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. This study investigates the expression of laminin-111 in laryngeal muscles during primary laryngeal innervation of Sprague Dawley rats. Adult larynges and embryos were sectioned for immunohistochemistry with ßIII-Tubulin, laminin subunit α-1 (LAMA1), NTN1, and α-bungarotoxin. Sections were processed for single-molecule inexpensive RNA fluorescence in situ hybridization analysis of LAMA1 mRNA. LAMA1 expression increased in all intrinsic laryngeal muscles, except the medial thyroarytenoid (MTA), at E20.5. At E20.5 there was increased expression in the lateral thyroarytenoid (LTA) and posterior cricoarytenoid (PCA) compared to the MTA. NTN1 upregulation was limited to the LTA and lateral cricoarytenoid (LCA) at E16.5 without any increase in the MTA or PCA. LAMA1 and NTN1 expression did not strictly follow expected patterns relative to the known timing of innervation and does not appear to be acting similarly to its role following RLN injury. These differences between developmental and post-injury innervation provide targets for investigations of therapeutics after nerve injury.

Proteomic Characterization of Senescent Laryngeal Adductor and Plantaris Hindlimb Muscles.

OBJECTIVES: The goals of this study were to 1) compare global protein expression in muscles of the larynx and hindlimb and 2) investigate differences in protein expression between aged and nonaged muscle using label-free global proteomic profiling methods. METHODS: Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed on thyroarytenoid intrinsic laryngeal muscle and plantaris hindlimb muscle from 10 F344xBN F1 male rats (5 old and 5 young). Protein expression was compared and pathway enrichment analysis performed for each muscle type (larynx and limb) and age group (old and young muscle). RESULTS: Over 1,000 proteins were identified in common across both muscle types and age groups using LC-MS/MS analysis. Significant age-related differences were seen across 107 proteins in plantaris hindlimb and in 19 proteins in thyroarytenoid laryngeal muscle. Bioinformatic and enrichment analysis demonstrated protein differences between the hindlimb and larynx may relate to immune and stress redox responses and RNA repair. CONCLUSION: There are clear differences in protein expressions between the laryngeal and hindlimb skeletal muscles. Initial analysis suggests differences between the two muscle groups may relate to stress responses and repair mechanisms. Age-related changes in the thyroarytenoid appear to be less obvious than in the plantaris. Further in-depth study is needed to elucidate how aging affects protein expression in the laryngeal muscles. LEVEL OF EVIDENCE: NA Laryngoscope, 132:148-155, 2022.

Metabolomic Expression of Laryngeal and Hindlimb Muscles in Adult versus Senescent Rats.

OBJECTIVES: (1) Determine the feasibility of obtaining a global, unbiased metabolomic profile on laryngeal muscle in a rat model; (2) evaluate the impact of biological aging on the laryngeal metabolome; and (3) characterize biochemical expression differences between aged and non-aged laryngeal and hindlimb muscle. METHODS: Thyroarytenoid laryngeal muscle and plantaris hindlimb muscle were harvested from 5 young adult (9 months old) and 5 older adult (32 months old) F344BN rats. Tissue was processed and analyzed using LC-MS methods. Detected metabolites were compared to widely used metabolite databases and KEGG pathway enrichment was performed on significant metabolites. RESULTS: The greatest differences in metabolite expression were between laryngeal and limb muscle with 126 different metabolites found between laryngeal and limb within the young group and 149 different metabolites within the old group. Significant hits between muscle groups highlighted amino acid differences between these tissues. There were more robust differences with age in limb muscle compared to laryngeal muscle. CONCLUSIONS: Amino acid metabolism is a key difference between muscles of the limbs and larynx. Due to the number of differentially expressed metabolites between the 2 muscle groups, caution should be exercised when applying skeletal limb muscle physiology and biology concepts to the vocal muscles in both aged and non-aged musculoskeletal systems. Mechanisms underlying less robust effects of age on laryngeal muscle compared to limb muscle require elucidation.

