Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge.

BACKGROUND: Voice disorders are a worldwide problem impacting human health, particularly for occupational voice users. Avoidance of surface dehydration is commonly prescribed as a protective factor against the development of dysphonia. The available literature inconclusively supports this practice and a biological mechanism for how surface dehydration of the laryngeal tissue affects voice has not been described. In this study, we used an in vivo male New Zealand white rabbit model to elucidate biological changes based on gene expression within the vocal folds from surface dehydration. Surface dehydration was induced by exposure to low humidity air (18.6% + 4.3%) for 8 h. Exposure to moderate humidity (43.0% + 4.3%) served as the control condition. Ilumina-based RNA sequencing was performed and used for transcriptome analysis with validation by RT-qPCR. RESULTS: There were 103 statistically significant differentially expressed genes identified through Cuffdiff with 61 genes meeting significance by both false discovery rate and fold change. Functional annotation enrichment and predicted protein interaction mapping showed enrichment of various loci, including cellular stress and inflammatory response, ciliary function, and keratinocyte development. Eight genes were selected for RT-qPCR validation. Matrix metalloproteinase 12 (MMP12) and macrophage cationic peptide 1 (MCP1) were significantly upregulated and an epithelial chloride channel protein (ECCP) was significantly downregulated after surface dehydration by RNA-Seq and RT-qPCR. Suprabasin (SPBN) and zinc activated cationic channel (ZACN) were marginally, but non-significantly down- and upregulated as evidenced by RT-qPCR, respectively. CONCLUSIONS: The data together support the notion that surface dehydration induces physiological changes in the vocal folds and justifies targeted analysis to further explore the underlying biology of compensatory fluid/ion flux and inflammatory mediators in response to airway surface dehydration.

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.

Dexamethasone Controlled Release on TGF-ß1 Treated Vocal Fold Fibroblasts.

OBJECTIVE: Corticosteroids may be beneficial in treating vocal fold scarring. Current drug delivery methods do not permit controlled corticosteroid release. Here we investigate the effects of poly-lactic-co-glycolic acid (PLGA) microparticles loaded with the corticosteroid dexamethasone in reducing collagen synthesis and inflammation in vocal fold fibroblasts treated with and without TGF-ß1. STUDY DESIGN: Experimental, in vitro study. METHODS: PLGA microparticles of differing molecular weight and terminating moieties were synthesized using a hydrogel template method. The release of dexamethasone was characterized from these microparticles over 4 days. Based on the release studies, ester-terminated low molecular weight PLGA microparticles were loaded with dexamethasone and applied to TGF-ß1 treated vocal fold fibroblasts for 4 days. Quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assays (ELISAs) were used to assess the effects of released dexamethasone on collagen synthesis and inflammatory mediators. RESULTS: COL3A1 and COL1A2 were significantly down-regulated after exposure to ester-terminated low molecular weight PLGA microparticles loaded with dexamethasone. The loaded microparticles also reduced interleukin-6 synthesis. CONCLUSION: These data show promise in using a PLGA microparticle-based delivery system to control dexamethasone release over 4 days. Our findings lay the groundwork for developing more effective treatments for vocal fold scarring.

Vocal fold ion transport and mucin expression following acrolein exposure.

The vocal fold epithelium is exposed to inhaled particulates including pollutants during breathing in everyday environments. Yet, our understanding of the effects of pollutants on vocal fold epithelial function is extremely limited. The objective of this study was to investigate the effect of the pollutant acrolein on two vocal fold epithelial mechanisms: ion transport and mucin (MUC) synthesis. These mechanisms were chosen as each plays a critical role in vocal defense and in maintaining surface hydration which is necessary for optimal voice production. Healthy, native porcine vocal folds (N = 85) were excised and exposed to an acrolein or sham challenge. A 60-min acrolein, but not sham challenge significantly reduced ion transport and inhibited cyclic adenosine monophosphate-dependent, increases in ion transport. Decreases in ion transport were associated with reduced sodium absorption. Within the same timeline, no significant acrolein-induced changes in MUC gene or protein expression were observed. These results improve our understanding of the effects of acrolein on key vocal fold epithelial functions and inform the development of future investigations that seek to elucidate the impact of a wide range of pollutant exposures on vocal fold health.

