Cell membrane integrity assessed by LDH leakage.

<p>Data represents LDH release in extracellular medium normalized by the average in the control group, #: p<0.01 as compared to controls by paired t-test.</p

Barrier permeability assay with or without acrolein exposure.

<p>Tissues were incubated with acrolein as the concentration indicated for 3 hours. (A). The TEER values were assessed by Ussing chamber system and associated voltage clamp. (B). The epithelial permeability was measured by fluorescent marker NaFI. Index of permeability represents the percentage of fluorescent marker passing though epithelium. Data represent Mean ±SE, n = 7, *: p < 0.05 as compared to controls.</p

Western blot analysis of occludin protein with or without acrolein exposure.

<p>Data represent Mean ± SE, n = 6, p = 0.337 as compared to controls. “C” represents control group and “A” represents acrolein treated group.</p

Expression levels of mRNAs encoding typical tight junctional proteins.

<p>Tissues were incubated with acrolein as the concentration indicated for 3 hours. The mRNA levels of occludin (A) and claudin3 (B) were determined by qPCR. Data represent Mean ± SE, n = 6 (p = 0.259 for occludin and p = 0.556 for claudin3) as compared to controls.</p

The metabolic activities of vocal fold tissue following acrolein exposure ex vivo.

<p>Epithelial metabolic activity was determined by the MTT assay. Tissues were incubated with acrolein as the concentration indicated for 3 hours. Data represent Mean ± SE, n = 7, *: p < 0.05, §: p < 0.001 as compared to controls.</p

Immunohistochemical study of lipid peroxidation marker with or without acrolein exposure.

<p>(A). A typical confocal image of vocal fold epithelia. 4-HNE was stained in red on the left panel; occludin was stained in green on the middle panel; and the merged signals in yellow was present on the right panel. (B). Quantification of signal intensities of 4-HNE and occludin. Data represent Mean ± SE, n = 4, *: p < 0.05 as compared to controls.</p

Figure 6

<p>A. Measurement of depth of the remaining viable cell surface. Objective measurement of the depth (Dp) of the remaining viable cell surface layer in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. Measurement bar represents 4 µm. B. Objective examination of depth of the remaining viable cell surface using TEM. Average depth (µm) of the remaining viable cell surface in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions for 30, 60, and 120 minutes. * Denotes a significant difference between groups (p<0.0167).</p

Figure 4

<p>A. SEM representative images for control, modal intensity, and raised intensity phonation. Representative 2×2 µm SEM images for the control, modal intensity, and raised intensity phonation conditions after 30, 60, and 120 minutes of phonation. Measurement bar represents 0.5 µm. B. Visual examination of epithelial surface microprojections. Classification of images by damage severity using routine visual examination of epithelial surface microprojections. Four categories of damage severity: extensive (black fill), moderate (dark gray fill), minimal (light gray fill), and normal (white fill) in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. C. Objective examination of epithelial surface microprojection density using SEM. The percentage of vocal fold surface covered by microprojections in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. * Denotes a significant difference between groups (p<0.01).</p

Figure 5

<p>A. Measurement of epithelial surface microprojection density and height. Objective measurement of the density (Dn) and height (H) of vocal fold epithelial surface microprojections in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. Measurement bar represents 1 µm. B. Objective examination of epithelial surface microprojection density using TEM. The number of microprojections per 10 µm field in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes. * Denotes a significant difference between groups (p<0.0167). C. Objective examination of epithelial surface microprojection height using TEM. Average height (µm) of microprojections in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes. * Denotes a significant difference between groups (p<0.0167).</p

Damage severity descriptions used for visual evaluation of microprojections.

<p>All images were categorized into four groups according to the degree of surface damage. (1) extensive – the cell surface was destroyed and cytoskeleton was exposed; (2) moderate – all microprojections were damaged and appeared flat or normal; (3) minimal – microprojections were slightly damaged; and (4) normal – microprojections showed no damage.</p