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

OBJECTIVES/HYPOTHESIS: The present study investigated the characteristics of early internal laryngeal muscle atrophy in recurrent laryngeal nerve injury (RLNI) rats. STUDY DESIGN: To observe the characteristics of early internal laryngeal muscle atrophy post RLNI. METHODS: Rats were divided into three groups: sham-operated control group (n = 20), recurrent laryngeal nerve transverse injury group (RLNTI, n = 50), and recurrent laryngeal nerve blunt contusion group (RLNBC, n = 50). Five weeks after RLNI, certain rats were sacrificed weekly, and their laryngeal tissues were harvested. The atrophic features of internal laryngeal muscles were detected using hematoxylin and eosin. NF-κB and MuRF-1 levels were tested using IHC. RESULTS: The atrophic degree and fibrosis of thyroarytenoid, posterior cricoarytenoid, and lateral cricoarytenoid muscles were related to the type of RLNI. The average myofiber cross-sectional areas increased before an obvious decrease in the RLNTI and RLNBC groups. Muscle recovery occurred in the RLNBC group starting 4 weeks after RLNI, but only a weak trend was observed in the RLNTI group in the 5th week. During the muscle atrophy process, MuRF-1 and NF-κB were upregulated early and were maintained at a high level, which showed a trend similar to muscle atrophy. However, NF-κB expression was opposite to MuRF-1 expression and muscle atrophy when the muscles recovered. CONCLUSION: The atrophy degree of internal laryngeal muscles was associated with the type of RLNI. The NF-κB/MuRF-1 signaling pathway was involved in internal laryngeal muscle atrophy after RLNI, which is different from skeletal muscle after denervation. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E1256-E1264, 2021.

Unraveling the molecular pathobiology of vocal fold systemic dehydration using an in vivo rabbit model.

Vocal folds are a viscoelastic multilayered structure responsible for voice production. Vocal fold epithelial damage may weaken the protection of deeper layers of lamina propria and thyroarytenoid muscle and impair voice production. Systemic dehydration can adversely affect vocal function by creating suboptimal biomechanical conditions for vocal fold vibration. However, the molecular pathobiology of systemically dehydrated vocal folds is poorly understood. We used an in vivo rabbit model to investigate the complete gene expression profile of systemically dehydrated vocal folds. The RNA-Seq based transcriptome revealed 203 differentially expressed (DE) vocal fold genes due to systemic dehydration. Interestingly, function enrichment analysis showed downregulation of genes involved in cell adhesion, cell junction, inflammation, and upregulation of genes involved in cell proliferation. RT-qPCR validation was performed for a subset of DE genes and confirmed the downregulation of DSG1, CDH3, NECTIN1, SDC1, S100A9, SPINK5, ECM1, IL1A, and IL36A genes. In addition, the upregulation of the transcription factor NR4A3 gene involved in epithelial cell proliferation was validated. Taken together, these results suggest an alteration of the vocal fold epithelial barrier independent of inflammation, which could indicate a disruption and remodeling of the epithelial barrier integrity. This transcriptome provides a first global picture of the molecular changes in vocal fold tissue in response to systemic dehydration. The alterations observed at the transcriptional level help to understand the pathobiology of dehydration in voice function and highlight the benefits of hydration in voice therapy.

Laryngeal muscle biology in the Pink1-/- rat model of Parkinson disease.

