Article Figures & Data
Figures
- Figure 1.
Cavitation device produces intense underwater sound pressure and well plate acceleration. A, B, Two ultrasonic transducers emit stimuli at 40 kHz creating underwater cavitation within the attached stainless steel tank. The signal was generated with a 300-W ultrasonic generator with inline rheostat for fine amplitude adjustment. B, inset, Larval zebrafish are exposed to acoustic stimulation, likely produced by cavitation, in labeled wells of a modified 24-well plate encased in glycerol resting atop 22 cm of water. C, Sound pressure levels measured via hydrophone show increasing intensity with increasing input voltage. D, Peak amplitude of well plate acceleration (measured from the top of the well plate) increases somewhat linearly with input voltage while average envelope exhibits a more modest increase. E, Fast Fourier transform of power spectrum produced by cavitation device shows broadband energy within the low-frequency range.
- Figure 2.
Acoustic stimulation results in exposure time-, intensity-, and post-exposure time-dependent reduction in DASPEI labeling, indicative of hair cell damage. A–C, Representative images of (A) unexposed larval zebrafish and (B, C) fish exposed to acoustic stimulation. Scale bar applies to all three images. Unexposed fish exhibit bright DASPEI staining indicative of a full complement of lateral line hair cells while fish exposed to 80 min of acoustic stimulation have diminished DASPEI labeling 72 h post-exposure. Two representative images of acoustically exposed fish are shown to depict the diversity of DASPEI labeling observed. D–F, Quantification of acoustic stimulation-induced hair cell loss. D, Fish exposed to 0.7 V show no reduction in DASPEI labeling. E, Fish exposed to 1.2 V of acoustic stimulation exhibit the greatest reduction in DASPEI labeling after 80 min of exposure and 72 h post-exposure. F, 1.7 V of acoustic stimulation produces similar DASPEI reduction to 1.2 V. Asterisks indicate significant differences from age-matched unexposed controls (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). Statistical analysis is shown in Table 1. N = 10–12 animals per treatment, values are mean ± SD.
- Figure 3.
Acoustic stimulation decreases lateral line hair cell number. Unexposed (A) and acoustically stimulated (B) O2 neuromasts from myo6b:EGFP transgenic larval zebrafish. Scale bar applies to both images. 1.2 V (C) and 1.7 V (D) of acoustic stimulation significantly reduces the number of hair cells in five anterior lateral line neuromasts at 48 and 72 h after cessation of noise. 1.2 V (E) and 1.7 V (F) reduces hair cell number in pLL neuromasts P1 and P2. Asterisks indicate significant difference from age-matched unexposed controls (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001). Statistical analysis is shown in Table 1. N = 10–12 animals per treatment, values are mean ± SD.
- Figure 4.
Acoustic stimulation produces an exposure time-dependent reduction in saccular hair cells. Unexposed (A) and acoustically stimulated (B) saccules from myo6b:EGFP transgenic zebrafish. There was no obvious spatial pattern to the damage in the acoustically exposed saccules. C, Treatment with 1.7 V of acoustic stimulation for 120 min significantly reduces saccular hair cell number when assessed 72 h post-exposure (one-way ANOVA; exposure time: F(2,30) = 11.89, p = 0.0002). Asterisks indicate significant difference from unexposed age-matched control (****p < 0.001). N = 10–12 animals per treatment, values represent mean ± SD.
- Figure 5.
Loading of the mechanotransduction dependent dye FM 1-43FX is not affected by acoustic stimulation in wild-type *AB zebrafish. A–C, Representative images of neuromasts loaded with FM 1-43FX. Unexposed (A) and acoustically stimulated (B, C) neuromasts are brightly labeled with FM 1-43FX. D, Quantified FM 1-43FX fluorescence (normalized to hair cell number) is not significantly different in unexposed control versus 72 h post-exposure suggesting that acoustic stimulation does not alter hair cell mechanotransduction (one-way ANOVA; post-exposure time: F(4,50) = 4.001, p = 0.0068). Acoustically-exposed fish exhibit highly variable FM 1-43FX loading. E, Hair cell survival is reduced 72 h after acoustic stimulation (one-way ANOVA; post-exposure time: F(4,43) = 17.19, p < 0.0001). Hair cells were labeled with anti-parvalbumin and quantified in fixed animals. Asterisks indicate significant difference from unexposed control (****p < 0.001). N = 8–12 animals per treatment and values represent mean ± SD.
- Figure 6.
A total of 80 min of acoustic stimulation reduces ribeye puncta and increases the frequency of orphaned ribeye puncta. A–A’’, In unexposed neuromasts, the presynaptic marker ribeye b (red) colocalizes with the postsynaptic marker MAGUK (green). B–C”, 72 h after acoustic stimulation, many orphaned ribeye b puncta are present (white arrows). Hair cells are labeled with DAPI (blue). D, Acoustic stimulation significantly reduces the number of synaptic ribbons per hair cell when assessed 72 h after acoustic stimulation (t test; p = 0.0079). E, Acoustic stimulation increased the number of synaptic ribbons lacking a neighboring MAGUK puncta (orphaned ribbons; t test; p = 0.0004). Asterisks indicate significant different from unexposed control (**p < 0.01, ***p < 0.005). N = 12 animals per treatment (three neuromasts per animal), values represent mean ± SD.
- Figure 7.
