Mechanotransduction in the Vocal Fold Microenvironment: A Narrative Review.

PURPOSE: The vocal fold tissues undergo nearly continuous and repeated cycles of injury and repair throughout the course of an individual's lifetime. It is well established that certain individuals are at greater risk of lesion development based on personality and behavioral classification. However, these characteristics alone do not wholly predict or explain lesion development or severity. In this review, we discuss current knowledge of mechanotransduction proteins and their potential relevance to tissue homeostasis in the vocal folds. METHOD: A review of literature surrounding mechanotransduction and tissue homeostasis as it relates to the vocal folds was conducted. Review of the literature included searches of PubMed, Google Scholar, and other various online peer-reviewed sources. Search terms pertained to mechanosensation, mechanotransduction, mechanically activated channels, mechanical cellular regulation, and other associated concepts and terms. Additional literature was identified through the reference lists of identified papers. Findings of this literature review were then applied to known physiology and pathophysiology of the vocal folds in order to speculate on the contribution of mechanically mediated mechanisms within the vocal fold. DISCUSSION AND CONCLUSION: Because the vocal folds are such mechanically active structures, withstanding nearly constant external forces, there is strong support for the idea that mechanically sensitive molecular pathways within the vocal fold tissue play a major role in tissue homeostasis in the presence of these considerable forces. As such, mechanotransduction within the vocal fold should be considered and targeted in future biological studies of vocal fold physiology.

Acute Effects of Systemic Glucocorticoids on the Vocal Folds in a Pre-Clinical Model.

OBJECTIVES/HYPOTHESIS: Systemic glucocorticoids (GC)s are employed to treat various voice disorders. However, GCs have varying pharmacodynamic properties with adverse effects ranging from changes in epithelial integrity, skeletal muscle catabolism, and altered body weight. We sought to characterize the acute temporal effects of systemic dexamethasone and methylprednisolone on vocal fold (VF) epithelial glucocorticoid receptor (GR) nuclear translocation, epithelial tight junction (ZO-1) expression, thyroarytenoid (TA) muscle fiber morphology, and body weight using an established pre-clinical model. We hypothesized dexamethasone and methylprednisolone will elicit changes in VF epithelial GR nuclear translocation, epithelial ZO-1 expression, TA muscle morphology, and body weight compared to placebo-treated controls. METHODS: Forty-five New Zealand white rabbits received intramuscular injections of methylprednisolone (4.5 mg; n = 15), dexamethasone (450 µg; n = 15), or volume matched saline (n = 15) into the iliocostalis/longissimus muscle for 6 consecutive days. Vocal folds from 5 rabbits from each treatment group were harvested at 1-, 3-, or 7 days following the final injection and subjected to immunohistochemistry for ZO-1 and GR as well as TA muscle fiber cross-sectional area (CSA) measures. RESULTS: Dexamethasone increased epithelial GR nuclear translocation and ZO-1 expression 1-day following injections compared to methylprednisolone (P = .024; P = .012). Dexamethasone and methylprednisolone increased TA CSA 1-day following injections (P = .011). Methylprednisolone decreased body weight 7 days following injections compared to controls (P = .004). CONCLUSIONS: Systemic dexamethasone may more efficiently activate GR in the VF epithelium with a lower risk of body weight loss, suggesting a role for more refined approaches to GC selection for laryngeal pathology.

Protein Substrate Alters Cell Physiology in Primary Culture of Vocal Fold Epithelial Cells.

The basement membrane interacts directly with the vocal fold epithelium. Signaling between the basement membrane and the epithelium modulates gene regulation, differentiation, and proliferation. The purpose of this study was to identify an appropriate simple single-protein substrate for growth of rabbit vocal fold epithelial cells. Vocal folds from 3 New Zealand white rabbits (Oryctolagus cuniculus) were treated to isolate epithelial cells, and cells were seeded onto cell culture inserts coated with collagen I, collagen IV, laminin, or fibronectin. Transepithelial electrical resistance (TEER) was measured, and phase contrast microscopy, PanCK, CK14, and E-cadherin immunofluorescence were utilized to assess for epithelial cell-type characteristics. Further investigation via immunofluorescence labeling was conducted to assess proliferation (Ki67) and differentiation (Vimentin). There was a significant main effect of substrate on TEER, with collagen IV eliciting the highest, and laminin the lowest resistance. Assessment of relative TEER across cell lines identified a larger range of TEER in collagen I and laminin. Phase contrast imaging identified altered morphology in the laminin condition, but cell layer depth did not appear to be related to TEER, differentiation, or morphology. Ki67 staining additionally showed no significant difference in proliferation. All conditions had confluent epithelial cells and dispersed mesenchymal cells, with increased mesenchymal cell numbers over time; however, a higher proportion of mesenchymal cells was observed in the laminin condition. The results suggest collagen IV is a preferable basement membrane substrate for in vitro vocal fold epithelial primary cell culture, providing consistent TEER and characteristic cell morphology, and that laminin is an unsuitable substrate for vocal fold epithelial cells and may promote mesenchymal cell proliferation.

