The Impact of Vocal Task on Voice Acoustics, Effort and Discomfort Following Submandibular Neuromuscular Electrical Stimulation in Healthy Adults.

BACKGROUND: Neuromuscular electrical stimulation (NMES) offers a potential adjuvant to traditional voice therapy for individuals with dysphonia. The type of vocal task to implement in conjunction with electrical stimulation to achieve maximal therapeutic benefit is unknown. The purpose of this study was to elucidate the impact of tasks on voice outcomes. METHODS: Nineteen vocally-healthy adult females, between 23 and 27 years of age (Ave: 23.8, SD: 1.13), participated in the study. 15 participants completed all three 30-minute sessions, and four completed at least one session. NMES was paired with three different voice conditions: high-pitched hum, low-pitched hum, and comfortable-pitched hum. Acoustic (average fundamental frequency and loudness; perturbation (jitter, shimmer, noise to harmonic ratio); Cepstral Spectral Index of Dysphonia; pitch range), perceived phonatory effort, and discomfort (delayed onset muscle soreness) measures were compared across conditions. RESULTS: Eight participants experienced discomfort following NMES. Three participants withdrew from the study due to discomfort, and one withdrew due to an unrelated oral surgery. NMES paired with high-pitch humming resulted in increased average fundamental frequency during sustained phonation and reading tasks, and increased Cepstral Spectral Index of Dysphonia during sustained phonation. Low-pitch humming resulted in a decreased noise to harmonic ratio. No statistically significant changes in perceived phonatory effort were noted. CONCLUSION: Almost half of the participants reported temporary discomfort. Task-specific differences in some outcomes were noted indicating that the nature of voice task performed with NMES must be considered when examining the impact of NMES on voice. Vocal tasks can impact discomfort and acoustic vocal outcomes of NMES.

Methodology for the establishment of primary porcine vocal fold epithelial cell cultures.

OBJECTIVE: A current lack of methods for epithelial cell culture significantly hinders our understanding of the role of the epithelial and mucus barriers in vocal fold health and disease. Our first objective was to establish reproducible techniques for the isolation and culture of primary porcine vocal fold epithelial cells. Our second objective was to evaluate the functional significance of cell cultures using an in vitro exposure to an inflammatory cytokine. METHODS: Epithelial cells were isolated from porcine vocal folds and expanded in culture. Characterization of cultures was completed by immunostaining with markers for pan-cytokeratin (epithelial cells), vimentin (stromal cells), von Willebrand factor (endothelial cell), and MUC1 and MUC4 (mucin) glycoproteins. Established epithelial cell cultures were then exposed to the inflammatory cytokine tumor necrosis factor alpha (TNF-α) for 24-hours, and transcript expression of MUC1 and MUC4 was evaluated. RESULTS: Reproducible, porcine vocal fold epithelial cell cultures, demonstrating cobblestone appearance characteristic of the typical morphology of epithelial cell cultures were created. Cells showed positive staining for pan-cytokeratin with limited expression of vimentin and von Willebrand factor. Epithelial cells also expressed MUC1 and MUC4. TNF-α significantly increased transcript expression of MUC4. CONCLUSION: Here, we present the first report of successful culture of primary porcine vocal fold epithelial cells. Cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus as well as the effects of common challenges, including inflammatory cytokines, on these barriers. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E355-E364, 2019.

Derivation of Epithelial Cells from Human Embryonic Stem Cells as an In Vitro Model of Vocal Mucosa.

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here, we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology, gene expression, and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and, therefore, offers a promising approach to studying vocal fold biology and biomechanics in health and disease.

Epidermal growth factor mediated healing in stem cell-derived vocal fold mucosa.

