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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. Level of Evidence: NA (prospective animal study) Laryngoscope, 134:4636–4641, 2024 [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Objectives: A holistic understanding of the many ways that systemic dehydration affects vocal fold biology is still evolving. There are also myriad physiologically relevant methodologies to induce systemic dehydration. To untangle the effects of systemic dehydration on vocal fold biology, we need to utilize realistic, clinically translatable paradigms of systemic dehydration in lab animals. Restricted access to water accommodates clinical translation. We investigated whether systemic dehydration via reduced water intake would negatively affect vocal fold biology. Study Design: Prospective, in vivo study design. Methods: Male Sprague Dawley rats (N = 13) were provided 4 mL/100 g of water/day for 5 days, whereas male control rats (N = 8) were given ad lib access to water. Following euthanasia, tissues were processed for histological staining, gene expression, and protein assays. Results: Renin gene expression level in kidneys increased significantly (P ≤.05), validating dehydration. Dehydration induced by restricted water access downregulated the gene expression of interleukin‐1α and desmoglein‐1 (P ≤.05). Hyaluronidase‐2 gene expression increased after dehydration (P ≤.05). The protein level of desmoglein‐1 decreased after dehydration (P ≤.05). Histological analyses suggested decreased hyaluronan (P ≤.05) in the water‐restricted rat vocal fold. Conclusion: Reduced daily water intake for just 5 days impairs vocal fold biology by disrupting inflammatory cytokine release, reducing plasma membrane integrity, and disrupting the hyaluronan network. This is the first study investigating the dehydrating effects of restricted water intake on vocal fold tissue in an in vivo model. Level of Evidence: NA (prospective animal study). Laryngoscope, 131:839–845, 2021 [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: Inhaled pollutants can contact vocal fold tissue and induce detrimental voice changes. Acrolein is a pollutant in cigarette smoke and can also be inhaled during the combustion of fossil fuels, animal fats, and plastics in the environment. However, the vocal fold pathological changes induced by acrolein and the underlying inflammatory pathways are not well understood. These biologic data are needed to understand why voice problems may result from pollutant exposure.Study Design: In vivo prospective design with experimental and control groups.Methods: Sprague-Dawley male rats (N = 36) were exposed to acrolein (3 ppm) or filtered air (control) through a whole-body exposure system for 5 hours/day, for 5 days/week, over 4 weeks. Histopathological changes, presence of edema, expression of proinflammatory cytokines and markers, and the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were investigated.Results: Histological evaluation and quantification demonstrated that subacute acrolein exposure induced significant vocal fold edema. Acrolein exposure also induced epithelial sloughing and cell death. Quantitative polymerase chain reaction showed a significant upregulation of genes encoding interferon regulatory factor and chitinase-3-like protein 3. Western blot revealed a 76.8% increase in phosphorylation of NF-κB P65 after subacute acrolein exposure.Conclusions: These findings suggest that 4-week exposures to 3 ppm acrolein induce vocal fold inflammation manifested as edema, related to the activation of NF-κB signaling. The edema may underlie the voice changes reported in speakers exposed to pollutants.Level Of Evidence: NA Laryngoscope, 129:E313-E317, 2019. [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: Dehydrated vocal folds are inefficient sound generators. Although systemic dehydration of the body is believed to induce vocal fold dehydration, this causative relationship has not been demonstrated in vivo. Here we investigate the feasibility of using in vivo proton density (PD)-weighted magnetic resonance imaging (MRI) to demonstrate hydration changes in vocal fold tissue following systemic dehydration in rats.Study Design: Animal study.Methods: Sprague-Dawley rats (n = 10) were imaged at baseline and following a 10% reduction in body weight secondary to withholding water. In vivo, high-field (7 T), PD-weighted MRI was used to successfully resolve vocal fold and salivary gland tissue structures.Results: Normalized signal intensities within the vocal fold decreased postdehydration by an average of 11.38% ± 3.95% (mean ± standard error of the mean [SEM], P = .0098) as compared to predehydration levels. The salivary glands experienced a similar decrease in normalized signal intensity by an average of 10.74% ± 4.14% (mean ± SEM, P = .0195) following dehydration. The correlation coefficient (percent change from dehydration) between vocal folds and salivary glands was 0.7145 (P = .0202).Conclusions: Ten percent systemic dehydration induced vocal fold dehydration as assessed by PD-weighted MRI. Changes in the hydration state of vocal fold tissue were highly correlated with that of the salivary glands in dehydrated rats in vivo. These preliminary findings demonstrate the feasibility of using PD-weighted MRI to quantify hydration states of the vocal folds and lay the foundation for further studies that explore more routine and realistic magnitudes of systemic dehydration and rehydration.Level Of Evidence: NA. Laryngoscope, 128:E222-E227, 2018. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1.Study Design: In vitro.Methods: Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor β1 (TGFβ1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin.Results: Fibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFβ1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture.Conclusions: These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.Level Of Evidence: NA Laryngoscope, 127:E185-E192, 2017. [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: The study objective was to investigate epithelial changes in response to direct, repeated, acidified pepsin exposures in an in vivo porcine model. We hypothesized that 12 acidified pepsin applications to simulate reflux would elicit a vocal fold response characterized by inflammation, epithelial proliferation, and increased intercellular space, as well as changes in the gene expression of epithelial junctional proteins, ion transporter proteins, and proinflammatory cytokines.Study Design: Prospective, in vivo study.Methods: Pigs received acidified pepsin (pH = 4) or saline (sham) applied directly to vocal folds. Larynges were collected following three exposures per week for 4 weeks. Vocal fold tissue morphology, collagen, and elastin were evaluated histologically. Gene expression of E-cadherin, zona occludens-1, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, interleukin-1β, tumor necrosis factor-α, and interferon-γ were measured. Ultrastructural alterations, epithelial intercellular space diameter, and microridge height were measured using transmission electron microscopy.Results: There were no significant differences in histology, gene transcripts, epithelial ultrastructure, intercellular space, and microridge height after acidified pepsin exposure.Conclusions: Twelve simulated reflux challenges were insufficient to elicit epithelial changes, demonstrating the resistance of healthy vocal folds to direct, repeated acidified pepsin exposures. These data increase our understanding of healthy vocal fold defenses and lay the groundwork for a prospective, uninjured, nonsurgical, laryngopharyngeal reflux model where pigs can be exposed directly to acidified pepsin. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis Ex vivo models are routinely used to investigate the barrier function of the vocal fold epithelium. However, there are limited reports on assays that can be used to investigate the effect of clinically relevant challenges on vocal fold epithelial tissue viability. Our objective was to determine the utility of two assays routinely used in cell culture-a cellular metabolic activity assay and a cell membrane integrity assay-to investigate the viability of ex vivo porcine vocal fold epithelium. Study Design Prospective, ex vivo animal study. Methods Porcine vocal folds were exposed to acrolein, hydrochloric acid, or hydrogen peroxide challenge. An untreated, sham challenge was included as a control. Assays including metabolic activity, cell membrane integrity, and histology were used to determine whether challenges reduced epithelial viability as compared to sham. Results Cell membrane integrity and metabolic activity assays detected reductions in viability following hydrochloric acid and hydrogen peroxide challenges but not acrolein challenge as compared to sham. No challenge produced significant changes in epithelial appearance as evidenced by light microscopy. Conclusions Metabolic activity and cell membrane integrity assays are valuable tools that can be used to evaluate the viability of ex vivo vocal fold epithelial tissue following clinically relevant challenges. As viability is reduced, the ability of epithelial tissue to maintain its barrier function is compromised. Accurate assessment of viability may provide us clues into understanding mechanisms underlying vocal fold epithelial injury and disease. Level of Evidence NA Laryngoscope, 125:E180-E185, 2015 [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis To investigate the effects of increasing time and magnitude doses of vibration exposure on transcription of the vocal fold's junctional proteins, structural alterations, and functional tissue outcomes. Study Design Animal