Simulated reflux decreases vocal fold epithelial barrier resistance

Abstract

Objectives/hypothesis: The vocal fold epithelium provides a barrier to the entry of inhaled and systemic challenges. However, the location of the epithelium makes it vulnerable to damage. Past research suggests, but does not directly demonstrate, that exposure to gastric reflux adversely affects the function of the epithelial barrier. Understanding the nature of reflux-induced epithelial barrier dysfunction is necessary to better recognize the mechanisms for vocal fold susceptibility to this disease. Therefore, we examined the effects of physiologically relevant reflux challenges on vocal fold transepithelial resistance and gross epithelial and subepithelial appearance.

Study design: Ex vivo, mixed design with between-group and repeated-measures analyses.

Methods: Healthy, native porcine vocal folds (N = 52) were exposed to physiologically relevant acidic pepsin, acid-only, or pepsin-only challenges and examined with electrophysiology and light microscopy. For all challenges, vocal folds exposed to a neutral pH served as control.

Results: Acidic pepsin and acid-only challenges, but not pepsin-only or control challenges significantly reduced transepithelial resistance within 30 minutes. Reductions in transepithelial resistance were irreversible. Challenge exposure produced minimal gross changes in vocal fold epithelial or subepithelial appearance as evidenced by light microscopy.

Conclusions: These findings demonstrate that acidic environments characteristic of gastric reflux compromise epithelial barrier function without gross structural changes. In healthy, native vocal folds, reductions in transepithelial resistance could reflect reflux-related epithelial disruption. These results might guide the development of pharmacologic and therapeutic recommendations for patients with reflux, such as continued acid-suppression therapy and patient antireflux behavioral education.

Fig. 1

Change in transepithelial resistance from baseline (ΔR_T) in response to simulated reflux and control challenges. (A) Acidic pepsin. (B) Acid-only. (C) Pepsin-only. Reversibility assessment of reductions in R_T following acidic pepsin and acid-only challenges is also represented (30_Rinse) in (A) and (B). Negative ΔR_T values suggest a decrease in resistance from baseline, whereas positive ΔR_T suggest an increase from baseline. ΔR_T for control challenges and pepsin-only challenge (C) remained positive, while ΔR_T for acidic pepsin (A) and acid-only (B) challenges decreased. Error bars represent standard errors.

Fig. 2

Light microphotographs of representative vocal fold samples obtained after 30 minutes of challenge exposure (hematoxylin and eosin stain, 40× magnification). (A) Control. (B) Acidic pepsin (pH 3). (C) Acid-only (pH 3). (D) Pepsin-only (pH 7). Ep = stratified squamous epithelium; LP = lamina propria; arrowhead = epithelial shedding; dashed oval = sub-basilar edema or vacuolization; * = interstitial edema.

Fig. 3

Raw transepithelial resistance (R_T) data from vocal folds (n = 4) treated with a series of acidic pH. Each vocal fold is represented by a different icon: (A) = ◆, (B) = ■, (C) = ●, and (D) = ▲. R_T decreased at pH3 and pH2 relative to baseline and pH4 for each vocal fold. The greatest reductions in R_T are observed at pH2.