Changes in vocal fold gene expression and histology after injection augmentation in a recurrent laryngeal nerve injury model.

OBJECTIVE: To investigate changes in neuroregenerative pathways with vocal fold denervation in response to vocal fold augmentation. METHODS: Eighteen Yorkshire crossbreed swine underwent left recurrent laryngeal nerve transection, followed by observation or augmentation with carboxymethylcellulose or calcium hydroxyapatite at two weeks. Polymerase chain reaction expression of genes regulating muscle growth (MyoD1, MyoG and FoxO1) and atrophy (FBXO32) were analysed at 4 and 12 weeks post-injection. Thyroarytenoid neuromuscular junction density was quantified using immunohistochemistry. RESULTS: Denervated vocal folds demonstrated reduced expression of MyoD1, MyoG, FoxO1 and FBXO32, but overexpression after augmentation. Healthy vocal folds showed increased early and late MyoD1, MyoG, FoxO1 and FBXO32 expression in all animals. Neuromuscular junction density had a slower decline in augmented compared to untreated denervated vocal folds, and was significantly reduced in healthy vocal folds contralateral to augmentation. CONCLUSION: Injection augmentation may slow neuromuscular degeneration pathways in denervated vocal folds and reduce compensatory remodelling in contralateral healthy vocal folds.

Evaluation of recurrent laryngeal neuropathy in domestic and feral horse populations in Australia using histologic and immunohistochemical analysis: A pilot study.

BACKGROUND: Little is known about potential differences in the left recurrent laryngeal nerve (Lrln) and left cricoarytenoideus dorsalis (LCAD) muscle between domestic and feral horse populations. If a difference exists, feral horses may provide a useful control population for research related to recurrent laryngeal neuropathy (RLN) and increase our understanding of potential population pressures influencing the incidence RLN. OBJECTIVES: The objective of this study was to compare the Lrln and LCAD of domestic and feral horses using histological and immunohistochemical techniques (IHC). METHODS: Sixteen horses, domestic (n = 8) and feral (n = 8), without clinical or ancillary examinations that were processed at an abattoir had the Lrln and LCAD muscle harvested immediately following death. Carcass weights were recorded. Subjective and morphometric histologic assessment were performed on Lrln sections. The LCAD was assessed for myosin heavy chain (fibre type proportion, diameter and grouping using IHC. RESULTS: Fibre-type grouping consistent with RLN was seen in both groups. Regenerating fibre clusters were more common in domestic compared to feral horses (p = 0.04). No other histologic differences occurred between groups. Muscle fibre typing demonstrated a lower mean percentage of type IIX fibres in the feral group compared to the domestic group (p = 0.03). There was no difference in type I or IIA proportions or mean diameter of any fibre type between the groups. CONCLUSIONS: The domestic population showed evidence of nerve regeneration suggesting RLN in this group, yet this was not supported by the higher proportion of type IIX muscle fibres compared to the feral population. Further evaluation to clarify the significance and wider occurrence of the differences is indicated.

A Systematic Review on Surgical Treatments for Sulcus Vocalis and Vocal Fold Scar.

OBJECTIVES/HYPOTHESIS: Vocal fold sulcus and scars are benign vocal fold lesions that present as a challenge to the laryngologist. A number of different surgical techniques have been proposed, aiming at restoring the lamina propria (LP), closing the glottal gap, or both. This study aimed to provide a systematic review of surgical treatment for sulcus and scar and to propose a new classification for these techniques. STUDY DESIGN: A literature search using MEDLINE and Google Scholar through August 2020. METHODS: Data on study design were retrieved and outcomes were classified as acoustic, aerodynamic, self-reported, perceptual, and stroboscopic. Methodological quality was assessed using the MINORs criteria. Each technique was classified as direct, indirect, or combined. RESULTS: Our search included 31 studies with a total of 617 patients. Direct techniques included dissection, graft interposition, or LP regeneration/scar degradation while indirect techniques aimed for glottal gap closure. Only one article performed a comparison between different types of techniques and only eight studied the five types of outcomes. No superiority of any technique was noted in our analysis. Self-reported outcomes were the most frequently improved. CONCLUSIONS: There seems to not exist a one-fits-all treatment for this clinical picture and no clear decision-making pattern. A recent trend toward sequential approaches, starting with less invasive procedures, can be observed. Laryngoscope, 132:822-830, 2022.

Thyroarytenoid Muscle Gene Expression in a Rat Model of Early-Onset Parkinson's Disease.

OBJECTIVES/HYPOTHESIS: Voice disorders in Parkinson's disease (PD) are early-onset, manifest in the preclinical stages of the disease, and negatively impact quality of life. The complete loss of function in the PTEN-induced kinase 1 gene (Pink1) causes a genetic form of early-onset, autosomal recessive PD. Modeled after the human inherited mutation, the Pink1-/- rat demonstrates significant cranial sensorimotor dysfunction including declines in ultrasonic vocalizations. However, the underlying genetics of the vocal fold thyroarytenoid (TA) muscle that may contribute to vocal deficits has not been studied. The aim of this study was to identify differentially expressed genes in the TA muscle of 8-month-old male Pink1-/- rats compared to wildtype controls. STUDY DESIGN: Animal experiment with control. METHODS: High throughput RNA sequencing was used to examine TA muscle gene expression in adult male Pink1-/- rats and wildtype controls. Weighted Gene Co-expression Network Analysis was used to construct co-expression modules to identify biological networks, including where Pink1 was a central node. The ENRICHR tool was used to compare this gene set to existing human gene databases. RESULTS: We identified 134 annotated differentially expressed genes (P < .05 cutoff) and observed enrichment in the following biological pathways: Parkinson's disease (Casp7, Pink1); Parkin-Ubiquitin proteasome degradation (Psmd12, Psmd7); MAPK signaling (Casp7, Ppm1b, Ppp3r1); and inflammatory TNF-α, Nf-κB Signaling (Casp7, Psmd12, Psmd7, Cdc34, Bcl7a, Peg3). CONCLUSIONS: Genes and pathways identified here may be useful for evaluating the specific mechanisms of peripheral dysfunction including within the laryngeal muscle and have potential to be used as experimental biomarkers for treatment development. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2874-E2879, 2021.

Development of a constant pressure perfused ex vivo model of the equine larynx.

Distal axonopathy is seen in a broad range of species including equine patients. In horses, this degenerative disorder of the recurrent laryngeal nerve is described as recurrent laryngeal neuropathy (RLN). The dysfunctional innervation of the cricoarytenoideus dorsalis muscle (CAD) leads to a loss of performance in affected horses. In general, ex vivo models of the larynx are rare and for equine patients, just one short report is available. To allow for testing new therapy approaches in an isolated organ model, we examined equine larynges in a constant pressure perfused setup. In order to check the vitality and functionality of the isolated larynx, the vessels´ reaction to norepinephrine (NE) and sodium nitroprusside (NP) as vasoactive agents was tested. Additionally, the contractility of the CAD was checked via electrical stimulation. To determine the extent of hypoxic alterations, lactate dehydrogenase (LDH) and lactate were measured and an immunofluorescent analysis of hypoxia-inducible factor (HIF-1α), a key transcription factor in hypoxia, was performed. For this, a hypoxia-induced cell culture for HIF-1α was developed. The application of NE led to an expected vasoconstriction while NP caused the expected vasodilation. During a perfusion period of 352 ±20.78 min, LDH values were in the reference range and lactate values slightly exceeded the reference range at the end of the perfusion. HIF-1α nuclear translocation could reliably be detected in the hypoxia-induced cell cultures, but not in sections of the perfused CAD. With the approach presented here, a solid basis for perfusing equine larynges was established and may serve as a tool for further investigations of equine larynx disorders as well as a transferrable model for other species.

Dual-wavelength fiber-optic technique to assist needle cricothyroidotomy.

