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(B)
6-well plate
speaker
Noise level (dB SPL)
(A)
(C)
control
120
(D)
75 dB
120
120
(E)
85 dB
95 dB
120
80
80
80
80
40
40
40
40
0.1 8
16
0.1 8 16
0.1
8 16
0.1 8
16
Frequency (kHz)
Ex vivo exposure of utricle to LFN for 1 hour
(F)
75 dB
control
85 dB
95 dB
Otoconial
membrane
(G)
(per 10 4 µm^2)
Area covered with otoconial membrane
15000
12000
9000
6000
3000
cont
75
85
95
LFN (dB)
Figure 1. Ex vivo exposure to LFN at 95 dB produced breakage of the otoconial membrane. (A)
Experimental setting. We used the same conditions as those used for in vivo LFN exposure in our recent
study (Negishi-Oshino et al., 2019). (B-E) Sound patterns of low frequency noise (LFN; 100 Hz). Control
(no exposure) (B), LFN at 75 dB (C), LFN at 85 dB (D) and LFN at 95 dB (E) are shown. Background
level of noise at 100 Hz in the control was 55 dB. Peak levels of sound with a frequency of 100 Hz are
indicated by arrows. (F) After ex vivo exposure of the utricle to LFN for 1 hour at 100 Hz, 75 dB (second
panel from the left), at 85 dB (third panel from the left), at 95 dB (fourth panel from the left) and without
exposure (control, first panel from the left), the otoconial membrane with a “cloud-like shape” in utricles
observed under a stereoscopic microscope are shown. Dotted lines show the edges of utricles and arrows
show a damaged area not covered with the otoconial membrane. Scale bar: 50 µm. (G) Area covered with
the otoconial membrane in utricles (per 10,000 µm2, mean ± SD, black bar: control, n = 10; dark gray
bar: LFN at 75 dB, n = 3; gray bar: LFN at 85 dB, n = 5; white bar: LFN at 95 dB, n = 5). Significant
differences (*p < 0.05) among the three groups were determined by Tukey’s post-hoc multiple
comparison tests.
Ex vivo exposure of utricle to LFN for 1 hour
75 dB
control
85 dB
95 dB
(A)
FM1-43FX
uptake
(B)
Phalloidin
staining
(C)
150
100
50
cont
75
Hair bundles
100
(per 10 4 µm^2)
200
(% of control)
(D)
FM1-43FX uptake
85
LFN (dB)
95
50
cont
75
85
95
LFN (dB)
Figure 2. Ex vivo exposure of the utricle to LFN at 95 dB decreased uptake of FM1-43FX in hair
cells. (A-D) After ex vivo exposure of the utricle to LFN for 1 hour at 100 Hz, 75 dB (second panels from
the left), at 85 dB (third panels from the left), at 95 dB (fourth panels from the left) and without exposure
(control, first panels from the left), (A) uptake of FM1-43FX by vestibular hair cells and (B) hair bundles
stained by fluorescein-phalloidin are shown. Scale bars: 50 µm. (C) Fluorescence intensity of FM1-43FX
incorporated by the utricles (% of control, mean ± SD) and (D) number of hair bundles (per 104 µm2,
mean ± SD) were determined [black bar: control, n = 10 (C), n = 3 (D); dark gray bar: LFN at 75 dB, n =
3 (C, D); gray bar: LFN at 85 dB, n = 5 (C), n = 3 (D); white bar: LFN at 95 dB, n = 5 (C), n = 6 (D)].
Significant differences (*p < 0.05) among the three groups were determined by Tukey’s post-hoc multiple
comparison tests.
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