Development of the rat larynx: a histological study.

OBJECTIVES/HYPOTHESIS: To evaluate and describe the cartilaginous and muscular development of the rat larynx. STUDY DESIGN: Histologic evaluation. METHODS: The larynges of Sprague Dawley rats of embryonic day (E) 13, 15, 17, 19, 21, postnatal day 0, 14, and adult of 250 gm were collected. Four larynges of each age were harvested, cut into 15-µm serial sections, stained with hematoxylin and eosin, and evaluated under light microscopy. Representative digital images were recorded and evaluated at the preglottic (supraglottic in humans), glottic, and postglottic (subglottic in humans) levels. RESULTS: Brachial arches were observed at E13. At E17, immature structures of the larynx, including skeletal muscle, cartilage, and the lumen were identifiable. Chondrification and muscle formation were clearly seen by E19. The muscular and cartilagenous components of the larynx were well established by E21. During the span between birth and adult maturation, the size of the larynx increased from a height of 1.10 mm to 2.90 mm, and from a width of 1.80 mm to 5.40 mm, and from a length of 1.38 mm to 4.77 mm in the stained section. Although developed at E21, the laryngeal structures continued to grow by approximately 30%. CONCLUSION: Rat laryngeal development parallels that in mice and humans. In the rat, at E17 immature structures of the larynx are identifiable, they are well developed at birth and grow by approximately 30% into adulthood. Understanding the chronology and morphology of the embryogenesis of the rat laryngeal musculature is essential and will allow for further evaluation of the embryologic innervation of these muscles.

Recurrent laryngeal nerve transection and anastomosis: rat laryngeal motoneuron survival and effect of the anastomosis site.

OBJECTIVES: We investigated the quantity of recurrent laryngeal nerve motoneurons (RLNMs) that survive after transection and anastomosis of the rat recurrent laryngeal nerve (RLN), as well as the impact of the anastomosis site on RLN regeneration. METHODS: Ten rats underwent right RLN transection and anastomosis. After 16 weeks, Fluoro-Ruby (FR) was applied to the RLN that was transected proximal or distal to the anastomosis site. The brain stems were harvested, and the nucleus ambiguus was evaluated for labeled RLNMs. The RLNM counts were compared to each other and to those from 3 control rats in which FR was applied to an acutely transected RLN. RESULTS: The number of RLNMs that were stained after RLN transection, anastomosis, and regeneration was consistent with the total number of RLNMs in the nucleus ambiguus of control rats. This finding confirms that most RLNMs survived after RLN transection and anastomosis. The quantity of labeled RLNMs was statistically similar whether the FR was applied proximal or distal to the anastomosis, implying that most of the viable axons that were present proximal to the anastomosis crossed into the distal nerve. CONCLUSIONS: Rat RLNMs survive nerve transection, anastomosis, and regeneration. The anastomosis site does not significantly impede axonal regeneration, and most of the axons traverse the anastomosis into the distal nerve.

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

OBJECTIVES/HYPOTHESIS: 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. STUDY DESIGN: Descriptive anatomic study. METHODS: 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 ß-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. RESULTS: 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. CONCLUSIONS: 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.