Bicarbonate availability for vocal fold epithelial defense to acidic challenge.

OBJECTIVES: Bicarbonate is critical for acid-base tissue homeostasis. In this study we investigated the role of bicarbonate ion transport in vocal fold epithelial defense to acid challenges. Acidic insults to the larynx are common in gastric reflux, carcinogenesis and metastasis, and acute inflammation. METHODS: Ion transport was measured in viable porcine vocal fold epithelium. First, 18 vocal folds were exposed to either the carbonic anhydrase antagonist acetazolamide or to vehicle. Second, 32 vocal folds were exposed to either a control buffer or a bicarbonate-free buffer on their luminal or basolateral surface or both. Third, 32 vocal folds were challenged with acid in the presence of bicarbonate-free or control buffer. RESULTS: The vocal fold transepithelial resistance was greater than 300 Ω*cm(2), suggesting robust barrier integrity. Ion transport did not change after exposure to acetazolamide (p > 0.05). Exposure to bicarbonate-free buffer did not compromise vocal fold ion transport (p > 0.05). Ion transport increased after acid challenge. This increase approached statistical significance and was the greatest for the control buffer and for the bicarbonate-free buffer applied to the basolateral surface. CONCLUSIONS: Bicarbonate secretion may contribute to vocal fold defense against acid challenge. Our data offer a potential novel role for bicarbonate as a therapeutic agent to reduce pH abnormalities in the larynx and prevent associated pathological changes.

Reducing the negative vocal effects of superficial laryngeal dehydration with humidification.

OBJECTIVES: Environmental humidification is a simple, cost-effective method believed to reduce superficial laryngeal drying. This study sought to validate this belief by investigating whether humidification treatment would reduce the negative effects of superficial laryngeal dehydration on phonation threshold pressure (PTP). Phonation threshold pressure data analysis may be vulnerable to bias because of lack of investigator blinding. Consequently, this study investigated the extent of PTP analysis reliability between unblinded and blinded investigators. METHODS: Healthy male and female adults were assigned to a vocal fatigue (n = 20) or control group (n = 20) based on their responses to a questionnaire. PTP was assessed after 2 hours of mouth breathing in low humidity (dehydration challenge), following a 5-minute break in ambient humidity, and after 2 hours of mouth breathing in high humidity (humidification). RESULTS: PTP significantly increased following the laryngeal dehydration challenge. After humidification, PTP returned toward baseline. These effects were observed in both subject groups. PTP measurements were highly correlated between the unblinded and blinded investigator. CONCLUSIONS: Humidification may be an effective approach to decrease the detrimental voice effects of superficial laryngeal dehydration. These data lay the foundation for future investigations aimed at preventing and treating the negative voice changes associated with chronic, surface laryngeal drying.

Water Versus Electrolyte Rehydration on Vocal Fold Osmotic and Oxidative Stress Gene Expression.

OBJECTIVES: Systemic dehydration may induce osmotic and oxidative stress in the vocal folds, but our knowledge of the biology and mitigation with rehydration is limited. The purpose of this experiment was to evaluate whether systemic dehydration induces vocal fold oxidative and osmotic stress and to compare the impact of rehydration by water intake versus electrolyte intake on osmotic and oxidative stress-related gene expression. METHODS: Four-month-old male Sprague-Dawley rats (N = 32) underwent water restriction. Rehydration was achieved with ad libitum access to water or electrolytes for 24 hours. Rats were divided into four groups: euhydration control, dehydration-only, dehydration followed by either water or electrolyte rehydration (n = 8/group). Gene expression was assessed via RT2 Gene Expression Profiler arrays. RESULTS: With respect to oxidative stress, 10 genes were upregulated and 2 were downregulated after vocal fold dehydration compared with the euhydrated control. Concerning osmotic stress, six genes were upregulated with dehydration only, six genes were upregulated following rehydration with water, whereas a single gene was upregulated with electrolyte rehydration. All genes with significantly different expression between the rehydration groups showed lower expression with electrolytes compared with water. CONCLUSIONS: The results support a potential role of oxidative and osmotic stresses in vocal folds related to systemic dehydration. The differences in stress-related gene expression in vocal fold tissue between rehydration with electrolytes or water, albeit modest, suggest that both rehydration options offer clinical utility to subjects experiencing vocal fold dehydration with preliminary evidence that electrolytes may be more effective than water in resolving osmotic stress. LEVELS OF EVIDENCE: NA (prospective animal study) Laryngoscope, 2024.

