Next-Generation Sequencing Application: A Systematic Approach for High-Quality RNA Isolation from Skeletal Muscles.

RNA extraction and analyses from tissues using bulk RNA-Sequencing (RNA-Seq) provide a more accurate picture of the gene expression compared to other molecular biology techniques for RNA quantification. Challenges associated with high-quality RNA extraction from skeletal muscles require a modification of standard protocols. Here, we describe a procedure for high-quality RNA isolation from intrinsic laryngeal muscles transferable to skeletal muscles with comparable technical and biological difficulties.

Transcriptome Analysis of Left Versus Right Intrinsic Laryngeal Muscles Associated with Innervation.

Objectives/hypothesis: Recurrent laryngeal nerve injury diagnosed as idiopathic or due to short-term surgery-related intubation exhibits a higher incidence of left-sided paralysis. While this is often attributed to nerve length, it is hypothesized there are asymmetric differences in the expression of genes related to neuromuscular function that may impact reinnervation and contribute to this laterality phenomenon. To test this hypothesis, this study analyzes the transcriptome profiles of the intrinsic laryngeal muscles (ILMs), comparing gene expression in the left versus right, with particular attention to genetic pathways associated with neuromuscular function.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation.

A comparison of confocal and epifluorescence microscopy for quantification of RNAScope and immunohistochemistry fluorescent images.

Background: Quantification of RNA expression and protein production in fluorescent stainings provides critical information concerning neurodevelopment. A trustable independent quantification technique requires acquisition of reliable images prior to image processing. There is uncertainty in existing literature regarding the use of confocal microscopy compared to standard epifluorescence microscopy, especially in the context of RNA in situ hybridization protocols.

Transcriptome analysis of left versus right intrinsic laryngeal muscles associated with innervation.

Objectives/hypothesis: Recurrent laryngeal nerve injury diagnosed as idiopathic or due to short-term surgery-related intubation exhibits a higher incidence of left-sided paralysis. While this is often attributed to nerve length, it is hypothesized there are asymmetric differences in the expression of genes related to neuromuscular function that may impact reinnervation and contribute to this laterality phenomenon. To test this hypothesis, this study analyzes the transcriptome profiles of the intrinsic laryngeal muscles (ILMs), comparing gene expression in the left versus right, with particular attention to genetic pathways associated with neuromuscular function.

Development and Application of Automated Vocal Fold Tracking Software in a Rat Surgical Model.

Objective: The rat is a widely used model for studying vocal fold (VF) function after recurrent laryngeal nerve injury, but common techniques for evaluating rat VF motion remain subjective and imprecise. To address this, we developed a software package, called RatVocalTracker1.0 (RVT1.

Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation.

Objectives: Recurrent laryngeal nerve (RLN) injury results in synkinetic reinnervation and vocal fold paralysis. Investigation of cues expressed in the developing brainstem that influence correct selective targeting of intrinsic laryngeal muscles may elucidate post-injury abnormalities contributing to non-functional reinnervation. Primary targets of interest were Hoxb1 and Hoxb2, members of the Hox family that create overlapping gradients in the developing brain, and their target Phox2b, a transcription factor necessary for cranial nerve branchio- and visceromotoneuron survival.

Corrigendum: A review of the peripheral proprioceptive apparatus in the larynx.

[This corrects the article DOI: 10.3389/fnana.2023.

A review of the peripheral proprioceptive apparatus in the larynx.

The larynx is an organ of the upper airway that participates in breathing, glutition, voice production, and airway protection. These complex functions depend on vocal fold (VF) movement, facilitated in turn by the action of the intrinsic laryngeal muscles (ILM). The necessary precise and near-instantaneous modulation of each ILM contraction relies on proprioceptive innervation of the larynx.

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

Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequencing (RNA-seq) application in certain skeletal muscle groups, including the intrinsic laryngeal muscles (ILMs). The present study identified critical factors contributing to substandard RNA extraction from the ILMs and established a suitable method that permitted high-throughput analysis. Here, standard techniques for tissue processing were adapted, and an effective means to control confounding effects during specimen preparation was determined.

Expression of Glial Cell-Derived Neurotrophic Factor Receptors Within Nucleus Ambiguus During Rat Development.

Objective: The nucleus ambiguus (NAmb) is a column of neurons in the medulla oblongata, involved in bulbar functions. Expression of Glial Cell-Derived Neurotrophic Factor (GDNF) and its receptors (GDNFR) is observed within the cell bodies during reinnervation following recurrent laryngeal nerve (RLN) injury. Little is known regarding GDNFR expression in the formation of the NAmb and the laryngeal innervation during embryogenesis.

Temporal expression of Laminin-111 in the developing rat larynx.

Laminin-111 is a basement membrane protein that participates in motor innervation and reinnervation. During axonal pathfinding, laminin-111 interacts with netrin-1 (NTN1) and changes its attractant growth cone properties into repulsion. While previous models of recurrent laryngeal nerve (RLN) transection show increased Laminin-111 and NTN1 production after injury, developmental expression in the larynx has not been defined.

Expression of trophic factors receptors during reinnervation after recurrent laryngeal nerve injury.

Objective: An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a poor functional recovery. Netrin-1 and glial cell-derived neurotrophic factor (GDNF) expression are up-regulated in laryngeal muscles during RLN regeneration, but the role of their receptors produced in the nucleus ambiguus is unknown. The aim of this work was to determine the timing of the production of Netrin-1 and GDNF receptors during RLN regeneration and correlate this with the previously identified timing of up-regulation of their trophic factors in the laryngeal muscles.

