-related damage in the cochlea.
In the present study, the labeling indices of Mn-SOD in the SGC and SV in HET mice were reduced to be ~50% of those of WT mice at either 4 or 16 months of age. The expression of Mn-SOD was declined with aging in the SGC in both WT and HET mice, whereas it was unchanged with aging in the SV. These results are in harmony with the report by Jiang et al. [46
] that immunoreactivity of Mn-SOD was decreased with aging in the SGC but not in the SV in CBA/J mice. These suggest that age-related decline of Mn-SOD expression differs among tissues even in the same organ, namely, the cochlea.
In the SGC, the expression of 8-OHdG was significantly increased with aging in both WT and HET mice and was significantly greater in HET mice at 16 months compared to WT mice. On the other hand, in the SV, the expression of 8-OHdG was significantly increased with aging only in HET mice, being significantly greater at 16 months in HET mice compared to WT mice. These findings suggest that, under normal condition, 8-OHdG accumulates steadily in the SGC but not significantly in the SV. This appears reasonable, considering that Mn-SOD is decreased with aging in the SGC but not in the SV. Under pathological situation that Mn-SOD is decreased by half, 8-OHdG may accumulate with aging more significantly in the SGC and even in the SV.
The expression of 4-HNE was increased with aging in the SGC in both WT and HET mice, and there was no significant difference in the expression level between these mice. The expression of 4-HNE did not significantly differ in the SV between WT and HET mice at any age, although it was slightly greater in HET mice than in WT mice at either 4 or 16 months of age and slightly increased with aging in both HET and WT mice. These findings suggest that, under normal condition, 4-HNE accumulates with aging in the SGC but not significantly so in the SV. It is unclear why reduction of Mn-SOD by half increased the expression of 8-OHdG but not 4-HNE in the SGC with aging. Downregulation of Mn-SOD might contribute to the augmentation of oxidative DNA stress more significantly than lipid peroxidation in the cochlear tissues.
Although we did not find significant differences of ABR threshold shift or the extent of degeneration of the SGC and SV between HET and WT mice under normal physiological condition, it is possible that reduction of Mn-SOD by half accelerates age-related cochlear damage under pathological condition. It has been reported that, when Mn-SOD heterozygote mice were administered hepatotoxic agent that promoted ROS generation, liver damage became prominent [47
], although liver-specific Mn-SOD homozygous knockout mice presented no obvious morphological or biochemical damages under normal environmental stress [37
]. This result implies that, even if tissue damage is not evident under normal condition, it can become prominent under the burden of oxidative stress. It is well known that inner ear damage by intense noise and ototoxic drugs is mediated through excessive generation of ROS [48
]. To verify the hypothesis, functional and morphological assessment should be carried out in Mn-SOD homozygous knockout mice under pathological oxidative stress. We are now conducting an experiment to investigate whether HET mice will exhibit acceleration of AHL compared to WT mice when they are raised in noisy environment.