Effects of Time-Doses and Cycle-Doses of Acute Vibration Exposure on Apoptotic Cell Death and TNF-α Signaling in the Vocal Fold Epithelium

Abstract

The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (3 levels) or a control group. Animals received 30 minutes (n = 5), 60 minutes (n = 5), or 120 minutes (n = 5) of modal intensity phonation or 120 minutes of vocal fold approximation without airflow (control; n = 5). Laryngeal tissue specimens were evaluated for apoptosis by immunofluorescence staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method and transmission electron microscopy (TEM). TNF-α gene transcript levels were measured using quantitative real-time polymerase chain reaction and TNF-α protein expression was evaluated using immunofluorescence staining. Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency and correlated with measures of apoptosis. Results revealed a significant main effect of time-dose of vibration on the mean intensity of TUNEL staining. Post-hoc testing indicated that TUNEL staining in the vocal fold epithelium of animals receiving 120 minutes of vibration was significantly higher than animals receiving 120 minutes of vocal fold approximation alone (control). There was no significant effect of time-dose on the mean area of epithelial cell nuclei using TEM images. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Findings illustrate that 120 minutes of in vivo modal intensity phonation is an apoptosis-inducing stimulus in the vocal fold epithelium. In contrast, shorter durations of vibration do not signal apoptosis. Morphological features of apoptosis using TEM images failed to agree with TUNEL results. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases.