Ignacio Hernandez-Morato | New York Medical College (original) (raw)
Papers by Ignacio Hernandez-Morato
Temporal expression of Laminin-111 in the developing rat larynx
Neuroscience Letters, Jun 1, 2022
Expression of Glial Cell‐Derived Neurotrophic Factor Receptors Within Nucleus Ambiguus During Rat Development
Laryngoscope, Oct 22, 2022
ObjectiveThe nucleus ambiguus (NAmb) is a column of neurons in the medulla oblongata, involved in... more ObjectiveThe 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. Understanding the timing and pattern of GDNFR expression in embryogenesis versus after RLN injury may provide insights into therapeutic targets for regeneration after RLN injury.Study DesignLaboratory experiment.MethodsRat brainstems at E14.5/E16.5/E18.5/E20.5/adult were stained for GDNFR: GFRα‐1/GFRα‐2/GFRα‐3/Ret. Islet1 and choline acetyltransferase were used as cell body markers. Sections were observed using fluorescent microscopy and quantified through manual cell counting.ResultsExpression of GFRα‐1, GFRα‐3, and Ret was identified within the NAmb, hypoglossal, and facial nuclei of the adult medulla. During development, GFRα‐1 immunoreactivity was seen at E20.5. GFRα‐2 expression was not observed at any timepoint. GFRα‐3 expression began at E16.5. Ret expression within nerve fibers in the NAmb were observed beginning at E14.5, but never in the cell bodies.ConclusionEmbryonic GDNFR expression in the NAmb differs from that of the adult after RLN injury. The developing brainstem experienced upregulation at discrete timepoints with signaling sustained through adulthood. In contrast, adult RLN‐transected rats experienced patterns of up and down regulation. GFRα‐1 may contribute to muscle targeting and neuromuscular junction maturation, GFRα‐3 may contribute to both, as well as axon guidance. It is likely that GDNF is functioning via a Ret‐independent pathway.Level of EvidenceNA Laryngoscope, 133:2240–2247, 2023
Laryngoscope, Feb 27, 2019
An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a po... more 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. Study Design: Laboratory experiment with rat model. Methods: The right RLN was transected and dextran amine tracer applied. At 7, 14, and 21 days postinjury (DPI), brainstems were removed and harvested. Immunostaining was performed for Netrin-1 (deleted in colorectal carcinoma [DCC], UNC5A) and GDNF receptors (rearranged during transfection [Ret], glycosylphosphatidylinositol-linked cell surface receptors [GFRα1, GFRα2, GFRα3]). The timing and type of receptor production relative to injury as well as their position in the nucleus ambiguus was analyzed. Results: Netrin-1 UNC5A receptors were minimal in the nucleus ambiguus during RLN regeneration. DCC, the receptor that plays an attract role, was immunopositive from 7 to 21 DPI. All GDNF receptors, except GFRα2, were clearly positive from 7 to 14 DPI. No differences of production were observed according to the position of the motor neurons in the nucleus ambiguus. Conclusion: An injury of the RLN leads to a higher production of Netrin-1 DCC and GDNF receptors in the nucleus ambiguus. The timing of receptor production is similar to up-regulation of their trophic factors in the laryngeal muscles.
bioRxiv (Cold Spring Harbor Laboratory), May 4, 2022
Transcriptome analysis of left-right intrinsic laryngeal muscles shows divergent expression of genes associated with innervation
Objectives/Hypothesis: Recurrent laryngeal nerve neuropathy diagnosed as idiopathic or due to sho... more Objectives/Hypothesis: Recurrent laryngeal nerve neuropathy 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. Study Design: Laboratory experiment. Methods: High-quality RNA was extracted from the left and right sides of the rat posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA), respectively. After high-throughput RNA-Sequencing (RNA-Seq), 88 samples were organized into 12 datasets acco...
Development and Application of Automated Vocal Fold Tracking Software in a Rat Surgical Model
The Laryngoscope
ObjectiveThe rat is a widely used model for studying vocal fold (VF) function after recurrent lar... more ObjectiveThe 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 (RVT1.0), to quantify VF motion and tested it on rats with iatrogenic unilateral vocal fold paralysis (VFP).MethodsA deep neural network was trained to identify the positions of the VFs and arytenoid cartilages (ACs) in transoral laryngoscope videos of the rat glottis. Software was developed to estimate glottic midline, VF displacement, VF velocity, and AC angle. The software was applied to laryngoscope videos of adult rats before and after right recurrent and superior laryngeal nerve transection (N = 15; 6M, 9F). All software calculated metrics were compared before and after injury and validated against manually calculated metrics.ResultsRVT1.0 accurately tracked and quantified VF displacement, VF velocity, a...
Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation
The Laryngoscope
Frontiers in Neuroanatomy
ABSTRACTProprioception plays a crucial role in laryngeal function. Further, dysfunctional proprio... more ABSTRACTProprioception plays a crucial role in laryngeal function. Further, dysfunctional proprioception likely contributes to disorders such as laryngeal dystonia, dysphagia and vocal fold paresis. Despite this, the physiology of laryngeal proprioception is not well-understood. Controversy remains over whether canonical proprioceptive organs, like muscle spindles (MS) even exist in the intrinsic laryngeal muscles (ILM). Vesicular Glutamate Transporter 1 (VGLUT1) expression has been described in the sensory afferents of MS. This study’s primary aim is to determine whether the ILM contain MS using VGLUT1. This is a novel approach, as prior studies have relied on morphology and myosin composition to study this question. Secondarily, we describe the pattern of VGLUT1 expression in the rat larynx, Larynges of 62 Sprague-Dawley rats distributed across 5 age groups (P3, P8, P11, P14-15, and adult), were sectioned and immunostained for VGLUT1 and beta-tubulin III. Other markers (S46, GNAT3...
Frontiers in Neuroanatomy
The larynx is an organ of the upper airway that participates in breathing, glutition, voice produ... more 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. Dysfunctional laryngeal proprioception likely contributes to disorders such as laryngeal dystonia, dysphagia, vocal fold paresis, and paralysis. While the proprioceptive system in skeletal muscle derived from somites is well described, the proprioceptive circuitry that governs head and neck structures such as VF has not been so well characterized. For over two centuries, researchers have investigated the question of whether canonical proprioceptive organs, muscle spindles, and Golgi tendon organs, exist in the ILM, with variable findings. The present work is a state-of-the-art review of the peri...
ABSTRACTObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervatio... more ABSTRACTObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervation after nerve injury. In contrast, little is known regarding the expression of GDNF receptors in the formation of the nucleus ambiguus (NA) and its innervation of the larynx during embryogenesis. Differences may suggest therapeutic targets after nerve injury.Study DesignLaboratory experiment.MethodsRat brainstems at E14, E16, E18, E20, adult (4 animals/timepoint) were sectioned and stained for GDNF receptors: GFRα-1, GFRα-2, GFRα-3, and Ret. Islet1 and ChAT were used as markers for motoneuron cell bodies. Sections were observed using Zeiss Axio Imager M2 Microscope and quantified using Image J.ResultsExpression of all four GDNF receptors was identified within the nucleus ambiguus, as well as hypoglossal and facial nuclei of the adult rat brainstem. During rat development, GFRα-3 and Ret exhibited upregulation within the nucleus ambiguus at E14 whereas GFRα-1 began showing upregulation at...
Temporal expression of Laminin-111 in the developing rat larynx
Neuroscience Letters
Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequen... more 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. The experimental procedure consistently provided sufficient intact total RNA (N = 68) and RIN ranging between 7.0 and 8.6, which was unprecedented using standard RNA purification protocols. This study confirmed the reproducibility of the workflow through repeated trials at different postnatal time points and across the distinctive ILMs. High-throughput diagnostics from 90 RNA samples indicated no sequencing alignment scores below 70%, validating ...
Otolaryngology–Head and Neck Surgery, 2019
ObjectiveRecurrent laryngeal nerve (RLN) injury causes vocal fold paralysis from which functional... more ObjectiveRecurrent 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 DesignAnimal experiment (rat model).SettingBasic science laboratory.MethodsThe 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 q...
Journal of Neuroscience Methods, 2016
h i g h l i g h t s • The development of a new protocol for specific brainstem motoneuron isolati... more h i g h l i g h t s • The development of a new protocol for specific brainstem motoneuron isolation from postnatal rats. • Abductor and adductor motoneurons were isolated in separate plates. • The utility of neural tracers for a long term neuron culture isolation was addressed. • The best period for cranial motoneuron isolation and culture in postnatal rats was determined.