The traditional needle cricothyroidotomy procedure is performed blindly without any medical equipment. Complications including posterior tracheal wall perforation, accidental vessel puncture, and missed tracheal puncture are reported. Therefore, we proposed a dual-wavelength fiber-optic technique based on the technique of near-infrared spectroscopy to assist operators performing needle cricothyroidotomy in a swine model. We embedded optical fibers in a 16-gauge intravenous needle catheter. Real-time data were displayed on an oscilloscope, and we used the program to analyze the data immediately. The change of optical density corresponding to 690-nm and 850-nm wavelengths and hemoglobin parameters (HbO2 and Hb concentrations) was analyzed immediately using the program in the laptop. Unique and significant optical differences were presented in this experiment. We could easily identify every different tissue by the change of optical density corresponding to 690-nm and 850-nm wavelengths and hemoglobin parameters (HbO2 and Hb concentrations). Statistical method (Kruskal-Wallis H test) was used to compare differences in tissues at each time-point, respectively. The p values in every tissue in optical density change corresponding to 690 nm and 850 nm were all < 0.001. Furthermore, the p values in every tissue in Hb and HbO2 were also all < 0.001. The results were statistically significant. This is the first and novel study to introduce a dual-wavelength embedded fibers into a standard cricothyroidotomy needle. This proposed system might be helpful to provide us real-time information of the advanced needle tip to decrease possible complications.

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.

The association of sulcus vocalis and benign vocal cord lesions: intraoperative findings.

OBJECTIVE: To investigate the presence of sulcus vocalis in patients who underwent phonomicrosurgery due to benign vocal cord lesions. METHODS: Between January 2013 and June 2018, the records of 133 patients who underwent operations for benign vocal fold pathology were retrospectively reviewed. Intraoperative findings of the patients were noted. Patients were divided into two groups: patients with only benign vocal fold lesions (BVFL) and patients with benign vocal fold lesions and accompanying sulcus vocalis (SV + BVFL). RESULTS: In total, 67 patients (50.4%; 38 females, 29 males) had BVFL and 66 (49.6%; 37 females, 29 males) had SV + BVFL. The patients in the SV + BVFL group were significantly younger than those in the BVFL group (p = 0.039). The sulcus was unilateral in 60.6% of the patients. The presence of sulcus vocalis was 49% and 47.6% in patients with a diagnosis of polyps (n = 51) and cysts (n = 42), respectively. A total of 12 of 13 patients with multiple benign vocal fold lesions had accompanying sulcus vocalis. CONCLUSION: Approximately half of the patients who underwent operations for benign vocal fold lesions had accompanying sulcus vocalis. More than half of the sulcus associated with benign lesions was unilateral.

Could the infiltration of the thyroarytenoid muscle define the pT2 glottic carcinoma?

BACKGROUND: The involvement of the thyroarytenoid (TA) muscle by glottic cancer may be related to an impaired vocal cord mobility, which is classified as cT2 disease. The primary endpoint was to evaluate the prognostic significance of TA muscle involvement in early glottic cancer treated with transoral laser microsurgery (TLM). METHODS: A review was conducted on a cohort of 209 patients consecutively treated with TLM for early glottic carcinoma. Univariate analysis was used to examine the prognostic meaning of clinical and pathological parameters. RESULTS: The statistical analysis showed that TA muscle infiltration correlated significantly with a worse prognosis in terms of recurrence rate and disease-free survival, and this was confirmed even in the subcohort with pT1a glottic cancer. CONCLUSIONS: Our preliminary findings suggest that it could be considered as a criterion for upstaging a glottic cancer from pT1 to pT2.

Radiographic muscle invasion not a recurrence predictor in HPV-associated oropharyngeal squamous cell carcinoma.