In Vivo Visualization and Quantification of Rat Laryngeal Blood Supply After Hydration Challenge.

OBJECTIVES: Systemic dehydration decreases total body blood volume; however, hemodynamic alterations at the level of local organs, such as the larynx, remain unclear. Here we sought to quantify superior thyroid artery (STA) blood flow after dehydration and rehydration using in vivo magnetic resonance angiography (MRA) and ultrasound imaging in a rat model. METHODS: Male Sprague-Dawley rats (N = 17) were included in this prospective, repeated measures design. Rats first underwent MRA to determine baseline STA cross-sectional area, followed by high-frequency in vivo ultrasound imaging to measure STA blood velocity at baseline. Next, rats were systemically dehydrated (water withholding), followed by rehydration (water ad-lib). Ultrasound imaging was repeated immediately after dehydration and following rehydration. The STA blood velocity and STA cross-sectional area were used to compute STA blood flow. Three rats served as temporal controls for ultrasound imaging. To determine if the challenges to hydration status affected the STA cross-sectional area, four rats underwent only MRA at baseline, dehydration, and rehydration. RESULTS: Systemic dehydration resulted in 10.5% average body weight loss. Rehydration resulted in average body weight gain of 10.9%. Statistically significant reductions were observed in STA mean blood flow rate after dehydration. Rehydration reversed these changes to pre-dehydration levels. No significant differences were observed in STA cross-sectional area with dehydration or rehydration. CONCLUSION: Systemic dehydration decreased blood flow in the superior thyroid artery. Rehydration restored blood flow in the STA. Change in hydration status did not alter the STA cross-sectional area. These preliminary findings demonstrate the feasibility of using ultrasound and MRA to quantify hemodynamic changes and visualize laryngeal blood vessels. LEVEL OF EVIDENCE: NA Laryngoscope, 134:779-785, 2024.

A Review of Factors Associated with Voice Problems in the Fitness Instructor Population.

There is a high prevalence of reported dysphonia symptomology in the fitness instructor population. This is concerning as these reported symptoms of dysphonia, aphonia, and vocal fatigue can significantly compromise quality of life. The purpose of this review is to explore key factors that may contribute to voice problems in the fitness instructor population. Voicing with concurrent phonation and exercise may be influenced by (1) the increased cardiovascular requirement during exercise, (2) the increased cognitive load associated with dual tasking, (3) the altered hydration state associated with prolonged exercise, and (4) the phonatory dose associated with continued voicing with loud background music. This manuscript will explore the literature on these key factors (ie, phonatory dose, dehydration, metabolic bioenergetics, cognitive load, and psychosocial stress) as they pertain to fitness instructors.

Comparative proteomic changes in rabbit vocal folds undergoing systemic dehydration and systemic rehydration.