Neuromuscular pathology is found in the larynx and pharynx in humans with Parkinson disease (PD); however, it is unknown when this pathology emerges. We hypothesized that pathology occurs in early (premanifest) stages. To address this, we used the Pink1-/- rat model of PD, which shows age-dependent dopaminergic neuron loss, locomotor deficits, and deficits related to laryngeal function. We report findings in the thyroarytenoid muscle (TA) in Pink1-/- rats compared with wild-type (WT) control rats at 4 and 6 mo of age. TAs were analyzed for force production, myosin heavy chain isoform (MyHC), centrally nucleated myofibers, neural cell adhesion molecule, myofiber size, and muscle section size. Compared with WT, Pink1-/- TA had reductions in force levels at 1-Hz stimulation and 20-Hz stimulation, increases in relative levels of MyHC 2L, increases in incidence of centrally nucleated myofibers in the external division of the TA, and reductions in myofiber size of the vocalis division of the TA at 6 mo of age. Alterations of laryngeal muscle biology occur in a rat model of premanifest PD. Although these alterations are statistically significant, their functional significance remains to be determined. NEW & NOTEWORTHY Pathology of peripheral nerves and muscle has been reported in the larynx and pharynx of humans diagnosed with Parkinson disease (PD); however, it is unknown whether differences of laryngeal muscle occur at premanifest stages. This study examined the thyroarytenoid muscles of the Pink1-/- rat model of PD for differences of muscle biology compared with control rats. Thyroarytenoid muscles of Pink1-/- rats at premanifest stages show differences in multiple measures of muscle biology.

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. 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. LEVEL OF EVIDENCE: NA. Laryngoscope, 129:2537-2542, 2019.

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. 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.

Magnetic resonance imaging quantification of dehydration and rehydration in vocal fold tissue layers.

Clinicians commonly recommend increased hydration to patients with voice disorders. However, effects on clinical voice outcome measures have been inconsistent. Hydration-induced change within different layers of vocal fold tissue is currently unknown. Magnetic Resonance Imaging (MRI) is a promising method of noninvasively measuring water content in vocal folds. We sought to image and quantify changes in water content within vocal fold mucosa and thyroarytenoid muscle after dehydration and rehydration. Excised porcine larynges were imaged using proton density (PD) weighted MRI (1) at baseline and (2) after immersion in one of five hypertonic, isotonic, or hypotonic solutions or in dry air. Larynges dehydrated in hypertonic solutions or dry air were rehydrated and imaged a third time. Scans revealed fluid-rich vocal fold mucosa that was distinct from muscle at baseline. Baseline normalized signal intensity in mucosa and muscle varied by left vs. right vocal fold (p < 0.01) and by anterior, middle, or posterior location (p < 0.0001). Intensity changes in the middle third of vocal fold mucosa differed by solution after immersion (p < 0.01). Hypertonic solutions dehydrated the middle third of mucosa by over 30% (p < 0.001). No difference from baseline was found in anterior or posterior mucosa or in muscle after immersion. No association was found between intensity change in mucosa and muscle after immersion. After rehydration, intensity did not differ by solution in any tissue, and was not different from baseline, but post-rehydration intensity was correlated with post-immersion intensity in both mucosa and muscle (p < 0.05), suggesting that degree of change in vocal fold water content induced by hypertonic solutions ex vivo persists after rehydration. These results indicate that PD-MRI can be used to visualize large mammalian vocal fold tissue layers and to quantify changes in water content within vocal fold mucosa and thyroarytenoid muscle independently.

Elementary School Teachers' Vocal Dose: Muscle Bioenergetics and Training Implications.

Purpose: Translating exercise-science methodology for determination of muscle bioenergetics, we hypothesized that the temporal voice-use patterns for classroom and music teachers would indicate a reliance on the immediate energy system for laryngeal skeletal-muscle metabolism. It was hypothesized that the music-teacher group would produce longer voiced segments than the classroom teachers. Method: Using a between- and within-group multivariate analysis-of-variance design (5 classroom teachers; 7 music teachers), we analyzed fundamental-frequency data-collected via an ambulatory phonation monitor-for length (seconds) of voiced and nonvoiced intervals. Data were collected for 7.5 hr during the workday, over the course of several workdays for each teacher. Results: Descriptive analyses of voiced and nonvoiced intervals indicated that over 99% of voiced segments for both groups were no longer than 3.15 s, supporting the hypothesis of reliance on the immediate energy system for muscle bioenergetics. Significant differences were identified between and within the classroom- and music-teacher groups, with the music-teacher group producing longer voiced segments overall. Conclusions: Knowledge of probable intrinsic laryngeal skeletal-muscle bioenergetics requirements could inform new interdisciplinary considerations for voice habilitation and rehabilitation.

