Analysis of Dye Distribution in Cadaveric Supraglottic Injections: Elucidating the Mechanism of Action for Supraglottic Botulinum Neurotoxin Injection.

Supraglottic botulinum neurotoxin (BoNT) injections have been introduced as an alternative treatment for adductor spasmodic dysphonia (AdSD) to reduce adverse effects associated with direct intramuscular vocal fold injections. However, the anatomical distribution pattern of BoNT following supraglottic injection and the predominantly affected intrinsic laryngeal muscles have not yet been clearly elucidated, owing to a lack of detailed anatomical investigations. Thirteen fresh human cadavers (seven males and six females; mean age, 77.0 years) were included in this study. After harvesting the larynx, a supraglottic injection was performed under direct anatomical visualization using a blue dye as a tracer. Injection volumes of 0.4 mL and 0.2 mL were administered bilaterally. Following passive simulation of laryngeal adduction and abduction, specimens were frozen at -80 °C and sectioned coronally. Dye distribution was analyzed with a primary focus on the thyroarytenoid (TA) muscle complex, including the aryepiglottic muscle fibers, main TA muscle, vocalis muscle, and diffusion to other intrinsic laryngeal muscles. In all specimens, the dye remained confined to the supraglottic soft tissues, with no extension to the structures outside the larynx. At the muscular level, the dye involvement was limited to the TA muscle complex. The dye was consistently observed along the aryepiglottic muscle fibers and extended toward the main TA muscle. Partial intramuscular spread into the main TA muscle was identified in 50% of specimens in both injection volume groups (0.4 and 0.2 mL). Minimal extension into the vocalis muscle was observed in 33% of specimens. In contrast, no dye diffusion into the lateral cricoarytenoid, posterior cricoarytenoid, or interarytenoid muscles was observed in any of the specimens. Although the extent of dye spread varied according to injection volume, the pattern of muscle involvement was similar between the two groups. The mean distance from the supraglottic injection site to the TA muscle was 12.9 ± 1.7 mm. Supraglottic BoNT injections preferentially involve the thyroarytenoid muscle complex, with limited extension into the vocalis muscle and no diffusion into the other intrinsic laryngeal muscles. These anatomical findings provide a plausible explanation for the balance between therapeutic efficacy and tolerability reported with supraglottic BoNT injection in adductor spasmodic dysphonia and support its relative safety with respect to unintended muscle paralysis.