Importance and Objective: This study presents an innovative method to develop a rabbit model for varying durations of voice rest through low-dose botulinum toxin at the thyroarytenoid muscle neuromuscular junction. The aim is to induce transient unilateral vocal fold paralysis of varying degrees by injecting different doses of botulinum toxin into the unilateral thyroarytenoid muscle, establishing rabbit models for different durations of voice rest.
Methods: The first phase evaluated the effects of botulinum toxin on normal vocal fold tissue. A total of 1U type A botulinum toxin was injected into the left thyroarytenoid muscle of four rabbits, with the contralateral side serving as a control. After 14 days, vocal folds were analyzed using hematoxylin-eosin (HE) staining, and ImageJ was used to measure epithelial layer parameters. The gray level co-occurrence matrix (GLCM) method was employed in MATLAB to extract texture features (Energy, Entropy, Inertia, Correlation, Inverse Difference Moment) to assess microstructural impacts.
In the second phase, four experimental groups (A, B, C, D), each containing four rabbits, received varying doses of botulinum toxin (0.025U, 0.0625U, 0.175U, 0.375U) in the left thyroarytenoid muscle. The contralateral side remained untreated. Vocal fold motion was tracked using Kinovea software to analyze movements under anesthesia, and the left-to-right vocal fold movement ratio was calculated.
Results: In the first phase, HE staining revealed no significant differences between treated and control sides (P > 0.05). GLCM analysis also showed no statistical differences (P > 0.05), confirming that 1U of botulinum toxin had no significant structural impact.
In the second phase, all groups exhibited no differences in movement before injection (day 0: P > 0.05, movement ratio near 1). Group A (0.025U) showed transient paralysis on day 1 (P < 0.001, ratio = 0.788), recovering by day 2 (P = 0.627, ratio = 1.000), indicating a 1-day voice rest model. Group B (0.0625U) showed paralysis on day 2 (P < 0.001, ratio = 0.893), recovering by day 3 (P = 0.952, ratio = 1.000), representing a 3-day model. Group C (0.175U) exhibited paralysis on days 1 and 5 (P < 0.001, ratios = 0.903 and 0.835), recovering by day 7 (P = 0.861, ratio = 1.000), suitable for a 7-day model. Group D (0.375U) showed paralysis on days 3 and 10 (P < 0.001, ratios = 0.68 and 0.95), with recovery by day 14 (P = 0.856, ratio = 1.001), establishing a 14-day model.
Conclusions: Injecting varying doses of botulinum toxin into the unilateral thyroarytenoid muscle can induce transient unilateral vocal fold paralysis, creating rabbit models for different durations of voice rest. This model may assist in researching optimal voice rest durations for vocal fold healing.