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. This study investigates the expression of certain neurotrophic factors in the laryngeal muscles during the course of axonal regeneration using RT-PCR. The timing of neurotrophic factor expression was correlated to the reinnervation of the laryngeal muscles by motor axons. Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Netrin-1 (NTN-1) increased their expression levels in laryngeal muscles after nerve section and during regeneration of RLN. The upregulation of trophic factors returned to control levels following regeneration of RLN. The expression levels of the neurotrophic factors were correlated with the innervation of regenerating axons into the denervated muscles. The results suggest that certain neurotrophic factor expression is strongly correlated to the reinnervation pattern of the regenerating RLN. These factors may be involved in guidance and neuromuscular junction formation during nerve regeneration. In the future, their manipulation may enhance the selective reinnervation of the larynx.

Thyroarytenoid muscle uptake and attenuation in PET/CT in elderly.

The objective of this study is to compare uptake and attenuation on positron emission tomography/computed tomography (PET/CT) imaging modality at the thyroarytenoid muscle between subjects aged less and older than 65 years old. The study design is retrospective chart review. The setting is academic medical center. PET/CT images of 60 patients aged less than 65 years old and 60 patients aged more than 65 years old were selected. Demographic data were collected. Both the groups were compared with respect to the maximum standardized uptake value (SUV max) and CT attenuation of bilateral thyroarytenoid muscles. The mean SUV max of the right thyroarytenoid muscle was 2.09 ± 0.8 in the group of patients aged less than 65 years old compared to 1.9 ± 0.6 in the group of patients aged more than 65 years old. For the left thyroarytenoid muscle, the mean SUV max in the first and second groups was, respectively, 2 ± 0.6 and 1.9 ± 0.6. The differences were not statistically significant. As for the CT attenuation, the mean value at the right thyroarytenoid muscle in the first and second groups was, respectively, 31.2 ± 0.8 HU and 20.8 ± 14.4 HU (p < 0.05). At the left thyroarytenoid muscle, the mean value was, respectively, 29.6 ± 9.9 and 22.8 ± 15 (p < 0.05). This study suggests that CT attenuation measurements can be used for objectively assessing the change in the density of aging thyroarytenoid muscle.

Enhancement of aging rat laryngeal muscles with endogenous growth factor treatment.

Clinical evidence suggests that laryngeal muscle dysfunction is associated with human aging. Studies in animal models have reported morphological changes consistent with denervation in laryngeal muscles with age. Life-long laryngeal muscle activity relies on cytoskeletal integrity and nerve-muscle communication at the neuromuscular junction (NMJ). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. We hypothesized that treatment with neurotrophin 4 (NTF4) would modify the morphology and functional innervation of aging rat laryngeal muscles. Fifty-six Fischer 344xBrown Norway rats (6- and 30-mo age groups) were used to evaluate to determine if NTF4, given systemically (n = 32) or directly (n = 24), would improve the morphology and functional innervation of aging rat thyroarytenoid muscles. Results demonstrate the ability of rat laryngeal muscles to remodel in response to neurotrophin application. Changes were demonstrated in fiber size, glycolytic capacity, mitochondrial, tyrosine kinase receptors (Trk), NMJ content, and denervation in aging rat thyroarytenoid muscles. This study suggests that growth factors may have therapeutic potential to ameliorate aging-related laryngeal muscle dysfunction.

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. 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. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E337-E342, 2016.

The effects of treadmill running on aging laryngeal muscle structure.