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

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.

Influence of Netrin-1 on reinnervation of laryngeal muscles following recurrent laryngeal nerve injury.

Following recurrent laryngeal nerve (RLN) injury, recovery results in poor functional restitution of the paralyzed vocal fold. Netrin-1 has been found to be upregulated in the rat posterior cricoarytenoid muscle (PCA) during nerve regeneration. We evaluated the effect of ectopic Netrin-1 in the PCA during RLN reinnervation.

Muscle specific nucleus ambiguus neurons isolation and culturing.

Background: Peripheral nerve injury leads to a regenerative state. However, the reinnervation process is highly non-selective. Growing axons are often misrouted and establish aberrant synapsis to abductor or adductor muscles.

Changes in neurotrophic factors of adult rat laryngeal muscles during nerve regeneration.

Injury to the recurrent laryngeal nerve (RLN) leads to the loss of ipsilateral laryngeal fold movement, with dysphonia, and occasionally dysphagia. Functional movement of the vocal folds is never restored due to misrouting of regenerating axons to agonist and antagonist laryngeal muscles. Changes of neurotrophic factor expression within denervated muscles occur after nerve injury and may influence nerve regeneration, axon guidance and muscle reinnervation.

Blockade of glial-derived neurotrophic factor in laryngeal muscles promotes appropriate reinnervation.

Objectives/hypothesis: Synkinetic reinnervation of the laryngeal muscles is one of the causes of the poor functional recovery after a recurrent laryngeal nerve (RLN) injury. Glial-derived neurotrophic factor (GDNF) is elevated in rat laryngeal muscles during RLN reinnervation. The specific aim of this investigation was to evaluate the effect of anti-GDNF on RLN reinnervation.

Differential expression of glial-derived neurotrophic factor in rat laryngeal muscles during reinnervation.

Objectives/hypothesis: Nonspecific, synkinetic reinnervation is one of the causes of poor functional recovery after a peripheral nerve lesion. Knowledge of the differential expression of neurotrophic factors that subserve axon guidance, as well as neuromuscular junction formation and maintenance in the denervated muscles, may allow appropriate interventions that will improve the functional nonsynkinetic reinnervation.

Study Design: Laboratory experiment.

Somatotopic changes in the nucleus ambiguus after section and regeneration of the recurrent laryngeal nerve of the rat.

Changes in motoneurons innervating laryngeal muscles after section and regeneration of the recurrent laryngeal nerve (RLN) are far from being understood. Here, we report the somatotopic changes within the nucleus ambiguus (Amb) after the nerve injury and relates it to the resulting laryngeal fold impairment. The left RLN of each animal was transected and the stumps were glued together using surgical fibrin glue.

Reorganization of laryngeal motoneurons after crush injury in the recurrent laryngeal nerve of the rat.

Motoneurons innervating laryngeal muscles are located in the nucleus ambiguus (Amb), but there is no general agreement on the somatotopic representation and even less is known on how an injury in the recurrent laryngeal nerve (RLN) affects this pattern. This study analyzes the normal somatotopy of those motoneurons and describes its changes over time after a crush injury to the RLN. In the control group (control group 1, n = 9 rats), the posterior cricoarytenoid (PCA) and thyroarytenoid (TA) muscles were injected with cholera toxin-B.

Somatotopy of the neurons innervating the cricothyroid, posterior cricoarytenoid, and thyroarytenoid muscles of the rat's larynx.

Neurons innervating the intrinsic muscles of the larynx are located within the nucleus ambiguus but the precise distribution of the neurons for each muscle is still a matter for debate. The purpose of this study was to finely determine the position and the number of the neurons innervating the intrinsic laryngeal muscles cricothyroid, posterior cricoarytenoid, and thyroarytenoid in the rat. The study was carried out in a total of 28 Sprague Dawley rats.

Morphogenesis of the human laryngeal ventricles.

Background: Two theories explain the origin of human laryngeal ventricles: (1) ventricles derive from the fifth pharyngeal pouches; (2) development independent from the pouches.

Methods: In all, there were 21 serially sectioned human embryos from stages 15 to 23, and 11 fetuses of 9 to 18 weeks. Computer-aided 3-dimentional reconstructions were made.

The central projections of the laryngeal nerves in the rat.

The larynx serves respiratory, protective, and phonatory functions. The motor and sensory innervation to the larynx controlling these functions is provided by the superior laryngeal nerve (SLN) and the recurrent laryngeal nerve (RLN). Classical studies state that the SLN innervates the cricothyroid muscle and provides sensory innervation to the supraglottic cavity, whereas the RLN supplies motor innervation to the remaining intrinsic laryngeal muscles and sensory innervation to the infraglottic cavity, but recent data suggest a more complex anatomical and functional organisation.

The clinical interest of the ary-thyro-cricoid fascicle.

The aim of this work was to study the prevalence and form of the ary-thyro-cricoid (ATC) muscular fascicle, a variable muscular slip connecting the oblique and/or transverse arytenoid muscles with the thyroarytenoid (TA) and/or lateral cricoarytenoid (LCA) muscles resembling a sphincter encircling the glottis. Thirty larynges obtained from necropsies of individuals with no known laryngeal pathology were dissected. The ATC fascicle was observed in 96.