Chronic exposure to low frequency noise at moderate levels causes impaired balance in mice

Abstract

We are routinely exposed to low frequency noise (LFN; below 0.5 kHz) at moderate levels of 60-70 dB sound pressure level (SPL) generated from various sources in occupational and daily environments. LFN has been reported to affect balance in humans. However, there is limited information about the influence of chronic exposure to LFN at moderate levels for balance. In this study, we investigated whether chronic exposure to LFN at a moderate level of 70 dB SPL affects the vestibule, which is one of the organs responsible for balance in mice. Wild-type ICR mice were exposed for 1 month to LFN (0.1 kHz) and high frequency noise (HFN; 16 kHz) at 70 dB SPL at a distance of approximately 10-20 cm. Behavior analyses including rotarod, beam-crossing and footprint analyses showed impairments of balance in LFN-exposed mice but not in non-exposed mice or HFN-exposed mice. Immunohistochemical analysis showed a decreased number of vestibular hair cells and increased levels of oxidative stress in LFN-exposed mice compared to those in non-exposed mice. Our results suggest that chronic exposure to LFN at moderate levels causes impaired balance involving morphological impairments of the vestibule with enhanced levels of oxidative stress. Thus, the results of this study indicate the importance of considering the risk of chronic exposure to LFN at a moderate level for imbalance.

Figure 1. Frequency distributions of noise used in this study.

Frequency distributions (means ± SD) of (A) low frequency noise (LFN; 0.1 kHz) and (B) high frequency noise (HFN; 16 kHz) are presented. Noise levels from a speaker at a distance of 10 cm in a soundproof room were measured by a noise level meter and calculated as average of five repeated measurements. Background levels measured in a soundproof room without noise-generating devices were subtracted from noise levels from the speaker. Whole noise levels of (A) LFN and (B) HFN measured by the noise level meter without FFT analyzing software were almost the same (70 dB SPL).

Figure 2. Exposure to LFN affects rotarod performance of ICR mice.

Before (A) and after (B) exposure to LFN (open triangles) and HFN (open squares), retention times (seconds, mean ± SD, n = 7) on the rotarod (at 30 rpm) were measured. Results for non-exposed mice (closed circles) are also plotted (mean ± SD, n = 7). Mice were allowed a maximum retention time of 120 seconds per trial. Significant difference (**, p<0.01; *, p<0.05) from the non-exposure group was analyzed by the Mann-Whitney U test.

Figure 3. Exposure to LFN affects gait pattern of ICR mice.

(A) After exposure to LFN (center panel) and HFN (right panel), front and back paws of mice were dipped in red or green paint, and mice walked across a box lined with paper. Non-exposed mice (left panel) are also shown. LFN-exposed mice display shorter stride length and winding gait patterns (center panel, arrows). (B) Quantification of stride length. Strides (mean ± SD) for seven mice (each group) were assessed. A total of 40–50 steps for each group were determined. Significant difference (**, p<0.01) from the non-exposure group was analyzed by the Mann-Whitney U test.

Figure 4. Decreased number of vestibular hair cells with increased levels of oxidative stress in LFN-exposed mice.

(A, B) Immunohistochemical analysis with anti-calbindin D28k for vestibules in LFN-exposed (B) and non-exposed mice (A). (C-F) Enhanced oxidative stress levels in vestibules of LFN-exposed mice. Vestibules of LFN-exposed (D, F) and non-exposed mice (C, E) were immunohistochemically stained by an anti-Ox-PC antibody (DLH3) (C, D) and anti-D-βeta-Asp antibody (E, F). Vestibules of LFN-exposed mice showed stronger signals (D, F, arrows) than those of non-exposed mice (C, E). Scale bars: 20 µm. (G-I) Percentage (means ± SD) of calbindin-positive hair cells (G) and positive areas of anti-Ox-PC antibody (H) and anti-D-βeta-Asp antibody (I) in vestibules from LFN-exposed mice (LFN, black bar, n = 7) and non-exposed mice (Cont, gray bar, n = 7). Significant difference (*, p<0.05) from non-exposed mice was analyzed by the Mann-Whitney U test.