The Laryngoscope, 2016
Objectives/Hypothesis: Synkinetic reinnervation of the laryngeal muscles is one of the causes of ... more 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. Methods: Anti-GDNF antibody was injected into the posterior cricoarytenoid (PCA) 3 days following RLN transection and anastomosis. Larynges were harvested at 7, 14, 28, 56, and 112 days post injury (DPI). Prior to sacrifice, the vocal fold mobility was assessed. Immunostaining to identify neuromuscular junctions was used to evaluate the extent of axonal reinnervation of the PCA, lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA). Results: After anti-GDNF injection into PCA, RLN reinnervation in all muscles was altered when compared to the controls. PCA innervation was delayed. At 7 DPI, only a few axons made synapses in the PCA. In contrast, axons prematurely innervated the LTA and MTA when compared to controls. Innervation was similar to controls at 56 and 112 DPI. Vocal fold motion was enhanced in 10 of 24 animals studied. Conclusions: After injection of anti-GDNF into the PCA, early arriving axons bypass the PCA and enter the LTA. Later arriving axons innervate the PCA and MTA. Vocal fold function is improved as compared to controls. Anti-GDNF injection into the PCA influences the pattern of reinnervation and may result in less synkinetic, more functional innervation.
Neuroscience Letters, 2017
h i g h l i g h t s • Recurrent laryngeal nerve injury leads to non-specific reinnervation of the... more h i g h l i g h t s • Recurrent laryngeal nerve injury leads to non-specific reinnervation of the larynx. • Netrin-1 plays an important role during laryngeal muscle reinnervation. • Injection of Netrin-1 reduces reinnervation of the posterior cricoarytenoid muscle. • Netrin-1 injection alters the motor reinnervation process of the larynx.
Endogenous alkaline phosphatase in the optic chiasm suggests a novel mechanism of retinal axonal guidance
Investigative Ophthalmology & Visual Science, 2018
Alkaline Phosphatase Enriched Exosomes as Guidance Cues in the Developing Avian Optic Chiasm
Investigative Ophthalmology & Visual Science, 2016
Expression of trophic factors receptors during reinnervation after recurrent laryngeal nerve injury
The Laryngoscope
An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a po... more 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.
Temporal expression of Laminin-111 in the developing rat larynx
Neuroscience Letters, Jun 1, 2022
Expression of Glial Cell‐Derived Neurotrophic Factor Receptors Within Nucleus Ambiguus During Rat Development
Laryngoscope, Oct 22, 2022
ObjectiveThe nucleus ambiguus (NAmb) is a column of neurons in the medulla oblongata, involved in... more ObjectiveThe 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. Understanding the timing and pattern of GDNFR expression in embryogenesis versus after RLN injury may provide insights into therapeutic targets for regeneration after RLN injury.Study DesignLaboratory experiment.MethodsRat brainstems at E14.5/E16.5/E18.5/E20.5/adult were stained for GDNFR: GFRα‐1/GFRα‐2/GFRα‐3/Ret. Islet1 and choline acetyltransferase were used as cell body markers. Sections were observed using fluorescent microscopy and quantified through manual cell counting.ResultsExpression of GFRα‐1, GFRα‐3, and Ret was identified within the NAmb, hypoglossal, and facial nuclei of the adult medulla. During development, GFRα‐1 immunoreactivity was seen at E20.5. GFRα‐2 expression was not observed at any timepoint. GFRα‐3 expression began at E16.5. Ret expression within nerve fibers in the NAmb were observed beginning at E14.5, but never in the cell bodies.ConclusionEmbryonic GDNFR expression in the NAmb differs from that of the adult after RLN injury. The developing brainstem experienced upregulation at discrete timepoints with signaling sustained through adulthood. In contrast, adult RLN‐transected rats experienced patterns of up and down regulation. GFRα‐1 may contribute to muscle targeting and neuromuscular junction maturation, GFRα‐3 may contribute to both, as well as axon guidance. It is likely that GDNF is functioning via a Ret‐independent pathway.Level of EvidenceNA Laryngoscope, 133:2240–2247, 2023
Laryngoscope, Feb 27, 2019
An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a po... more 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. Study Design: Laboratory experiment with rat model. Methods: The right RLN was transected and dextran amine tracer applied. At 7, 14, and 21 days postinjury (DPI), brainstems were removed and harvested. Immunostaining was performed for Netrin-1 (deleted in colorectal carcinoma [DCC], UNC5A) and GDNF receptors (rearranged during transfection [Ret], glycosylphosphatidylinositol-linked cell surface receptors [GFRα1, GFRα2, GFRα3]). The timing and type of receptor production relative to injury as well as their position in the nucleus ambiguus was analyzed. Results: Netrin-1 UNC5A receptors were minimal in the nucleus ambiguus during RLN regeneration. DCC, the receptor that plays an attract role, was immunopositive from 7 to 21 DPI. All GDNF receptors, except GFRα2, were clearly positive from 7 to 14 DPI. No differences of production were observed according to the position of the motor neurons in the nucleus ambiguus. Conclusion: An injury of the RLN leads to a higher production of Netrin-1 DCC and GDNF receptors in the nucleus ambiguus. The timing of receptor production is similar to up-regulation of their trophic factors in the laryngeal muscles.