BACKGROUND: A considerable body of clinical evidence suggests that systemic dehydration can negatively affect voice production, leading to the common recommendation to rehydrate. Evidence for the corrective benefits of rehydration, however, is limited with mixed conclusions, and biological data on the underlying tissue changes with rehydration is lacking. In this study, we used a rabbit model (n = 24) of acute (5 days) water restriction-induced systemic dehydration with subsequent rehydration (3 days) to explore the protein-level changes underlying the molecular transition from euhydration to dehydration and following rehydration using LC-MS/MS protein quantification in the vocal folds. We show that 5-day water restriction led to an average 4.3% decrease in body weight with relative increases in anion gap, Cl-, creatinine, Na+, and relative decreases in BUN, iCa2+, K+, and tCO2 compared to control (euhydrated) animals. A total of 309 differentially regulated (p < 0.05) proteins were identified between the Control and Dehydration groups. We observed a noteworthy similarity between the Dehydration and Rehydration groups, both well differentiated from the Control group, highlighting the distinct timelines of resolution of the clinical symptoms of systemic dehydration and the underlying molecular changes. SIGNIFICANCE: Voice disorders are a ubiquitous problem with considerable economic and psychological impact. Maintenance of proper hydration is commonly prescribed as a general vocal hygiene practice. There is evidence that dehydration negatively impacts phonation, but our understanding of the state of vocal folds in the context of systemic dehydration are limited, particular from a molecular perspective. Further, ours is a novel molecular study of the short-term impact of rehydration on the tissue. Given the relatively minimal difference in vocal fold proteomic profiles between the Dehydration and Rehydration groups, our data demonstrate a complex physiological response to acute systemic dehydration, and highlight the importance of considering persistent underlying molecular pathology despite the rapid resolution of clinical measures. This study sets a foundation for future research to confirm the nature of potential beneficial outcomes of clinical recommendations related to hydration.

Impact of Rehydration Following Systemic Dehydration on Vocal Fold Gene Expression.

OBJECTIVE: Biological data on the beneficial effects of vocal fold rehydration are lacking. This study aimed to examine the effects of acute systemic dehydration on vocal fold gene expression and determine whether rehydration would reverse these changes. METHODS: Male New Zealand White rabbits (N = 24, n = 8/group) provided the animal model. Systemic dehydration was induced by 5 days of water volume restriction. Rehydration was provided by ad-lib water for 3 days following dehydration. Euhydrated rabbits were used as the control group. Vocal fold tissue was dissected. Seventeen genes were selected based on physiological function and role in supporting vocal fold structure, oxidative stress, hemodynamics, and extracellular matrix turnover. Relative gene expression was assessed by RT-qPCR. RESULTS: Rehydration following systemic dehydration can modulate gene expression, with expression patterns suggesting that rehydration reverses dehydration-induced changes in over half of the tested genes. CLIC5 (chloride intracellular channel 5) and EFEMP1 (EGF containing fibulin extracellular matrix protein 1) genes were significantly upregulated in the dehydration group compared with the euhydrated control. A1BG (alpha-1B-glycoprotein) and IL1RAP (interleukin 1 receptor accessory protein) were downregulated by rehydration compared with the dehydration group. CONCLUSION: This study provides molecular evidence for a transcriptional response to rehydration following acute systemic dehydration in the vocal folds. These data are the first to study gene expression following realistic dehydration and rehydration paradigms and provide biological data to support clinical recommendations to increase water intake after acute dehydration. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3499-3505, 2023.

The effects of vocal exertion on lung volume measurements and acoustics in speakers reporting high and low vocal fatigue.

PURPOSE: Vocal exertion is common and often results in reduced respiratory and laryngeal efficiency. It is unknown, however, whether the respiratory kinematic and acoustic adjustments employed during vocal exertion differ between speakers reporting vocal fatigue and those who do not. This study compared respiratory kinematics and acoustic measures in individuals reporting low and high levels of vocal fatigue during a vocal exertion task. METHODS: Individuals reporting low (N = 20) and high (N = 10) vocal fatigue participated in a repeated measures design study over 2 days. On each day, participants completed a 10-minute vocal exertion task consisting of repeated, loud vowel productions at elevated F0 sustained for maximum phonation time. Respiratory kinematic and acoustic measures were analyzed on the 1st vowel production (T0), and the vowels produced 2 minutes (T2), 5 minutes (T5), 7 minutes (T7), and 10 minutes (T10) into the vocal exertion task. Vowel durations were also measured at each time point. RESULTS: No differences in respiratory kinematics were observed between low and high vocal fatigue groups at T0. As the vocal exertion task progressed (T2-T10), individuals reporting high vocal fatigue initiated phonation at lower lung volumes while individuals with low vocal fatigue initiated phonation at higher lung volumes. As the exertion task progressed, total lung volume excursion decreased in both groups. Differences in acoustic measures were observed, as individuals reporting high vocal fatigue produced softer, shorter vowels from T0 through T10. CONCLUSIONS: Individuals reporting high vocal fatigue employed less efficient respiratory strategies during periods of increased vocal demand when compared with individuals reporting low vocal fatigue. Individuals reporting high vocal fatigue had shorter maximum phonation time on loud vowels. Further study should examine the potential screening value of loud maximum phonation time, as well as the clinical implications of the observed respiratory patterns for managing vocal fatigue.