BACKGROUND: The goal of vocal fold wound healing is the reconstitution of functional tissue, including a structurally and functionally intact epithelium. Mechanisms underlying reepithelialization in vocal folds are not known, although it is suspected that healing involves the interplay between several growth factors. We used a three-dimensional human embryonic stem cell-derived model of vocal fold mucosa to examine the effects of one growth factor, exogenous epidermal growth factor (EGF), on wound healing. MATERIALS AND METHODS: A scratch wound was created in the in vitro model. Rate of wound healing, epidermal growth factor receptor (EGFR) activation, and cell proliferation after injury were analyzed with and without application of both exogenous EGF and an EGFR inhibitor, gefitinib. RESULTS: Wound repair after injury was significantly hastened by application of exogenous EGF (13.3 µm/h, ± 2.63) compared with absence of exogenous EGF (7.1 µm/h ± 2.84), but inhibited with concurrent addition of Gefitinib (5.2 µm/h, ± 2.23), indicating that EGF mediates wound healing in an EGFR-dependent manner. Immunohistochemistry revealed that EGFR activation occurred only in the presence of exogenous EGF. Although not statistically significant, increased density of Ki67 staining in the epithelium adjacent to the scratch wound was observed after treatment with EGF, suggesting a tendency for exogenous EGF to increase epithelial cell proliferation. CONCLUSIONS: Exogenous EGF increases the rate of wound healing in an EGFR-dependent manner in a three-dimensional stem cell-derived model of vocal fold mucosa. This model of wound healing can be used to gain insight into the mechanisms that regulate vocal fold epithelial repair after injury.

Epithelial cells are active participants in vocal fold wound healing: an in vivo animal model of injury.

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFß1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFß1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFß1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFß1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.

Structural and functional vocal fold epithelial integrity following injury.

OBJECTIVES/HYPOTHESIS: An intact epithelium is an important part of vocal fold defense. Damage to the epithelium can compromise vocal fold homeostasis and protection of the host tissue from viral and bacterial invasion. Elucidating the effects of damage on epithelial architectural and barrier integrity provides insight into the role of epithelium in protecting vocal folds. Using an animal model, we evaluated the time course of structural and functional epithelial restoration following injury. STUDY DESIGN: Prospective, controlled animal study. METHODS: Forty rats underwent surgery to remove vocal fold mucosa unilaterally. Larynges were harvested at five time intervals between 3 to 90 days postinjury and were prepared for histological and permeability analyses. RESULTS: Rapid restoration of structural integrity was demonstrated by return of a multilayerd epithelium, intercellular junctions, and basement membrane at 5 days postinjury. Atypical epithelial permeability was observed up to 5 weeks postinjury. CONCLUSION: Restoration of epithelial barrier integrity lags epithelial structural restoration. Consequently, epithelial regeneration cannot be equated with return of functional barrier integrity. Rather, ongoing leakiness of regenerated epithelium indicates that vocal folds remain at risk for damage, pathogen invasion, and remodeling postinjury. LEVEL OF EVIDENCE: N/A. Laryngoscope, 124:2764-2769, 2014.

Classification for animal vocal fold surgery: resection margins impact histological outcomes of vocal fold injury.

OBJECTIVES/HYPOTHESIS: Extent of vocal fold injury impacts the nature and timing of wound healing and voice outcomes. However, depth and extent of the lesion created to study wound healing in animal models vary across studies, likely contributing to different outcomes. Our goal was to create a surgery classification system to enable comparison of postoperative outcomes across animal vocal fold wound-healing studies. STUDY DESIGN: Prospective, controlled animal study. METHODS: Rats underwent one of three types of unilateral vocal fold surgeries classified by depth and length of resection. The surgeries were: for subepithelial injury, resection of epithelium and superficial layer of the lamina propria at the midmembranous portion of the vocal fold; for transmucosal injury, resection of epithelium and lamina propria; and for transmuscular injury, resection of epithelium, lamina propria, and superficial portion of the vocalis muscle. Wound healing was evaluated histologically at various time points up to 35 days postinjury. RESULTS: Complete healing occurred by 14 days postsurgery for subepithelial injury, and by day 35 for transmucosal injury. Injury remained present at day 35 for transmuscular injury. CONCLUSIONS: Timing and completeness of healing varied by extent and depth of resection. Scarless healing occurred rapidly following subepithelial injury, whereas scarring was observed at 5 weeks after transmuscular injury. The proposed classification system may facilitate comparison of surgical outcomes across vocal fold wound-healing studies. LEVELS OF EVIDENCE: N/A.

