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Fig 1.

CYP46A1 expression and cholesterol content in aged mouse cochlea.

(A) Representative confocal images in the xy projection of whole mounts organ of Corti from the medial region immunolabeled for CYP46A1 (green) and calretinin (red) from young (top) and old (bottom) C57BL/6J mice (scale bar = 30 μm). Filled arrows indicate the SCs’ area. Arrowheads point to surviving OHCs in aged mice. IHCs and OHCs’ area are also indicated. (B) Quantitative data obtained from young (n = 5) and aged (n = 6) mice. CYP46A1 fluorescence intensity (a.u., arbitrary units) was measured in IHCs (left), OHCs (middle), and SCs (right) at 2 months and 2 years of age at 3 regions of the cochlea: apical, medial, and basal end (Young: n = 43 IHCs, 78 OHCs, and 144 SCs at the apical; 25 IHCs, 29 OHCs, and 51 SCs at the medial; and 12 IHCs, 31 OHCs, and 24 SCs at the basal region. Old: n = 22 IHCs, 28 OHCs, and 55 SCs at the apical; 24 IHCs, 17 OHCs, and 79 SCs at the medial; and 28 IHCs, 13 OHCs, and 69 SCs at the basal region). Examples of ROIs that correspond to each cellular type (IHC, OHC, and SC) were drawn. In aged mice, there was a significant increase in CYP46A1 immunolabeling in IHCs, OHCs, and SCs compared to young ears at all the cochlear regions (except at the apical end in SCs). n.s. = not significant. Asterisks represent the statistical significance (t test, ** = p < 0.01, *** = p < 0.001 **** = p < 0.0001). (C) Representative confocal images of whole mounts organ of Corti from the medial region of the cochlea stained for filipin to label cholesterol (green) and phalloidin for visualizing actin in stereocilia (blue) from young (top) and old (bottom) C57BL/J mice (scale bar = 30 μm). Rectangles indicate the region of the zoomed-in areas shown on the right using a 63× objective (scale bar = 8 μm). Pink dotted lines represent the diameter of individual IHC and OHC. (D) Filipin staining quantified with a pixel intensity graph (a.u., arbitrary units) along a line (shown as dotted pink lines in panel C) crossing either individual OHCs or IHC in young (n = 3) and aged (n = 6) mice at all the regions of the cochlea. In aged mice, there was a significant reduction in cholesterol content in IHCs (top panel) and surviving OHCs (bottom panel) compared to young ears (Young: n = 104 IHCs and 200 OHCs; Old: n = 52 IHCs and 22 OHCs). Asterisks represent the statistical significance (t test, **** = p < 0.0001). The data underlying this figure can be found at https://osf.io/xpemk/. IHC, inner hair cell; OHC, outer hair cell; ROI, region of interest; SC, supporting cell.

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Fig 2.

Auditory function in control, efavirenz, and efavirenz plus phytosterols-treated mice.

(A) Representative ABR waveforms from control (black), efavirenz-treated (red), and efavirenz-treated plus Phy (blue) mice recorded at 16 kHz tone bursts at increasing sound pressure levels (dB SPL). Thresholds are indicated by dashed traces and were determined by the presence of peak 1. Scale bar applies to the 3 recordings. ABR thresholds showed a slight increase in those mice treated with efavirenz that partially recovered when supplemented with Phy. Mean ABR thresholds (B) and ABR peak 1 (P1) amplitudes at 80 dB SPL (C) for control (n = 6), treated with efavirenz (n = 6), and treated with efavirenz together with Phy (n = 6) mice at different test frequencies. We included controls at 12 weeks; the age after the treatment (black; n = 6) and controls at 8 weeks, before starting the treatment (gray; n = 6). (D) DPOAEs thresholds for control (12 weeks; black; n = 6 and 8 weeks; gray; n = 6), treated with efavirenz (n = 6), and treated with efavirenz together with Phy (n = 6) mice at different test frequencies. DPOAEs thresholds showed a significant increase in those mice treated with efavirenz that partially recovered when supplemented with Phy. (E) Mean DPOAEs amplitudes versus level functions with f2 = 16 kHz in the 3 groups of mice. Group means ± SEM are shown (B-E). (F) Examples of fast Fourier transform traces showing the cubic DPOAE amplitude (2f1-f2), at f1 = 13.34 kHz; f2 = 16 kHz with f2 input level of 80 dB SPL. Scale bar of 50 dB SPL applies to the 3 recordings. Asterisks represent the statistical significance (Kruskal–Wallis test, followed by Dunn’s post-tests * = p < 0.05). The data underlying this figure can be found at https://osf.io/xpemk/. ABR, auditory brainstem response; Phy, phytosterols; SPL, sound pressure level.

