Ethyl Cinnamate-Based Tissue Clearing Technique on Vocal Fold Mucosa with Vasculature Model.

OBJECTIVE: The tissue clearing technique functions as a method to analyze the orientation and distribution of microvascular networks with cellular resolution by labeling antibodies within the lamina propria. This study aims to provide a methodology and procedure to adapt the ethyl cinnamate organic tissue clearing procedure specifically to the unique properties of canine vocal fold mucosa and target vascular components of tissues via the CD31 antibody. METHODS: This study developed a novel method and procedure, including isolation of mucosa from muscle tissue, decreased dehydration temperature from room temperature to 6 ℃, optimization of antibody staining to 1:200, and post-processing of imaging that addressed the shortcomings of ethyl cinnamate clearing as applied to vocal fold tissue. RESULTS: The results demonstrate the ability for 3D reconstruction of vocal fold microvascular networks based on successful segmentation with available tools, as evidenced by high-resolution imaging that captures the orientation of CD31-positive vasculature and distribution of proteins. Multiple procedural improvements resulted in successful immunolabeling and clearing capacity. CONCLUSION: The results establish that the ethyl cinnamate organic tissue clearing procedure, as outlined in this study, adapted to the unique properties of vocal fold mucosa, can be applied to the larynx to visualize the direction and orientation of CD31-positive vasculature in the vocal fold mucosa of the lamina propria. This method can be utilized in future research to view other physiological components of the vocal fold mucosa, such as specialized cells like epithelial cells and macrophages, when applied to target other antibodies.