Medialization Laryngoplasty: A Review for Speech-Language Pathologists.

Purpose The purpose of this study is to familiarize speech-language-pathologists with the current state of the science regarding medialization laryngoplasty in the treatment of voice disorders, with emphasis on current evidence-based practice, voice outcomes, and future directions for research. Method A literature review was performed in PubMed and Embase using the keywords vocal fold/cord and laryngoplasty, thyroplasty, augmentation, or laryngeal framework. Articles published between 2010 and 2020 were reviewed for data about clinical applications, technical approach, voice-related outcomes, and basic science or clinical innovations with the potential to improve patient care. A synthesis of data was performed from articles meeting the outlined search criteria. Conclusions As key members in the multidisciplinary care of voice disorders, speech-language pathologists need to be informed of current research in medialization laryngoplasty, a procedure commonly used for patients with glottic insufficiency. Advances in anesthetic technique, office-based procedures, and the development of materials with increased bio-tolerability over the past decade have led to innovations in treatment and improved patient outcomes. Recent applications of computational and bioengineering approaches have the potential to provide new directions in the refinement of currently available techniques and the improvement of patient-based treatment outcomes.

Different Vibratory Conditions Elicit Different Structural and Biological Vocal Fold Changes in an In-Vivo Rabbit Model of Phonation.

OBJECTIVE: Vibration of the vocal folds can disrupt the tissue and induce structural, functional, and molecular changes; the presence or absence of contact between the vocal folds during vibration can affect the type and extent of these changes. The purpose of this study was to characterize vocal fold changes following 2 hours of contact phonation or phonation without vibratory contact. METHODS: Six New Zealand white breeder rabbits underwent 120 minutes of phonation with or without vibratory contact, and four served as nonphonated controls. The larynx was exposed and current was applied to the cricothyroids bilaterally to achieve vocal fold adduction while humidified airflow was delivered to induce vocal fold vibration. Laryngeal position, airflow, and stimulation levels were adjusted to obtain phonation with or without contact, and phonation was elicited for 120 minutes. Following excision, larynges were stained using Hematoxylin & Eosin, Elastica van Gieson, and Grocott's Methenamine Silver, or labeled with immunofluorescent markers for E-cadherin, CD31, CD11b, and Vimentin. All images were captured using a Nikon 90i microscope and analyzed using ImageJ. RESULTS: Differences between vibratory conditions and control samples were observed. There was more extensive epithelial thinning, reduced epithelial integrity and increased vascularity in the contact phonation group, while both phonatory groups demonstrated a decreased presence of mucous on the luminal surface and a decrease in elastin band thickness and lamina propria depth. Neither condition showed differences in inflammatory cell presence compared to control tissue. CONCLUSIONS: By showing that these two vibratory conditions result in structural changes of different types and magnitude, we have provided the first empirical evidence that vocal fold tissue is sensitive to differences in forces, and that changes in vibratory pattern can elicit different downstream biological changes within the tissue. The differences described herein are an important step toward understanding the vocal folds' potential for differential response to phonotraumatic damage following different vibratory behaviors.

A Comparison of the Localization of Integral Membrane Proteins in Human and Rabbit Vocal Folds.

OBJECTIVES: This study's objective was to identify and compare the localization of Aquaporin (AQP) 1, 4, 7, Na+/K + -ATPase, E-cadherin, zona occludin (ZO)-1, and occludin in human and rabbit vocal folds (VF)s to inform the design of future studies to explore the function of these proteins in the regulation of VF homeostasis. METHODS: Four human larynges and five New Zealand white rabbit larynges were used. Samples were immunolabeled for primary antibodies against AQP1, AQP4, AQP7, the alpha subunit of Na+/K + -ATPase, E-cadherin, and ZO-1 and occludin and then captured digitally using a Nikon Eclipse 90i microscope and Hamamatsu C10600 Camera. Two raters familiar with human and rabbit VF histology identified positive labeling in tissue structures, including the apical epithelium, basal epithelium/basement membrane, and lamina propria (LP). RESULTS: Samples from both species showed positive labeling for AQP1 in the basal epithelium/basement membrane, superficial LP, and deep/intermediate LP. Aquaporin 4, Aquaporin 7, Na+/K + -ATPase, and E-cadherin were primarily localized to the epithelium of both species. Zona occludin-1 was primarily localized apical epithelium and the superficial LP of both species. Occludin was primarily present in the apical epithelium in rabbit samples but not human. CONCLUSION: These data provide evidence of the presence of key ion transport channels and cell adhesion proteins in human and rabbit VFs. Aquaporin 1, 4, 7, Na+/K + -ATPase, E-cadherin, and ZO-1 were similarly localized in both species. These findings will be useful to investigators interested in the exploration of VF homeostasis and barrier integrity in future studies. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1265-E1271, 2021.