Determining Medical Urgency of Voice Disorders Using Auditory-Perceptual Voice Assessments Performed by Speech-Language Pathologists.

OBJECTIVE: This study examined whether speech-language pathologist auditory-perceptual voice assessments can predict the medical urgency of voice disorders. METHODS: Twenty speech-language pathologists (SLPs) evaluated 25 voice samples recorded during initial voice evaluations. Voice samples represented a range of dysphonia severity (mild-severe) balanced across patient diagnoses. Diagnoses included: benign lesions, laryngeal cancer, non-organic voice disorders, laryngeal edema (associated with LPR), and laryngeal paralysis or paresis. Laryngeal cancer and severe unilateral laryngeal paralysis were considered urgent disorders. While blinded to patient information, SLPs rated severity of voice quality, predicted patient diagnosis, and determined whether the patient should be seen urgently by a laryngologist. SLPs were then given basic medical history information and rated medical urgency of voice disorder a second time. RESULTS: On average, SLPs correctly identified 65% of urgent voices and 87% of nonurgent voices when blinded to patient information. Accuracy improved significantly to 86% for urgent voices with medical history information (P < .001) and decreased to 77% for nonurgent voices. Accuracy was better when severity of voice quality was severe for urgent voices and mild for nonurgent voices (P < .001). SLPs indicated that patient smoking history and severity of dysphonia were most influential in their decision making. Diagnostic accuracy of auditory-perceptual assessments was poor. CONCLUSIONS: SLPs identified 86% of medically urgent voice disorders when auditory perceptual assessments were combined with medical history information. Further work is needed to determine what medical history information is most crucial to rating accuracy and what speech tasks might best separate urgent and nonurgent patients.

The role of systemic dehydration in vocal fold healing: Preliminary findings.

Rationale: Systemic dehydration negatively alters the expression of vocal fold inflammatory and cell junction markers. These biological changes can have downstream effects on the healing processes of injured vocal folds. In the dermis, reduced hydration prolongs inflammation and delays healing. It is unknown whether this biological effect is observed in vocal fold tissue. Objective: To investigate the effects of systemic dehydration on vocal fold healing outcomes following acute, bilateral vocal fold injury in a rodent model. Methods: Eighteen systemic dehydrated and 18 euhydrated adult male Sprague Dawley rats experienced bilateral vocal fold injuries or no injury (N = 9/group). Vocal fold gene expression levels of inflammatory mediators and epithelial cell junction markers were measured 24 h post-injury. Results: Pro-inflammatory gene markers (IL-1ß; TNF-α) were differentially expressed in response to systemic dehydration with vocal fold injury compared to non-injury. Epithelial cell junction markers (Cadherin-3, Desmoglein-1) also exhibited divergent trends following systemic dehydration, but these data were not statistically significant. Conclusions: Systemic dehydration may affect cellular vocal fold healing processes within 24 h. These findings lay the groundwork for further investigation of how hydration status can affect vocal fold tissue recovery and influence clinical care.

Furosemide-induced systemic dehydration alters the proteome of rabbit vocal folds.

