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.

Postnatal Development of the Mouse Larynx: Negative Allometry, Age-Dependent Shape Changes, Morphological Integration, and a Size-Dependent Spectral Feature

“…Developmental biology of the larynx in the mouse and rat is highly relevant to both healthy and disordered human activity, as the rodent larynx is a model for studying laryngeal neuromuscular (e.g., Haney et al, 2020;Montalbano et al, 2019) and connective tissue (e.g., Lungova et al, 2015;Peterson et al, 2013;Welham et al, 2015) maturation and injury. Additional applications of rodent models relevant for human health include chronic electronic a stimulation of the rat thyroarytenoid muscle for voice disorder therapy (McMullen et al, 2011) and characterizing dysphagia in a rat model for Parkinson disease (Russell et al, 2013).…”

Characteristics of Early Internal Laryngeal Muscle Atrophy After Recurrent Laryngeal Nerve Injuries in Rats

“…22 Montalbano et al found that glial-derived neurotrophic factor (GDNF) and netrin-1 were upregulated significantly in nonpooled laryngeal muscles and peaked 7 days after RLNI. 25 The results of Wang et al and Hernandez-Morato et al suggested that GNDF and Netrin-1 may be linked to laryngeal reinnervation post RLN injury. 26,27 The mechanism may be that GDNF enhances axon growth and myotube innervation, and netrin-1 could alter the motor reinnervation process of the larynx.…”

An optimized method for high-quality RNA extraction from distinctive intrinsic laryngeal muscles in the rat model

“…Identifying factors that hinder global transcriptome analysis of the rat ILMs was rarely supported in the literature before the present study. Moreover, gene expression assessment of the ILMs previously relied upon low-throughput profiling technologies (e.g., qRT-PCR, microarrays) and histological studies due to their lower requisites for RNA quality (McCullen 2009) [12][13][14][15][16][17][18][19] . Whole-transcriptome RNA-Seq provides a broader characterization and distribution of mRNA transcripts and their isoforms and thus requires highly undegraded RNA samples 20 .…”