bioRxiv (Cold Spring Harbor Laboratory), May 4, 2022
Transcriptome analysis of left-right intrinsic laryngeal muscles shows divergent expression of genes associated with innervation
Objectives/Hypothesis: Recurrent laryngeal nerve neuropathy diagnosed as idiopathic or due to sho... more Objectives/Hypothesis: Recurrent laryngeal nerve neuropathy 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. Study Design: Laboratory experiment. Methods: High-quality RNA was extracted from the left and right sides of the rat posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA), respectively. After high-throughput RNA-Sequencing (RNA-Seq), 88 samples were organized into 12 datasets acco...
Development and Application of Automated Vocal Fold Tracking Software in a Rat Surgical Model
The Laryngoscope
ObjectiveThe rat is a widely used model for studying vocal fold (VF) function after recurrent lar... more ObjectiveThe 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 (RVT1.0), to quantify VF motion and tested it on rats with iatrogenic unilateral vocal fold paralysis (VFP).MethodsA deep neural network was trained to identify the positions of the VFs and arytenoid cartilages (ACs) in transoral laryngoscope videos of the rat glottis. Software was developed to estimate glottic midline, VF displacement, VF velocity, and AC angle. The software was applied to laryngoscope videos of adult rats before and after right recurrent and superior laryngeal nerve transection (N = 15; 6M, 9F). All software calculated metrics were compared before and after injury and validated against manually calculated metrics.ResultsRVT1.0 accurately tracked and quantified VF displacement, VF velocity, a...
Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation
The Laryngoscope
Frontiers in Neuroanatomy
ABSTRACTProprioception plays a crucial role in laryngeal function. Further, dysfunctional proprio... more ABSTRACTProprioception plays a crucial role in laryngeal function. Further, dysfunctional proprioception likely contributes to disorders such as laryngeal dystonia, dysphagia and vocal fold paresis. Despite this, the physiology of laryngeal proprioception is not well-understood. Controversy remains over whether canonical proprioceptive organs, like muscle spindles (MS) even exist in the intrinsic laryngeal muscles (ILM). Vesicular Glutamate Transporter 1 (VGLUT1) expression has been described in the sensory afferents of MS. This study’s primary aim is to determine whether the ILM contain MS using VGLUT1. This is a novel approach, as prior studies have relied on morphology and myosin composition to study this question. Secondarily, we describe the pattern of VGLUT1 expression in the rat larynx, Larynges of 62 Sprague-Dawley rats distributed across 5 age groups (P3, P8, P11, P14-15, and adult), were sectioned and immunostained for VGLUT1 and beta-tubulin III. Other markers (S46, GNAT3...
Frontiers in Neuroanatomy
The larynx is an organ of the upper airway that participates in breathing, glutition, voice produ... more 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. Dysfunctional laryngeal proprioception likely contributes to disorders such as laryngeal dystonia, dysphagia, vocal fold paresis, and paralysis. While the proprioceptive system in skeletal muscle derived from somites is well described, the proprioceptive circuitry that governs head and neck structures such as VF has not been so well characterized. For over two centuries, researchers have investigated the question of whether canonical proprioceptive organs, muscle spindles, and Golgi tendon organs, exist in the ILM, with variable findings. The present work is a state-of-the-art review of the peri...
ABSTRACTObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervatio... more ABSTRACTObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervation after nerve injury. In contrast, little is known regarding the expression of GDNF receptors in the formation of the nucleus ambiguus (NA) and its innervation of the larynx during embryogenesis. Differences may suggest therapeutic targets after nerve injury.Study DesignLaboratory experiment.MethodsRat brainstems at E14, E16, E18, E20, adult (4 animals/timepoint) were sectioned and stained for GDNF receptors: GFRα-1, GFRα-2, GFRα-3, and Ret. Islet1 and ChAT were used as markers for motoneuron cell bodies. Sections were observed using Zeiss Axio Imager M2 Microscope and quantified using Image J.ResultsExpression of all four GDNF receptors was identified within the nucleus ambiguus, as well as hypoglossal and facial nuclei of the adult rat brainstem. During rat development, GFRα-3 and Ret exhibited upregulation within the nucleus ambiguus at E14 whereas GFRα-1 began showing upregulation at...
Temporal expression of Laminin-111 in the developing rat larynx
Neuroscience Letters
Challenges related to high-quality RNA extraction from post-mortem tissue have limited RNA-sequen... more 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. The experimental procedure consistently provided sufficient intact total RNA (N = 68) and RIN ranging between 7.0 and 8.6, which was unprecedented using standard RNA purification protocols. This study confirmed the reproducibility of the workflow through repeated trials at different postnatal time points and across the distinctive ILMs. High-throughput diagnostics from 90 RNA samples indicated no sequencing alignment scores below 70%, validating ...
Otolaryngology–Head and Neck Surgery, 2019
ObjectiveRecurrent laryngeal nerve (RLN) injury causes vocal fold paralysis from which functional... more ObjectiveRecurrent 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 DesignAnimal experiment (rat model).SettingBasic science laboratory.MethodsThe 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 q...
Journal of Neuroscience Methods, 2016
h i g h l i g h t s • The development of a new protocol for specific brainstem motoneuron isolati... more h i g h l i g h t s • The development of a new protocol for specific brainstem motoneuron isolation from postnatal rats. • Abductor and adductor motoneurons were isolated in separate plates. • The utility of neural tracers for a long term neuron culture isolation was addressed. • The best period for cranial motoneuron isolation and culture in postnatal rats was determined.
The Laryngoscope, 2016
Objectives/Hypothesis: Synkinetic reinnervation of the laryngeal muscles is one of the causes of ... more 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. Methods: Anti-GDNF antibody was injected into the posterior cricoarytenoid (PCA) 3 days following RLN transection and anastomosis. Larynges were harvested at 7, 14, 28, 56, and 112 days post injury (DPI). Prior to sacrifice, the vocal fold mobility was assessed. Immunostaining to identify neuromuscular junctions was used to evaluate the extent of axonal reinnervation of the PCA, lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA). Results: After anti-GDNF injection into PCA, RLN reinnervation in all muscles was altered when compared to the controls. PCA innervation was delayed. At 7 DPI, only a few axons made synapses in the PCA. In contrast, axons prematurely innervated the LTA and MTA when compared to controls. Innervation was similar to controls at 56 and 112 DPI. Vocal fold motion was enhanced in 10 of 24 animals studied. Conclusions: After injection of anti-GDNF into the PCA, early arriving axons bypass the PCA and enter the LTA. Later arriving axons innervate the PCA and MTA. Vocal fold function is improved as compared to controls. Anti-GDNF injection into the PCA influences the pattern of reinnervation and may result in less synkinetic, more functional innervation.
Neuroscience Letters, 2017
h i g h l i g h t s • Recurrent laryngeal nerve injury leads to non-specific reinnervation of the... more h i g h l i g h t s • Recurrent laryngeal nerve injury leads to non-specific reinnervation of the larynx. • Netrin-1 plays an important role during laryngeal muscle reinnervation. • Injection of Netrin-1 reduces reinnervation of the posterior cricoarytenoid muscle. • Netrin-1 injection alters the motor reinnervation process of the larynx.
Endogenous alkaline phosphatase in the optic chiasm suggests a novel mechanism of retinal axonal guidance
Investigative Ophthalmology & Visual Science, 2018
Alkaline Phosphatase Enriched Exosomes as Guidance Cues in the Developing Avian Optic Chiasm
Investigative Ophthalmology & Visual Science, 2016
Expression of trophic factors receptors during reinnervation after recurrent laryngeal nerve injury
The Laryngoscope
An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a po... more 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.