The number of TUNEL+ hair cells increases 72 h after exposure to acoustic stimulation. A, Representative images from unexposed control fish labeled with anti-parvalbumin and processed with the apoptotic maker TUNEL show no TUNEL+ cells within the neuromast. B, 72 h after noise exposure, TUNEL+ hair cells are present within the IO1 neuromast (arrow). C, TUNEL+ hair cells are significantly increased over unexposed controls 72 h post-exposure recovery in IO1, IO2, and IO3 neuromasts (two-way ANOVA; post-exposure time: F(3,71) = 12.09, p < 0.0001; acoustic stimulation: F(1,71) = 9.081, p = 0.0036; interaction: F(3,71) = 2.872, p = 0.0423). D, TUNEL+ parv- cells (non-hair cells, arrowhead) in unexposed and acoustic stimulation exposed fish are not significantly different over 72 h of recovery, suggesting that acoustic stimulation specifically damages hair cells (two-way ANOVA; post-exposure time: F(3,72) = 21.91, p < 0.0001; acoustic stimulation: F(1,72) = 0.3405, p = 0.5613; interaction: F(3,72) = 1.345, p = 0.2666). Asterisks indicate significant difference from unexposed control (**p < 0.01). N = 7–12 fish per treatment (three neuromasts per fish) and values represent mean ± SD.
- Figure 8.
Acoustic stimulation-exposed fish exhibit complete hair cell regeneration. Eighty minutes of 1.7-V acoustic stimulation produces a reduction in DASPEI labeling by 72 h that is completely reversed by 96 h post-exposure (two-way ANOVA; post-exposure time: F(4,99) = 7.68, p < 0.0001). Asterisks indicate significant difference from age-matched unexposed control (****p < 0.001). N = 10–12 animals per treatment, values represent mean ± SD.
- Figure 9.
Hair cells in the defective mechanotransduction mutant mariner line are resistant to acoustic stimulation damage. Unexposed F1 progeny from mariner heterozygotes exhibit DASPEI scores centered on 100%. Exposed F1 fish are distributed in two distinct groups that are similar to the predicted Mendelian distribution (inset), where roughly 25% of fish do not exhibit hair cell damage. N = 13–16 animals per treatment, data presented as absolute values.
- Figure 10.
Acoustic stimulation-induced hair cell damage is inhibited by protein synthesis and caspase inhibition. A, A 4-h pulse with the protein synthesis inhibitor cycloheximide immediately after acoustic stimulation reduces hair cell damage when assessed 72 h after acoustic stimulation (two-way ANOVA; cycloheximide: F(3,83) = 10.58, p < 0.0001). B, 72-h treatment with the pan-caspase inhibitor Z-VAD starting immediately after acoustic stimulation exposure robustly protects hair cells from damage (two-way ANOVA; Z-VAD: F(2,64) = 13.9, p < 0.0001). Asterisks indicate significant difference from unexposed (0’, 0 µM cycloheximide/Z-VAD) controls (**p < 0.01, ****p < 0.001). N = 11–12 animals per treatment, values represent mean ± SD.
- Figure 11.
Treatment with antioxidants protects lateral line hair cells from acoustic stimulation. A, 72-h treatment with D-methionine, an antioxidant that prevents NIHL in mammals, robustly protects lateral line hair cells from acoustic stimulation (two-way ANOVA; D-methionine: F(2,69) = 14.92, p < 0.0001). B, A mini-screen of five antioxidants and the glutathione inhibitor buthionine sulfoximine (negative control) reveals novel hair cell protectants that have hair cell survival scores higher than vehicle (DMSO) control (one-way ANOVA; antioxidant: F(7,82) = 12.28, p < 0.0001). Asterisks indicate significant difference from unexposed, age-matched controls (A) and DMSO-only noise exposed controls (B; **p < 0.01, ****p < 0.001). N = 11–13 fish per treatment, values represent mean ± SD.
Tables
- Table 1.
Statistical analysis of hair cell survival, as determined by DASPEI assessment (Fig. 2) or hair cell counts (Fig. 3)
Assessment method Voltage Variable F score p value DASPEI 0.7 V Exposure time F(12,211) = 1.54 p = 0.2396 Recovery time F(6,211) = 1.34 p < 0.0001 Interaction F(12,211) = 1.54 p = 0.1131 DASPEI 1.2 V Exposure time F(6,216) = 25.26 p < 0.0001 Recovery time F(2,216) = 144.0 p < 0.0001 Interaction F(12,216) = 6.23 p < 0.0001 DASPEI 1.7 V Exposure time F(6,216) = 19.72 p < 0.0001 Recovery time F(2,216) = 38.83 p < 0.0001 Interaction F(12,216) = 4.33 p < 0.0001 Hair cell counts 1.2 V aLL Exposure time F(2,91) = 13.39 p < 0.0001 Recovery time F(2,91) = 12.30 p < 0.0001 Interaction F(4,91) = 4.42 p = 0.0026 Hair cell counts 1.7 V aLL Exposure time F(2,93) = 10.87 p < 0.0001 Recovery time F(2,93) = 4.97 p = 0.0089 Interaction F(4,93) = 3.02 p = 0.0217 Hair cell counts 1.2 V pLL Exposure time F(2,89) = 4.90 p = 0.0095 Recovery time F(2,89) = 12.30 p < 0.0001 Interaction F(4,89) = 3.27 p = 0.0151 Hair cell counts 1.7 V pLL Exposure time F(2,91) = 9.85 p = 0.0001 Recovery time F(2,91) = 3.62 p = 0.0308 Interaction F(4,91) = 6.50 p = 0.0001 All data were analyzed by two-way ANOVA.
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