Continuous Rate Infusion of Ketamine Hydrochloride and Dexmedetomidine for Maintenance of Anesthesia during Laryngotracheal Surgery in New Zealand White Rabbits (Oryctolagus cuniculus).

New Zealand white rabbits (Oryctolagus cuniculus) are an established in vivo model for the study of structural and functional consequences of vocal-fold vibration. Research design requires invasive laryngotracheal procedures, and the presence of laryngospasms or pain responses (or both) hinder phonation-related data collection. Published anesthesia regimens report respiratory depression and muscle tone changes and have been unsuccessful in mitigating autonomic laryngeal responses in our protocol. Infusion of ketamine hydrochloride and dexmedetomidine hydrochloride in pediatric medicine provides effective analgesia and sedation for laryngotracheal procedures including intubation and bronchoscopy; however, data evaluating the use of ketamine-dexmedetomidine infusion in rabbits are unavailable. This study reports a new infusion regimen, which was used in 58 male New Zealand white rabbits that underwent a nonsurvival laryngotracheal procedure to induce phonotraumatic vocal-fold injury. Animals were sedated by using ketamine hydrochloride (20 mg/kg IM) and dexmedetomidine (0.125 mg/kg IM). Maintenance anesthesia was provided by using continuous rate intravenous infusion of ketamine hydrochloride (343 µg/kg/min) and dexmedetomidine (1.60 µg/kg/min). A stable plane of anesthesia with no autonomic laryngeal response (laryngospasm) was achieved in 32 of the 58 rabbits (55%). Laryngospasms occurred in 25 of 58 animals (43%) and were controlled in 20 cases (80%) by providing 0.33 mL 2% topical lidocaine, incremental increase in infusion rate, or both. Continuous rate infusion of ketamine hydrochloride-dexmedetomidine with prophylactic topical lidocaine provides a predictable and adjustable surgical plane of anesthesia, with minimal confounding respiratory and autonomic laryngeal responses, during extended-duration laryngotracheal surgery in rabbits. This regimen should be considered as an alternative to injection maintenance for prolonged, invasive procedures.

Vibratory function and healing outcomes after small intestinal submucosa biomaterial implantation for chronic vocal fold scar.

OBJECTIVES/HYPOTHESIS: Vocal fold scar is a major cause of dysphonia, and optimal treatments do not currently exist. Small intestinal submucosa (SIS) is a biomaterial developed for the treatment of a variety of pathologies. The purpose of this study was to investigate the effects of SIS implantation on tissue remodeling in scarred vocal folds using routine staining, immunohistochemistry, and high-speed videoendoscopy (HSV). STUDY DESIGN: Prospective, blinded group analysis. METHODS: Thirteen New Zealand White rabbits underwent a vocal fold scarring procedure followed by microflap elevation with or without SIS implantation. Seven months later, they underwent a phonation procedure with HSV and laryngeal harvest. Alcian blue and elastica van Gieson staining and immunohistochemistry for collagen types I and III were used to evaluate histological healing outcomes. Dynamic functional remodeling of the scarred vocal fold in the presence of SIS implants was evaluated using HSV imaging to capture restoration of vibratory amplitude, amplitude ratio, and left-right phase symmetry. RESULTS: Density of collagen I was significantly decreased in SIS versus microflap-treated vocal folds. No differences were found between groups for hyaluronic acid, elastin, or collagen type III. Organization of elastin in the subepithelial region appeared to affect amplitude of vibration and the shape of the vocal fold edge. CONCLUSIONS: SIS implantation into chronic scar reduced the density of collagen I deposits. There was no evidence of a negative impact or complication from SIS implantation. Regardless of treatment type, organization of elastin in the subepithelial region may be important to vibratory outcomes. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:901-908, 2018.

Recovery of Vocal Fold Epithelium after Acute Phonotrauma.