Vocal fold epithelial barrier in health and injury: a research review.

PURPOSE: Vocal fold epithelium is composed of layers of individual epithelial cells joined by junctional complexes constituting a unique interface with the external environment. This barrier provides structural stability to the vocal folds and protects underlying connective tissue from injury while being nearly continuously exposed to potentially hazardous insults, including environmental or systemic-based irritants such as pollutants and reflux, surgical procedures, and vibratory trauma. Small disruptions in the epithelial barrier may have a large impact on susceptibility to injury and overall vocal health. The purpose of this article is to provide a broad-based review of current knowledge of the vocal fold epithelial barrier. METHOD: A comprehensive review of the literature was conducted. Details of the structure of the vocal fold epithelial barrier are presented and evaluated in the context of function in injury and pathology. The importance of the epithelial-associated vocal fold mucus barrier is also introduced. RESULTS/CONCLUSIONS: Information presented in this review is valuable for clinicians and researchers as it highlights the importance of this understudied portion of the vocal folds to overall vocal health and disease. Prevention and treatment of injury to the epithelial barrier is a significant area awaiting further investigation.

Human embryonic stem cell-derived epithelial cells in a novel in vitro model of vocal mucosa.

A satisfactory in vitro model of vocal fold mucosa does not exist, thus precluding a systematic, controlled study of vocal fold biology and biomechanics. We sought to create a valid, reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design, hESCs were differentiated into cells that coexpressed the simple epithelial cell marker, keratin 18 (K18), and the transcription factor, p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air-liquid interface. After that time, the engineered mucosa demonstrated a stratified, squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified, squamous epithelial markers, keratin 13 (K13) and 14 (K14), as well as tight junctions, adherens junctions, gap junctions, and desmosomes. Despite the presence of components critical for epithelial structural integrity, the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity, this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered, terminally differentiated epithelium.

Localization of label-retaining cells in murine vocal fold epithelium.

PURPOSE: Epithelial homeostasis is critical for vocal fold health, yet little is known about the cells that support epithelial self-renewal. As a known characteristic of stem cells is that they are slow-cycling in vivo, the purpose of this prospective controlled study was to identify and quantify slow-cycling cells or putative stem cells in murine vocal fold epithelium. METHOD: Twelve mice were administered daily intraperitoneal injections of a nucleotide dye, bromodeoxyuridine (BrdU), over 7 consecutive days. Under this pulse-chase paradigm, slow-cycling cells retain the dye (label-retaining cells; LRCs) while more rapidly cycling cells lose dye to dilution during multiple cell divisions. The percentage of label-retaining cells (%LRCs) was calculated following a chase period of 2, 4, and 8 weeks postinjections. RESULTS: The %LRCs decreased significantly from 9.4% at 2 weeks to 3.1% at 8 weeks following injections (p < .05). No statistically significant differences in the quantity of BrdU-positive cells were measured between the anterior, mid-membranous, or cartilaginous regions of the vocal fold (p > .05). CONCLUSION: These findings are consistent with the presence and first report of a small population of putative stem cells along the length of murine vocal fold epithelium.

The role of hydration in vocal fold physiology.

PURPOSE OF REVIEW: Increased vocal fold hydration is a popular target in the prevention and management of voice disorders. Current intervention strategies focus on enhancing both systemic (internal) and superficial (surface) hydration. We review relevant bench and human research on the role of hydration in vocal fold physiology. RECENT FINDINGS: Bench and human studies provide converging evidence that systemic and superficial dehydration are detrimental to vocal fold physiology. Dehydration challenges increase the viscous properties of excised vocal fold tissue. Systemic, superficial, and combined drying challenges increase aerodynamic and acoustic measures of voice production in speakers. Emerging theoretical and clinical data suggest that increasing both systemic and superficial hydration levels may benefit voice production; however, robust evidence for positive outcomes of hydration treatments is lacking. SUMMARY: Increased systemic and superficial vocal fold hydration as a component of vocal hygiene may improve overall health and efficiency of the vocal apparatus. However, continued exploration of biological mechanisms regulating vocal fold hydration is needed to optimize clinical hydration interventions. Specifically, the development of hydration treatments that maximize positive phonatory outcomes will necessitate understanding of the signaling pathways linking systemic and superficial hydration.

A meta-analysis of outcomes of hydration intervention on phonation threshold pressure.

OBJECTIVES: Vocal fold hydration is purported to promote optimal biomechanical characteristics of vocal fold mucosa, increase efficiency of vocal fold oscillation, and enhance voice quality. The purpose of this work was to determine the magnitude and consistency of the effect of vocal fold hydration on vocal fold function across published clinical studies. METHODS: We completed a comprehensive meta-analysis of the effects of superficial and systemic vocal fold hydration on phonation threshold pressure (PTP), a measure of efficiency of voice production. RESULTS: We identified 34 studies that examined the effects of hydration on vocal function. Of these studies, 14 examined the effects of hydration on PTP. Nine of these articles met the criteria for inclusion in this analysis. We observed an average effect size of 0.33, indicating that, overall, hydration treatment demonstrated a tendency to reduce PTP. However, this decrease in phonatory effort did not reach significance at the 95% confidence level. The effects of hydration intervention varied considerably across studies (-0.19 to 3.96). We considered that two factors, pitch level of the task and vocal health of participants, may have contributed to this variability in findings. However, our analysis found that these factors could not account for differences in effect size. CONCLUSION: To understand the variability in outcomes across studies, the role of factors that may impact the effects of hydration, such as the amount, type, and duration of intervention, must be determined. Only then can we obtain data to guide best clinical practice for protecting and rehabilitating vocal function.

Vocal fold surface hydration: a review.

Vocal fold surface liquid homeostasis contributes to optimal vocal physiology. In this paper we review emerging evidence that vocal fold surface liquid is maintained in part by salt and water fluxes across the epithelium. Based on recent immunolocalization and electrophysiological findings, we describe a transcellular pathway as one mechanism for regulating superficial vocal fold hydration. We propose that the pathway includes the sodium-potassium pump, sodium-potassium-chloride cotransporter, epithelial sodium channels, cystic fibrosis transmembrane regulator chloride channels, and aquaporin water channels. By integrating knowledge of the regulating mechanisms underlying ion and fluid transport with observations from hydration challenges and treatments using in vitro and in vivo studies, we provide a theoretical basis for understanding how environmental and behavioral challenges and clinical interventions may modify vocal fold surface liquid composition. We present converging evidence that clinical protocols directed at facilitating vocal fold epithelial ion and fluid transport may benefit healthy speakers, those with voice disorders, and those at risk for voice disorders.

The cystic fibrosis transmembrane conductance regulator and chloride-dependent ion fluxes of ovine vocal fold epithelium.

PURPOSE: Ion-driven transepithelial water fluxes participate in maintaining superficial vocal fold hydration, which is necessary for normal voice production. The authors hypothesized that Cl(-) channels are present in vocal fold epithelial cells and that transepithelial Cl(-) fluxes can be manipulated pharmacologically. METHOD: Immunohistochemical assays were used to identify cystic fibrosis transmembrane regulator Cl(-) channels in ovine vocal fold mucosae (n = 2). Electrophysiological responses of vocal fold mucosae (n = 80) to Cl(-) channel inhibitors and secretagogues were evaluated in an ovine model using a randomized controlled experimental design. RESULTS: Cystic fibrosis transmembrane regulator channels were localized to the plasma membranes of epithelial cells. The Cl(-) transport inhibitor, diphenylamine-2-carboxylate, elicited a 30% decrease in mean short-circuit current (I(sc); n = 10). The secretagogue, isobutylmethylxanthine, yielded a 31.7% increase in mean I(sc) (n = 10). Another secretagogue, uridine triphosphate, elicited a 48.8% immediate and 17.3% sustained increase in mean I(sc) (n = 10). No sustained increases occurred following application of secretagogues to mucosae bathed in a low Cl(-) environment (n = 10), suggesting that responses were Cl(-) dependent. CONCLUSIONS: The authors provide structural and functional evidence for the presence of a transepithelial pathway for Cl(-) fluxes. Pharmacological manipulation of this pathway may offer a mechanism for maintaining superficial vocal fold hydration.

Perceptual ratings of vocal characteristics and voicing features in untreated patients with unilateral vocal fold paralysis.

UNLABELLED: This study used visual analog scales to obtain perceptual ratings of features of voice production in subjects with unilateral vocal fold paralysis (UVFP), including clarity of laryngeal articulation, consistency of loudness across the utterance and the voiced/voiceless distinction. Recordings of repeated /i/, /isi/, and /izi/ from subjects diagnosed with UVFP and control subjects were randomly re-recorded, and then rated by five listeners. Significant differences in ratings (Smirnov test, p < 0.01) were found between groups for "aphonia", "severity", "clarity of articulation", "overall loudness", "consistency of loudness" and "amount of effort". Four of five raters agreed on the accuracy of /s/ or /z/ productions for only 54% of the samples from the subjects with UVFP. Voiceless and voiced cognates were equally likely to be rated as inaccurate. Results suggested that these variables were sensitive to changes in voice production resulting from paralysis, and may be useful in measuring treatment outcomes and spontaneous recovery of function. LEARNING OUTCOMES: As a result of reading this manuscript the reader will (1) gain an understanding of types of perceptual scales and how to develop the set of vocal characteristics to be used in distinguishing patients with UVFP and those without, (2) learn which vocal characteristics listeners are able to use to successfully distinguish between patients with UVFP and those without and (3) understand the possible role for perceptual ratings in tracking changes in vocal characteristics in subjects over time following treatment or spontaneous recovery of function.

The role of auditory feedback in sustaining vocal vibrato.

Vocal vibrato and tremor are characterized by oscillations in voice fundamental frequency (F0). These oscillations may be sustained by a control loop within the auditory system. One component of the control loop is the pitch-shift reflex (PSR). The PSR is a closed loop negative feedback reflex that is triggered in response to discrepancies between intended and perceived pitch with a latency of approximately 100 ms. Consecutive compensatory reflexive responses lead to oscillations in pitch every approximately 200 ms, resulting in approximately 5-Hz modulation of F0. Pitch-shift reflexes were elicited experimentally in six subjects while they sustained /u/ vowels at a comfortable pitch and loudness. Auditory feedback was sinusoidally modulated at discrete integer frequencies (1 to 10 Hz) with +/- 25 cents amplitude. Modulated auditory feedback induced oscillations in voice F0 output of all subjects at rates consistent with vocal vibrato and tremor. Transfer functions revealed peak gains at 4 to 7 Hz in all subjects, with an average peak gain at 5 Hz. These gains occurred in the modulation frequency region where the voice output and auditory feedback signals were in phase. A control loop in the auditory system may sustain vocal vibrato and tremorlike oscillations in voice F0.

Vocal warm-up increases phonation threshold pressure in soprano singers at high pitch.

Vocal warm-up is thought to optimize singing performance. We compared effects of short-term, submaximal, vocal warm-up exercise with those of vocal rest on the soprano voice (n = 10, ages 19-21 years). Dependent variables were the minimum subglottic air pressure required for vocal fold oscillation to occur (phonation threshold pressure, Pth), and the maximum and minimum phonation fundamental frequency. Warm-up increased Pth for high pitch phonation (p = 0.033), but not for comfortable (p = 0.297) or low (p = 0.087) pitch phonation. No significant difference in the maximum phonation frequency (p = 0.193) or minimum frequency (p = 0.222) was observed. An elevated Pth at controlled high pitch, but an unchanging maximum and minimum frequency production suggests that short-term vocal exercise may increase the viscosity of the vocal fold and thus serve to stabilize the high voice.