Embryologic innervation of the rat laryngeal musculature—a model for investigation of recurrent laryngeal nerve reinnervation

Abstract

Optimal management of vocal fold paralysis would entail recurrent laryngeal nerve (RLN) reinnervation resulting in normal vocal fold motion. Unfortunately, RLN reinnervation currently results in a nonfunctional vocal fold due to synkinetic reinnervation. Therapeutic interventions that guide regenerating axons back to the appropriate muscle would prevent synkinesis and restore vocal fold and glottal function. The initial step toward developing these therapies is the elucidation of the embryologic innervation of the larynx. This study aimed to identify the age of occurrence, timing, and pattern of embryologic innervation of the rat larynx, hypothesizing that differences in these parameters exist between distinct laryngeal muscles. Descriptive anatomic study. The larynx of rats aged embryologic day (E) 15, 16, 17, 19, and 21 were harvested and then sectioned. Two rats were used for each age. Sections were colabeled with neuronal class III β-tubulin polyclonal antibody to identify the presence of axons and alexa 488 conjugate α-bungarotoxin to identify the presence of motor endplates. The age at which axons and motor endplates were first present was noted. The position and pattern of the axons and motor endplates was recorded in relation to each other as well as the musculoskeletal anatomy of the larynx. The time at which axons appeared to innervate the medial thyroarytenoid (MTA) muscle, lateral thyroarytenoid (LTA) muscle, and the posterior cricoarytenoid (PCA) muscle was documented. Findings in the rat suggest the RLN reaches the larynx and begins branching by E15. Axons branch dorsally first and reach the PCA muscle before the other muscles. Branching toward the MTA muscle occurs only after axons have reached the LTA muscle. By E19, RLN axons have been guided to and selected their respective muscles with formation of neuromuscular junctions (NMJs) in the PCA, LTA and MTA muscles, though the formation of NMJs in the MTA muscle was comparatively delayed. This study describes the embryologic innervation of the rat larynx and suggests that there are distinct differences in the age of occurrence, timing, and pattern of innervation of the PCA, LTA, and MTA muscles of the rat. These findings lay the foundation for studies investigating the role of guidance cues in RLN axon guidance and the utility of these cues in the treatment of RLN injury via the stimulation of functional, nonsynkinetic reinnervation.