We investigated the timeline of tissue repair of vocal fold epithelium after acute vibration exposure using an in vivo rabbit model. Sixty-five New Zealand white breeder rabbits were randomized to 120 min of modal- or raised-intensity phonation. After the larynges were harvested at 0, 4, 8, and 24 h, and at 3 and 7 days, the vocal fold tissue was evaluated using electron microscopy and quantitative real-time polymerase chain reaction. There was an immediate decrease in the microprojection depth and height following raised-intensity phonation, paired with upregulation of cyclooxygenase-2. This initial 24-h period was also characterized by the significant downregulation of junction proteins. Interleukin 1ß and transforming growth factor ß1 were upregulated for 3 and 7 days, respectively, followed by an increase in epithelial cell surface depth at 3 and 7 days. These data appear to demonstrate a shift from inflammatory response to the initiation of a restorative process in the vocal fold epithelium between 24 h and 3 days. Despite the initial damage from raised-intensity phonation, the vocal fold epithelium demonstrates a remarkable capacity for the expeditious recovery of structural changes from transient episodes of acute phonotrauma. While structurally intact, the return of functional barrier integrity may be delayed by repeated episodes of phonotrauma and may also play an important role in the pathophysiology of vocal fold lesions.

In vivo measurement of vocal fold surface resistance.

OBJECTIVES/HYPOTHESIS: A custom-designed probe was developed to measure vocal fold surface resistance in vivo. The purpose of this study was to demonstrate proof of concept of using vocal fold surface resistance as a proxy of functional tissue integrity after acute phonotrauma using an animal model. STUDY DESIGN: Prospective animal study. METHODS: New Zealand White breeder rabbits received 120 minutes of airflow without vocal fold approximation (control) or 120 minutes of raised intensity phonation (experimental). The probe was inserted via laryngoscope and placed on the left vocal fold under endoscopic visualization. Vocal fold surface resistance of the middle one-third of the vocal fold was measured after 0 (baseline), 60, and 120 minutes of phonation. After the phonation procedure, the larynx was harvested and prepared for transmission electron microscopy. RESULTS: In the control group, vocal fold surface resistance values remained stable across time points. In the experimental group, surface resistance (X% ± Y% relative to baseline) was significantly decreased after 120 minutes of raised intensity phonation. This was associated with structural changes using transmission electron microscopy, which revealed damage to the vocal fold epithelium after phonotrauma, including disruption of the epithelium and basement membrane, dilated paracellular spaces, and alterations to epithelial microprojections. In contrast, control vocal fold specimens showed well-preserved stratified squamous epithelia. CONCLUSIONS: These data demonstrate the feasibility of measuring vocal fold surface resistance in vivo as a means of evaluating functional vocal fold epithelial barrier integrity. Device prototypes are in development for additional testing, validation, and for clinical applications in laryngology. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E364-E370, 2017.

Structurally and functionally characterized in vitro model of rabbit vocal fold epithelium.

In this paper, we describe a method for primary culture of a well differentiated electrically tight rabbit vocal fold epithelial cell multilayer and the measurement of transepithelial electrical resistance (TEER) for the evaluation of epithelial barrier function in vitro. Rabbit larynges were harvested and enzymatically treated to isolate vocal fold epithelial cells and to establish primary culture. Vocal fold epithelial cells were co-cultured with mitomycin C-treated feeder cells on collagen-coated plates. After 10-14 days in primary culture, cells were passaged and cultured until they achieved 70-90% confluence on collagen-coated plates. Epithelial cells were then passaged onto collagen-coated cell culture inserts using 4.5cm2 membrane filters (1.0µm pore size) with 10% fetal bovine serum or 30µg/mL bovine pituitary extract to investigate the effects of growth-promoting additives on TEER. Additional experiments were performed to investigate optimal seeding density (1.1, 2.2, 4.4, or 8.9×105 cells/cm2), the effect of co-culture with feeder cells, and the effect of passage number on epithelial barrier function. Characterization of in vitro cultures was performed using hematoxylin and eosin staining and immunostaining for vocal fold epithelial cell markers and tight junctions. Results revealed higher TEER in cells supplemented with fetal bovine serum compared to bovine pituitary extract. TEER was highest in cells passaged at a seeding density of 2.2×104 cells/cm2, and TEER was higher in cells at passage two than passage three. Ultrastructural experiments revealed a well-differentiated epithelial cell multilayer, expressing the epithelial cell markers CK13, CK14 and the tight junction proteins occludin and ZO-1.

Acute exposure to vibration is an apoptosis-inducing stimulus in the vocal fold epithelium.

Clinical voice disorders pose significant communication-related challenges to patients. The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (30, 60, 120min) or a control group (120min of vocal fold adduction and abduction). Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency. Laryngeal tissue specimens were evaluated for apoptosis and gene transcript and protein levels of TNF-α. Results revealed that terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was significantly higher after 120min of vibration compared to the control. Transmission electron microscopy (TEM) revealed no significant effect of time-dose on the mean area of epithelial cell nuclei. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Our findings suggest that apoptosis can be induced in the vocal fold epithelium after 120min of modal intensity phonation. In contrast, shorter durations of vibration exposure do not result in apoptosis signaling. However, morphological features of apoptosis are not observed using TEM. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases.