Recurrent Laryngeal Nerve Reinnervation in Rats Posttransection: Neurotrophic Factor Expression over Time

Abstract

Objective Recurrent laryngeal nerve (RLN) injury causes vocal fold paralysis from which functional recovery is typically absent due to nonselective reinnervation. This study investigates expression of axon guidance cues and their modulators relative to the chronology of reinnervation by examining the expression of glial-derived neurotrophic factor (GDNF), netrin 1, and laminin 111 (LAMA1) in nonpooled laryngeal muscles. This study is the first to describe the post-RLN injury expression pattern of LAMA1, a target of particular interest as it has been shown to switch netrin 1–mediated growth cone attraction to repulsion. Study Design Animal experiment (rat model). Setting Basic science laboratory. Methods The right RLNs of 64 female Sprague-Dawley rats were transected, with sacrifice at 1, 3, 7, 21, 28, and 56 days postinjury (DPI). Single-animal messenger RNA was isolated from the ipsilateral posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA) for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Immunostaining for LAMA1 expression was performed in the same muscles. Results LAMA1 was elevated in the PCA at 3 to 56 DPI, LTA at 7 DPI, and MTA at 14 and 28 DPI. This correlates with the chronology of laryngeal reinnervation. Using a new protocol, single-animal muscle qRT-PCR possible and expression results for GDNF and netrin 1 were similar to previous pooled investigations. Conclusion Reliable qRT-PCR is possible with single rat laryngeal muscles. The expression of netrin 1 and LAMA1 is chronologically coordinated with muscle innervation in the LTA and MTA. This suggests that LAMA1 may influence netrin 1 to repel axons and delay LTA and MTA reinnervation.