OBJECTIVES/HYPOTHESIS: Age-related changes in laryngeal muscle structure and function may contribute to deficits in voice and swallowing observed in elderly people. We hypothesized that treadmill running, an exercise that increases respiratory drive to upper airway muscles, would induce changes in thyroarytenoid muscle myosin heavy chain (MHC) isoforms that are consistent with a fast-to-slow transformation in muscle fiber type. STUDY DESIGN: Randomized parallel group controlled trial. METHODS: Fifteen young adult and 14 old Fischer 344/Brown Norway rats received either treadmill running or no exercise (5 days/week/8 weeks). Myosin heavy chain isoform composition in the thyroarytenoid muscle was examined at the end of 8 weeks. RESULTS: Significant age and treatment effects were found. The young adult group had the greatest proportion of superfast-contracting MHCIIL isoform. The treadmill running group had the lowest proportion of MHCIIL and the greatest proportion of MHCIIx isoforms. CONCLUSION: Thyroarytenoid muscle structure was affected both by age and treadmill running in a fast-to-slow transition that is characteristic of exercise manipulations in other skeletal muscles. LEVEL OF EVIDENCE: NA. Laryngoscope, 126:672-677, 2016.

Neurotrophin expression and laryngeal muscle pathophysiology following recurrent laryngeal nerve transection.

Laryngeal palsy often occurs as a result of recurrent laryngeal or vagal nerve injury during oncological surgery of the head and neck, affecting quality of life and increasing economic burden. Reinnervation following recurrent laryngeal nerve (RLN) injury is difficult despite development of techniques, such as neural anastomosis, nerve grafting and creation of a laryngeal muscle pedicle. In the present study, due to the limited availability of human nerve tissue for research, a rat model was used to investigate neurotrophin expression and laryngeal muscle pathophysiology in RLN injury. Twenty-five male Sprague-Dawley rats underwent right RLN transection with the excision of a 5-mm segment. Vocal fold movements, vocalization, histology and immunostaining were evaluated at different time-points (3, 6, 10 and 16 weeks). Although vocalization was restored, movement of the vocal fold failed to return to normal levels following RLN injury. The expression of brain­derived neurotrophic factor and glial cell line-derived neurotrophic factor differed in the thyroarytenoid (TA) and posterior cricoarytenoid muscles. The number of axons did not increase to baseline levels over time. Furthermore, normal muscle function was unlikely with spontaneous reinnervation. During regeneration following RLN injury, differences in the expression levels of neurotrophic factors may have resulted in preferential reinnervation of the TA muscles. Data from the present study indicated that neurotrophic factors may be applied for restoring the function of the laryngeal nerve following recurrent injury.

Increased Expression of MuRF1 Is Associated with Radiation-induced Laryngeal Muscle Atrophy.

BACKGROUND: Laryngeal muscles play an important role in breathing, sound production and trachea protection against food. Laryngeal dysfunctions during radiotherapy for head and neck cancers are common. In the present study, we aimed to investigate the early effect of radiation on the laryngeal muscles in vivo and possible mechanisms involved in this process. MATERIALS AND METHODS: Eight-week-old female C57bl/ mice received neck irradiation with a single dose of 25 Gy and bilateral thyroarytenoid (TA) muscles of mice were collected at day 3, 7 and 10 post-irradiation for evaluating muscle size, myosins, myosin heavy chain (MyHC) composition and MuRF1 protein levels. RESULTS: A significant reduction in the size of muscle fibers and myosins in the TA muscles were observed at days 3, 7, 10 after radiation (p<0.05). The loss of IIB myosin was more severe than that of IIA/X myosins at day 7 post-irradiation (75% vs. 64%). MuRF1 protein level was markedly increased at day 7 and 10 after radiation (p<0.05). CONCLUSION: Radiation induced an acute muscle fiber atrophy and myosin loss in the intrinsic laryngeal muscles. MuRF1 may play an important role in the radiation-induced protein degradation in the laryngeal muscles and warrants further investigation.