OBJECTIVE: To determine whether muscle invasion evident on pretreatment imaging in p16 + oropharyngeal squamous cell carcinoma (OPSCC) correlates with recurrence. STUDY DESIGN: Retrospective review. METHODS: Two-hundred and seventy-six patients with p16 + OPSCC treated at a tertiary referral center from 2003 to 2015 were analyzed. All scans were reviewed by a dedicated neuroradiologist with subspecialty expertise in head and neck imaging. Radiographic evidence of muscle invasion to the genioglossus, hyoglossus, medial pterygoid, and prevertebral muscles was analyzed. Local and regional recurrence rates were compared between the muscle invasion and no muscle invasion groups. RESULTS: One hundred and ninety patients met inclusion criteria with adequate follow-up data and pretreatment imaging. Patients were predominantly male (87.5% male) and smokers (65.6% smokers) with a mean age of 56.7 (standard deviation: 9.0 years). Most commonly invaded muscles in the muscle invasion group were hyoglossus (57.8%) and genioglossus (56.3%). There was no statistically significant difference in primary site or nodal recurrence between the combined group, including definite or possible muscle invasion and the group without muscle invasion (P = 0.205 and P = 0.569, respectively). Additionally, no statistically significant difference was present in recurrence-free and disease-specific survival between the two groups at 3- and 5-year follow-up (P > 0.05). CONCLUSION: Radiographic evidence of muscle invasion does not appear to be a predictor of human papilloma virus (+) OPSCC recurrence. Additional studies are needed to validate our findings. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:871-876, 2019.

Volumetric analysis of vocal fold atrophy via magnetic resonance imaging.

OBJECTIVE: To quantitatively test the hypothesis that older patients have increased thyroarytenoid muscle atrophy by comparing thyroarytenoid muscle volumes across different age groups. METHODS: A retrospective chart review was conducted. The study included 111 patients with no history of laryngeal pathology. Two investigators reviewed magnetic resonance imaging studies of these patients and manually traced the thyroarytenoid muscles on multiple slices bilaterally. Thyroarytenoid muscle volumes were then computed using imaging analysis software. Patients were stratified into three age groups (18-50 years, 51-64 years, and 65 years or older) for comparison. RESULTS: Intra- and inter-rater reliabilities were excellent for all measurements (intraclass correlation co-efficient > 0.90). There was no statistically significant difference in the mean volumes of left and right thyroarytenoid muscles in all age and gender groups. CONCLUSION: Given the lack of statistically significant difference in thyroarytenoid muscle volume between age groups on magnetic resonance imaging, the prevailing assumption that age-related thyroarytenoid muscle atrophy contributes to presbyphonia should be re-examined.

[Clinical anatomic study on the segment and adjacent of tract of congenital pyriform sinus fistula].

Objective: To investigate the anatomic tract of congenital pyriform sinus fistula (CPSF). Methods: A total of 90 patients with CPSF undergoing open surgery between August, 2007 and March, 2017 at the Department of Guangdong General Hospital were retrospectively analyzed. Results: The tracts of all the fistulas actually walked far different from those of theoretical ones. A whole fistula may be divided into 4 segments according to adjacent anatomy of CPSF. The posterior inner segment to the thyroid cartilage was initial part of the fistula. It originated from the apex of pyriform sinus, then piercing out of the inferior constrictor of pharynx inferiorly near the inferior cornu of the thyroid cartilage (ICTC), and descended between the lateral branch of the superior laryngeal nerve and the recurrent laryngeal nerve. The ICTC segment was the second part of the fistula, firstly piercing out of the inferior constrictor of pharynx and/or cricothyroid muscle, and then entering into the upper pole of thyroid. The relationship between fistula and ICTC could be divided into three types: type A (medial inferior to ICTC) accounting for 42.2% (38/90); type B (penetrate ICTC) for 3.3% (3/90); and type C (lateral inferior to ICTC) for 54.5% (49/90). The internal segment in thyroid gland was the third part of fistula, walking into the thyroid gland and terminating at its upper pole (92.2%, 83/90) or deep cervical fascia near the upper pole of thyroid (7.8%, 7/90). The lateral inferior segment to thyroid gland was the last part of the fisula, most of which are iatrogenic pseudo fistula, and started from the lateral margin of thyroid gland. Conclusions: CPSF has a complicated pathway. Recognition of the tract and adjacent anatomy of CPSF will facilitate the dissection and resection of CPSF in open surgery.

The influence of kinesiotaping on the loosening of the laryngeal muscles in hyperfunctional dysphones.

INTRODUCTION: Hyperfunctional dysfunction is one of the most common functional dysphonia, cha-racterized by voice insufficiency with excessive tightening of the muscles inside and outside the larynx during phonation. To make the treatment process more effective, new ways of rehabilitation are constantly being sought and developed. The aim of this work is to evaluate the effectiveness of laryngotaping - an innovative method of taping around the larynx and neck muscles. MATERIAL AND METHOD: 10 patients with diagnosed hyperfunctional dysphonia participated in the study. Using the kinesiotaping principles, for 7 days, the suprahyoid and infrahyoid muscles, ster-nocleidomastoid muscles as well as the thyroid cartilage were taped. Before and after the therapy, the patients completed the VHI voice self-evaluation questionnaire. The evaluation of the larynx according to the L. Mathienson scale was also assessed palpation. RESULTS: Analyzing the results of the VHI questionnaire and evaluation of palpation evaluation of the larynx before and after the therapy, statistically significant differences were observed. The results on average decreased by half, which is the desired effect of therapy. DISCUSSION: The results confirm the positive impact of kinesiotaping around the larynx. However, more research is needed on a larger group of patients to fully evaluate the therapeutic effect. CONCLUSIONS: 1. Laryngotaping is an effective way to normalize muscle tone, and thus to improve the quality of the voice. 2. The presented studies require continuation, however, positive reception of the introduced therapy by patients encourages further research on a larger group of patients.

Laryngeal Neuropathy in Adult Goats With Copper Deficiency.

The aim of this study was to elucidate the cause of a neurological syndrome characterized by stridor in adult goats with clinical signs of copper deficiency. The main clinical signs consisted of apathy, emaciation, pale mucous membranes, mucous nasal discharge, dyspnea, severe achromotrichia, diffuse alopecia, torpor, ataxia, and stridor. When the goats were forced to move, the stridor increased. In a herd of 194 Toggenburg goats, 10 adult goats with clinical signs of copper deficiency were removed from the herd and divided into 2 groups: group 1, which consisted of 4 nannies and 1 buck with stridor, and group 2, which consisted of 4 nannies and 1 buck without stridor. Group 3, used as a control, consisted of 5 adult goats from another flock without any clinical signs of disease. The mean serum copper concentrations were 1.3 ± 0.3 µmol/L in group 1, 8.1 ± 1.1 µmol/L in group 2, and 11.3 ± 2.2 µmol/L in group 3. The mean serum iron concentrations were 42.3 ± 14.2 µmol/L in group 1, 39.1 ± 8.2 µmol/L in group 2, and 20.6 ± 6.1 µmol/L in group 3. The main histological lesions in goats from group 1 were axonal degeneration of the recurrent laryngeal nerves and atrophy of the muscles of vocal folds and of the dorsal cricoarytenoid and right and left cricothyroid muscles. Goats with ataxia had neuronal degeneration and necrosis of cerebellar Purkinje cells and of the cranial cervical ganglion. We concluded that the stridor was caused by axonal degeneration of the recurrent laryngeal nerves due to the severe copper deficiency.

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. The right RLN was transected and Netrin-1 was injected into the PCA (2.5, 5, 10, 15, 20µg/ml). At 7 days post injury fluorescent retrograde tracer was injected into the PCA and Thyroarytenoid (TA) muscles. At 9 days tissues were harvested. Immunostaining showed reinnervation patterns in the laryngeal muscles and labelled motoneurons in the nucleus ambiguus. Lower concentrations of Netrin-1 (2.5 and 5µg/ml) showed no significant changes in laryngeal muscles reinnervation. Higher concentrations of Netrin-1 significantly reduced motor end plate innervation. The most effective dose was 10µg/ml showing reduced number of innervated motor endplates in the PCA. The somatotopic organization of the nucleus ambiguus was altered in all concentrations of Netrin-1 injection. These findings indicate that injection of Netrin-1 into the PCA changes the reinnervation pattern of the RLN.