Comparison of Stress-Mediated Vocal Fold Epithelial Effects in Conventionally-Raised and Germ-Free Mice

Up to 25% of patients with voice disorders report concurrent psychosocial stress, which has been shown to cause laryngeal microbiome dysbiosis. Microbiome dysbiosis underlies disease pathophysiology in other organs, leading to uncontrolled inflammation via epithelial barrier dysfunction. There is a need to delineate microbial versus nonmicrobial epithelial stress pathways in the vocal folds (VF) in order to clarify how psychosocial stress may contribute to VF epithelial disease.

C57BL/6 conventionally-raised (CR) and germ-free (GF) adult mice were equally allocated to stressed and unstressed groups (n = 64, 32 per mouse-type/group, equal sexes). Stressed animals completed a validated restraint stress protocol. Following stress, larynges were collected. Outcomes of VF epithelial barrier dysfunction were compared across stressed, unstressed GF, and CR mice. Immunofluorescence/Immunohistochemistry determined protein expression of epithelial adherens junctions, mucins, and proliferation in mid-membranous VF tissue. Transmission electron microscopy assessed VF epithelial permeability.

Regardless of microbiota status, stressed animals had reduced protein expression of VF epithelial adherens junctions, mucins (MUC1, 4) and increased epithelial permeability. Presence of microbiota was associated with stress-induced increases epithelial proliferation, observed in CR, but not GF mice.

Presence of microbiota was associated with stress-induced increase in VF epithelial proliferation. This increased proliferation may represent an adaptive response to damaged cells to maintain epithelial integrity. Collectively, these findings provide a foundation for identifying VF epithelial targets that could be leveraged in probiotic-based therapies.

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