Whole-body dehydration (i.e., systemic dehydration) leads to vocal fold tissue dehydration. Furosemide, a common diuretic prescribed to treat hypertension and edema-associated conditions, induces systemic dehydration. Furosemide also causes voice changes in human speakers, making this method of systemic dehydration particularly interesting for vocal fold dehydration studies. Our objective was to obtain a comprehensive proteome of vocal folds following furosemide-induced systemic dehydration. New Zealand White rabbits were used as the animal model and randomly assigned to euhydrated (control) or furosemide-dehydrated groups. Systemic dehydration, induced by injectable furosemide, was verified by an average body weight loss of -5.5% and significant percentage changes in blood analytes in the dehydrated rabbits compared to controls. Vocal fold specimens, including mucosa and muscle, were processed for proteomic analysis using label-free quantitation LC-MS/MS. Over 1600 proteins were successfully identified across all vocal fold samples; and associated with a variety of cellular components and ubiquitous cell functions. Protein levels were compared between groups showing 32 proteins differentially regulated (p ≤ 0.05) in the dehydrated vocal folds. These are mainly involved with mitochondrial translation and metabolism. The downregulation of proteins involved in mitochondrial metabolism in the vocal folds suggests a mechanism to prevent oxidative stress associated with systemic dehydration. SIGNIFICANCE: Voice disorders affect different population demographics worldwide with one in 13 adults in the United States reporting voice problems annually. Vocal fold systemic hydration is clinically recognized for preventing and treating voice problems and depends on optimal body hydration primarily achieved by water intake. Herein, we use the rabbit as a translatable animal model, and furosemide as a translatable method of systemic dehydration, to reveal a comprehensive proteomic profile of vocal fold mucosa and muscle in response to systemic dehydration. The significant subset of proteins differentially regulated due to furosemide-induced dehydration offer novel insights into the molecular mechanisms of systemic dehydration in the vocal folds. These findings also deepen our understanding of changes to tissue biology after diuretic administration.

Eliciting and Characterizing Porcine Vocalizations: When Pigs Fly.

BACKGROUND/OBJECTIVES: While voice-related therapeutic interventions are often researched preclinically in the porcine model, there are no well-established methods to induce porcine glottic phonation. Described approaches, such as training animals to phonate for positive reinforcement are time-consuming and plagued by inherent variability in the type of phonation produced and contamination of background noise. Thus, a reliable method of assessing glottic phonation in the porcine model is needed. METHODS: In this study, we have created a novel pulley-based apparatus with harness for "pig-lifting" with surrounding acoustic insulation and high-directional microphone with digital recorder for recording phonation. Praat and Matlab were used to analyze all porcine vocalizations for fundamental frequency (F0), intensity, duration of phonation and cepstral peak prominence (CPP). Glottic phonation was detected using F0 (≥2000 hz), duration (≥3 seconds) and researcher perceptual judgment. Partial-glottic phonations were also analyzed. Reliability between researcher judgment and acoustic measures for glottic phonation detection was high. RESULTS: Acoustic analysis demonstrated that glottic and partial-glottic phonation was consistently elicited, with no formal training of the minipigs required. Glottic vocalizations increased with multiple lifts. Glottic phonation continued to be elicited after multiple days but became less frequent. Glottic and partial-glottic phonations had similar CPP values over the 6 experimental days. CONCLUSION: Our cost-effective, reliable method of inducing and recording glottic phonation in the porcine model may provide a cost effective, preclinical tool in voice research.

Restoration Strategies Following Short-Term Vocal Exertion in Healthy Young Adults.

Purpose This study aims to investigate the effects of a 10-min vocal exertion task on voice and respiratory measures, to determine whether restorative strategies can mitigate these effects after cessation of exertion, and to assess whether these strategies continue to reduce these detrimental effects when vocal exertion is resumed. Method A prospective, repeated-measures design was used. On consecutive days, 20 participants (equal men and women) completed two vocal exertion tasks separated by 10 min of restoration strategies: vocal rest or controlled phonation (low-level tissue mobilization using straw phonation). Voice and respiratory data were collected at baseline, following the first exertion task, after restoration strategies, and after the second exertion task. Outcome measures included (a) vocal effort, (b) phonation threshold pressure, (c) maximum and minimum fundamental frequencies, (d) cepstral peak prominence of connected speech, (e) lung volume initiation and termination, (f) percent vital capacity expended per syllable, and (g) number of syllables per breath group. Results A worsening of phonation threshold pressure (p < .001), vocal effort (p < .001), and increase of minimum fundamental frequency (p = .007) were observed after vocal exertion. Lung volume initiation (p < .001) and lung volume termination (p < .001) increased. These changes were largely reversed by restoration strategies, but only controlled phonation prevented exertion-induced changes in respiratory kinematic measures on a subsequent vocal exertion task. Conclusions Exertion-induced voice changes occur rapidly and may be mitigated by either controlled phonation or vocal rest. Controlled phonation is recommended as a superior strategy due to evidence of a protective effect on a successive vocal exertion task.