Changes in calsequestrin, TNF-α, TGF-ß and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps, diaphragm and intrinsic laryngeal muscles.

In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium-related protein), tumour necrosis factor (TNF-α; pro-inflammatory cytokine), tumour growth factor (TGF-ß; pro-fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF-α, TGF-ß and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF-α and TGF-ß serve as markers of dystrophy primarily for the diaphragm.

Transition of myosin heavy chain isoforms in human laryngeal abductors following denervation.

The objective of this study was to investigate the myofiber subtype transition of human posterior cricoarytenoid (PCA) muscle after the injury to recurrent laryngeal nerve (RLN). PCA muscle specimens were obtained from 38 bilateral vocal fold paralysis patients underwent arytenoidectomy. According to the duration of RLN injury, all the cases were divided into five denervation groups: 6-12 months, 1-2, 2-3, 3-6, and >6 years. The normal PCA muscles from total laryngectomy patients were chosen as controls. Immunofluorescence was adopted to detect the expression level of myosin heavy chain (MHC)-I and MHC-II in PCA muscle. Quantitative real-time PCR was also used to assess the transcriptional level of MHC subtypes (MHC-I, MHC-IIa, MHC-IIb, MHC-IIx, embryonic-MHC, and peri-natal-MHC). Immunofluorescence showed that MHC-I-positive myofibers in denervation groups were much lower than control group, respectively, while MHC-II-positive myofibers were significantly higher than control group (P < 0.05). With the extension of denervation, the number of MHC-I-positive myofibers gradually decreased, while MHC-II gradually increased and peaked in 1- to 2-year group. Transcriptional level of MHC-I, MHC-IIa, and MHC-IIb in denervation groups significantly down-regulated compared with the control (P < 0.05), respectively. However, MHC-IIx, embryonic-MHC, and peri-natal-MHC significantly up-regulated in all denervation groups, and the highest level was in 1- to 2-year denervation group. Data from the present study demonstrated that the maximum transition of MHC subtypes in human PCA muscles occurred in 1-2 years after denervation, suggesting that laryngeal reinnervation before the occurrence of irreversible transition of MHC subtypes could maintain the structural integrity of laryngeal PCA muscles.

Modulation of satellite cells activity and MyoD in rat thyroarytenoid muscle after reinnervation.

OBJECTIVES/HYPOTHESIS: To examine modulation of M-cadherin, a marker for satellite cells (SCs); and MyoD, which may indicate the myogenic activity following recurrent laryngeal nerve (RLN) denervation and immediate reinnervation; and to elucidate the correlation between their modulations and establishment of neuromuscular junctions (NMJs) in the reinnervated rat thyroarytenoid (TA) muscle. STUDY DESIGN: Quantitative real-time polymerase chain reaction qPCR and histologic assessment of the TA muscle following RLN transection and anastomosis. METHODS: Rats were divided into three groups: 1) denervation alone (DNV) (n = 60), 2) denervation with anastomosis (ANS) (n = 60), and 3) sham-operated controls (n = 12). Animals were sacrificed at 3 days and 1, 3, and 5 weeks after treatment. TA muscles harvested from 40 animals from each DNV and ANS group; all of sham group were subjected to qPCR for assessment of the modulation of M-cadherin and MyoD; and the remaining larynges of DNV and ANS group were used for histologic analysis. RESULTS: The expression levels of messenger RNAs (mRNAs) encoding M-cadherin and MyoD in the TA muscle of the DNV group were significantly higher (P < 0.05) than in the control throughout the study period. These mRNA levels in the ANS group were significantly higher (P < 0.05) at ≤ 1 week than in the controls but fell to control levels at ≥ 3 weeks. In the ANS group, recovery of muscle area and NMJs structure occurred by 3 weeks. CONCLUSION: These data suggested that NMJ formation following reinnervation might prompt recovery of M-cadherin and MyoD mRNA expression to the quiescent level of SCs.