Oxidative imbalance in the aging inner ear
Introduction
Studies in a variety of organisms and species support oxidant stress as a factor in the pathology of aging. Levels of reactive oxygen species (ROS) increase in essentially all tissues during aging [7], [8], [27], [28], and mitochondrial DNA deletions, consequences of ROS action, also accumulate progressively [4], [29]. Such biochemical findings are extended by genetic and molecular biological evidence. Mutations in the nematode C. elegans leading to lifespan extension typically also conferred resistance to multiple forms of stress, including oxidant stress [10], [11]. Some of the single-gene mutations that prolonged lifespan in flies [9] also increased stress resistance, and extra copies of genes encoding the antioxidant enzymes superoxide dismutase (SOD) and catalase extended the life span in drosophila[18]. Finally, dietary restriction (reduced caloric intake with nutritional maintenance) enhanced the resistance of the nervous system to age-related diseases by inducing neurotrophic factors and stress proteins that protect neurons against ROS [14].
Oxidative stress may also be part of the aging process in the cochlea. Mitochondrial deletions increase with age and correlate with hearing loss in rats [22], [23]. Furthermore, age-related hearing loss developed earlier and was more severe in mice with SOD genes deleted [15], [16], [17]. However, little is known about the type of reactive oxygen species that may be involved in the aging process in the inner ear, which tissues and cells (sensory cells and nerve fibers versus supporting structures) are affected and whether the time course of their emergence correlates with the appearance of morphological and functional deficits.
The current study investigates indicators of oxidative stress in the aging cochlea and their distribution in the organ of Corti, the stria vascularis, and the spiral ganglion. The CBA/J mouse was chosen because it carries the ahl-resistant gene [5] thus not rendering it prone to accelerated premature hearing loss as, for example, C57/BL6 or BALB strains. The ages selected for the study represent distinct stages in auditory development and deterioration of the mouse [24]. The auditory system is mature at 3 months of age and has not yet undergone major pathological and pathophysiological changes by 12 months. At 18 months, a moderate hearing loss at high frequencies (24 kHz) can be expected in about 1/3 of the animals, a condition aggravated by 23 months of age when severe hearing loss is seen in 75% of animals. On the cell and tissue level, we studied markers of oxidant stress including glutathione-conjugated proteins, products of H2O2-mediated oxidation, and 4-hydroxynonenal and 3-nitrotyrosine, products of hydroxyl radical and peroxynitrite action. Conversely, antioxidant capacity was followed by analyzing superoxide dismutase and apoptosis-inducing factor histochemically and by Western blotting.
Section snippets
Materials
Enhanced chemiluminescence (ECL) for Western blotting detection was purchased from Amersham Pharmacia Biotech (Piscataway, NJ); mouse monoclonal antibody against glutathione-conjugated protein from ViroGen (Watertown, MA); monoclonal antibody to 3-nitrotyrosine and polyclonal antibody to 4-hydroxynonenal from Alexis Biochemicals (San Diego, CA); polyclonal anti-manganese superoxide dismutase from Stressgen (Victoria, Canada); polyclonal anti-AIF from Santa Cruz Biotechnology (Santa Cruz, CA).
Oxidative stress increases in the aging cochlea
The extent of glutathione-conjugation to proteins is considered a measure of oxidative stress, especially due to H2O2[30]. Glutathionylated proteins, including a very prominent conjugate with actin at 42 kDa, increased in cochlear extracts with age. Densitometry confirmed a significant 3-fold increase at 18 months compared to young mice (Fig. 1).
Next, we used immunohistochemistry to localize 4-hydroxynonenal (4-HNE), a marker for lipid peroxydation [3], and 3-nitrotyrosine (3-NT), a
Discussion
An oxidative imbalance is clearly evident in the aging mouse cochlea. Such an imbalance can result from increased production of reactive oxygen species or weakened defense systems, and both mechanisms may participate here. All markers of oxidant stress, lipid peroxidation, glutathionylation and nitrosylation of proteins increase whereas the measures of antioxidant defenses, AIF and SOD2, decrease with age.
While mitochondrial deletions have previously been detected in the aging inner ear [19],
Acknowledgements
This research was supported by a pilot grant from the American Claude Pepper Center at the University of Michigan, program project grant AG-025164 from the National Institute on Aging, and core grant P30 DC-05188 from the National Institute on Deafness and Other Communication Disorders, NIH.
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Present address: Otorhinolaryngological Hospital of First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, PR China.