Mitigating the Effects of Acute Vocal Exertion in Individuals With Vocal Fatigue.

OBJECTIVES/HYPOTHESIS: To investigate the effects of acute vocal exertion on individuals with vocal fatigue and to determine whether semi-occluded vocal tract exercises (SOVTEs) are more effective than vocal rest in mitigating acute effects. STUDY DESIGN: Prospective, repeated-measures design. METHODS: On consecutive days, 10 individuals (6 males, 4 females) with scores indicating vocal fatigue on the Vocal Fatigue Index completed two 10-minute vocal exertion tasks. Vocal rest or SOVTEs were interspersed in counterbalanced order between exertion tasks. Respiratory kinematic, acoustic, aerodynamic, and self-perceptual measures were collected at baseline, following vocal exertion, following SOVTE/vocal rest, and following the second exertion task. RESULTS: Acute vocal exertion worsened phonation threshold pressure (P < .001) and vocal effort (P < .001) and reduced maximum fundamental frequency (P < .001). Speech was terminated at lower lung volumes following vocal exertion (decreased lung volume termination [LVT], P < .001). Exertion-induced changes in vocal effort and LVT were significantly reversed by both vocal rest and SOVTE. Detrimental changes in voice measures reoccurred following the second vocal exertion task. SOVTE and vocal rest protected against changes in respiratory kinematics when vocal exertion was resumed. CONCLUSIONS: Vocal exertion impacted laryngeal, respiratory, and self-perceptual measures in individuals with vocal fatigue. Both SOVTE and vocal rest partially mitigated changes in voice measures and prompted more efficient respiratory strategies that were maintained when vocal exertion resumed. These data increase our understanding of how individuals with vocal fatigue respond to vocal exertion tasks and offer preliminary guidance for optimal clinical recommendations. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:2732-2739, 2021.

Dehydration and Estrous Staging in the Rat Larynx: an in vivo Prospective Investigation.

OBJECTIVE: This novel study sought to untangle the association between hydration state and the estrous cycle in the vocal folds, since the voice is reported to negatively change in speakers during the estrous cycle and with dehydration. We hypothesized that there would be alterations in vocal fold tissue morphology depending on hydration state and that these changes would vary with the estrous cycle. STUDY DESIGN: Prospective, in vivo study design. METHODS: Female Sprague Dawley rats (n = 30) were used in this study. Sixteen rats were systemically dehydrated to an average of 10% reduction in body weight by withholding water (range of body weight loss: 8%-13%). Fourteen rats were assigned to euhydrated, control condition. Estrous stage of female Sprague Dawley rats (n = 30) was determined via cytological evaluation of vaginal smears. Following euthanization, larynges were prepared for histological staining with hematoxylin and eosin, Masson's trichrome and alcian blue (pH 2.5). To quantify hyaluronan, alcian blue staining was completed pre- and posthyaluronidase incubation. The change in staining percent was quantified with image analysis algorithms and reported as the hyaluronan quantity. Relative collagen distribution (index of dehydration), hyaluronan quantity, and tissue morphology were the outcome measures. RESULTS: Systemic dehydration was associated with changes in hyaluronan quantity in the rat vocal fold lamina propria. Dehydration did not significantly affect the collagen distribution nor the tissue morphology. Estrous stage alone does not impact the quantity of vocal fold hyaluronan, alter tissue morphology, or change collagen distribution. CONCLUSION: Decreases in hyaluronan quantity in the lamina propria of the rat vocal fold may play a role in tissue fluid balance during systemic dehydration. Future studies will expand this work to investigate additional components of the vocal fold extracellular matrix to fully elucidate the impact of hydration state on the vocal fold.