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Fig 3.

Cholesterol content in OHCs from control, efavirenz, and efavirenz plus phytosterols-treated mice.

(A) Representative confocal images of whole mounts organ of Corti from the OHCs’ area of the basal region stained with phalloidin (blue; to visualize actin in stereocilia) and filipin (green; to label cholesterol) in control, treated with efavirenz, and treated with efavirenz plus Phy mice. Pink line represents approximately 16 μm, i.e., the width of 2 consecutive OHCs (scale bars = 8 μm). (B) Pixel intensities plot obtained from all the sections (Control: n = 14 cells from 6 animals; efavirenz: n = 12 cells from 6 animals and efavirenz together with phytosterols: n = 28 cells from 6 animals) along the width of 2 consecutive OHCs from each of the 3 rows at the basal region of the cochlea shows that after efavirenz treatment, there was a reduction in cholesterol and a slight increase in filipin staining when supplemented with Phy. Pink line underneath the x-axis represents the width of 2 consecutive OHCs (C) Relative intensity bar graph showing the changes in filipin staining in the 3 groups of mice at the 3 regions of the cochlea (apical, medial, and basal). There was a significant reduction in cholesterol levels after efavirenz treatment. Treatment with efavirenz together with Phy showed an increase in filipin staining at the 3 regions compared with mice treated with efavirenz alone. However, when compared to controls, there was a significant reduction in filipin staining at the 3 regions of the cochlea (Control: n = 80 OHCs at the apical; 140 OHCs at the medial; and 70 OHCs at the basal region from 6 mice; efavirenz: n = 200 OHCs at the apical; 70 OHCs at the medial; and 65 OHCs at the basal region from 6 mice and efavirenz together with phytosterols: n = 92 OHCs at the apical; 115 OHCs at the medial; and 77 OHCs at the basal region from 6 mice). Group means ± SEM are shown. Asterisks represent the statistical significance (one-way ANOVA, followed by Tukey’s test, **** = p < 0.0001). The data underlying this figure can be found at https://osf.io/xpemk/. OHC, outer hair cell; Phy, phytosterols.

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Fig 4.

Prestin expression in OHCs from control, efavirenz, and efavirenz plus phytosterols-treated mice.

(A) Representative confocal images of whole mounts organ of Corti from the medial region immunolabeled for calretinin (red) and prestin (green) in control, treated with efavirenz, and treated with efavirenz plus Phy mice (top) (scale bar = 16 μm). Cochlear whole mount yz projection of 3 OHCs from each row (bottom) (scale bar = 8 μm). (B) Mean pixel intensity plot along the width of one individual OHC from each of the 3 rows at the medial region of the cochlea as a function of distance in the 3 groups of mice (bottom panel). High-magnification image of one OHC from the selected area indicated by a white rectangle on the confocal image in A (top panel). The pink and orange line represent approximately 8 μm, the width of one OHC (pink ends indicate the region corresponding to the lateral wall, and orange the cytoplasm of the cell). (C) Ratio between the average fluorescence intensity in the lateral wall and the cytoplasm to visualize the change in distribution of prestin within each OHC at the medial region of the cochlea (Control: n = 24 cells from 6 mice; efavirenz: n = 25 cells from 6 mice and efavirenz together with phytosterols: n = 38 cells from 6 mice). (D) Bar graph showing the mean in the fluorescence intensity of prestin at the 3 cochlear regions (apical, medial, and basal). The intensity was measured by tracing a line through 3 consecutive OHCs. (Control: n = 110 OHCs at the apical, 60 OHCs at the medial, and 76 OHCs at the basal region; efavirenz: n = 75 OHCs at the apical, 90 OHCs at the medial, and 60 OHCs at the basal region and efavirenz together with phytosterols: n = 110 OHCs at the apical, 105 OHCs at the medial, and 80 OHCs at the basal region). After efavirenz treatment, there was a reduction in prestin labeling in the OHC lateral wall. Supplementation with Phy increased the presence of prestin in the OHCs membrane compared with mice following efavirenz treatment alone. Group means ± SEM are shown. Asterisks represent the statistical significance (one-way ANOVA, followed by Tukey’s test, **** = p < 0.0001). The data underlying this figure can be found at https://osf.io/xpemk/. OHC, outer hair cell; Phy, phytosterols.

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Fig 4 Expand