study. Methods 100 New Zealand White breeder rabbits were studied. Dependent variables were measured in response to increasing time doses (30, 60, or 120 minutes) and magnitude doses (control, modal intensity, and raised intensity) of vibration exposure. Messenger RNA expression of occludin, zonula occluden-1 (ZO-1), E-cadherin, β-catenin, interleukin 1β, cyclooxygenase-2, transforming growth factor β-1, and fibronectin were measured. Tissue structural alterations were assessed using transmission electron microscopy (TEM). Transepithelial resistance was used to measure functional tissue outcomes. Results Occludin gene expression was downregulated in vocal folds exposed to 120-minute time doses of raised-intensity phonation, relative to control, and modal-intensity phonation. ZO-1 gene expression was upregulated following a 120-minute time dose of modal-intensity phonation, compared to control, and downregulated after a 120-minute time dose of raised-intensity phonation, compared to modal-intensity phonation. E-cadherin gene expression was downregulated after a 120-minute time dose of raised-intensity phonation, compared to control and modal-intensity phonation. TEM revealed extensive desquamation of the stratified squamous epithelial cells with increasing time and magnitude doses of vibration exposure. A general observation of lower transepithelial resistance measures was made in tissues exposed to raised-intensity phonation compared to all other groups. Conclusions This study provides evidence of vocal fold tissue responses to varying time and magnitude doses of vibration exposure. Level of Evidence NA Laryngoscope, 124:2770-2778, 2014 [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis Vocal fold surface fluid (VFSF) is important in hydration and defense of underlying epithelial cells. The objective of this study was to quantify changes in the ionic composition of VFSF after altering the humidity of inhaled air. We tested the hypothesis that low humidity exposure would increase the concentration of VFSF sodium (Na+) and chloride (Cl−) ions but that high humidity exposure would decrease the concentration of VFSF Na+ and Cl− ions as compared to the low humidity challenge. Study Design Prospective design. Methods Eighteen healthy adults participated in this study. VFSF was collected from each subject at baseline and following exposure to low humidity and high humidity environments. VFSF Na+ concentration was assessed using inductively coupled plasma mass spectrometry. VFSF Cl− concentration was measured with indirect potentiometry. All analyses were completed by personnel blinded to the hypothesis being tested. Results The low humidity environment increased Na+ concentration in the majority of the subjects. Data for changes in Cl− concentrations were variable. Overall the data did not reach statistical significance ( P > .05). Subjective impressions suggested that VFSF collection was more difficult in low humidity as compared to the high humidity and baseline conditions. Conclusions This study is the first attempt to measure the ionic concentration of VFSF. The results from the current study have important implications for future programmatic research quantifying the effects of pollutants and laryngopharyngeal reflux on VFSF composition, epithelial hydration, and vocal fold defense. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: Vocal fold epithelium is exposed to reactive oxygen species from the inhaled environment and from tissue inflammation. The objective of this study was to explore the functional and structural consequences of reactive oxygen species exposure on vocal fold epithelium. Study Design: In vitro, prospective study design. Methods: Hydrogen peroxide (H2O2), a common reactive oxygen species, was utilized in this study. Freshly excised, viable porcine vocal fold epithelia (N = 32) were exposed to H2O2 or sham challenge for 2 hours. Electrophysiology, western blotting, and light microscopy were used to quantify the functional and structural effects of reactive oxygen species on vocal fold epithelia. Results: Exposure to reactive oxygen species did not significantly alter transepithelial resistance. There was a small, nonsignificant trend for decreased concentration of epithelial junctional complex protein with reactive oxygen species challenge. Minimal changes to the gross structural appearance of vocal fold epithelia were also noted. Conclusions: The stratified squamous epithelia of the vocal folds effectively defend against an acute reactive oxygen species challenge. The current study lays the groundwork for future investigations on the effects of reactive oxygen species on vocal fold epithelia that are compromised from phonotrauma. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: Given the recent emergence of encouraging efficacy data regarding the utility of intralesional glucocorticoid (GC) injection for a variety of vocal fold pathologies, we sought to describe the location and expression pattern of the GC receptors within the vocal folds and quantify the effects of GCs on vocal fold fibroblasts. Study Design: In vitro, in vivo. Methods: Immunolocalization of the GC receptor was performed on normal rat vocal fold tissue. Receptor expression was also assayed in our human vocal fold fibroblast cell line. These cells were then treated with exogenous dexamethasone (DM) to quantify the effects of GCs on receptor expression, proliferation, transforming growth factor (TGF)-β-induced collagen secretion, and matrix protease synthesis. Results: Positive immunostaining for the GC receptor was found throughout the vocal fold with particularly strong staining in the epithelium and capillaries. Human vocal fold fibroblasts constitutively express the GC receptor, but this expression decreased in response to exogenous DM. DM also decreased fibroblast proliferation and TGF-β-induced collagen synthesis. DM also abrogated TGF-β-mediated effects on enzymes related extracellular matrix turnover. Conclusions: Our data are the first to provide mechanistic insight regarding the recently published favorable data regarding the utility of GCs in patients with vocal fold scar. Although further investigation is warranted, both the accessibility of this class of agents and the amenability to office-based procedures are likely to direct patient care models. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: In response to chronic cigarette smoke exposure, a subset of patients present with edematous vocal folds, characteristically referred to as Reinke’s edema. This phenotype differs from the tissue changes associated with prolonged smoke exposure in the lower airway, and the mechanism underlying Reinke’s edema remains poorly described. We hypothesize that the effects of smoke are diffuse and involve both the epithelium and mucosa. Study Design: In vitro, ex vivo experiment. Methods: Transepithelial resistance (RT) was quantified in an ex vivo, viable, porcine vocal fold model. Excised tissue was exposed to cigarette smoke condensate (CSC) and RT was computed at baseline and 1 and 4 hours after exposure. In vitro, human vocal fold fibroblasts were exposed to CSC. Cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase-1, and 15-hydroxyprostaglandin dehydrogenase mRNA expression were assessed at 4 hours. Prostaglandin E2 (PGE2) synthesis was quantified via immunoassay following 24 hours of CSC exposure. Results: CSC had no effect on RT. CSC did, however, induce COX-2 mRNA expression as well as its downstream lipid mediator PGE2. PGE2 metabolism appears to be regulated via both synthetic and degradative enzymes in response to cigarette smoke. Conclusions: In vitro, CSC initiates an inflammatory response in vocal fold fibroblasts. However, in isolation, the epithelial resistance is not altered by CSC, at least acutely. These data may suggest a role for the interaction between the inflammatory response in the mucosa and compromised epithelial barrier function, as has been shown in other tissues. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: We investigated the hypothesis that 30 minutes of raised intensity phonation alters transcript levels of vocal fold intercellular tight junction proteins and disrupts the vocal fold epithelial barrier. Study Design: Prospective animal study. Methods: Eighteen New Zealand white breeder rabbits were randomly assigned to receive 30 minutes of raised intensity phonation or approximation of the vocal folds without phonation. Quantitative polymerase chain reaction (qPCR) was used to investigate transcript levels of the epithelial intercellular tight junction proteins, occludin and zonula occludin-1 (ZO-1), and the adherens junction proteins β-catenin and E-cadherin. Structural alterations to the vocal fold epithelium were further examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: Mann-Whitney U revealed significantly decreased occludin (P = .016) and β-catenin (P = .016) gene expression from rabbits undergoing raised intensity phonation compared with control. There were no significant differences in Z0-1 and E-cadherin gene expression between groups (P > .025). SEM revealed significant obliteration, desquamation, and evidence of microhole formation in rabbit vocal folds exposed to raised intensity phonation compared with control, whereas TEM revealed dilated intercellular morphology between groups. Conclusions: Results provide support for the hypothesis that a transient episode of raised intensity phonation alters transcript levels of vocal fold intercellular tight junction proteins and disrupts integrity of the epithelial barrier. The loss of barrier integrity may have significant consequences on epithelial defenses and compromise protection of the underlying mucosa from damage secondary to prolonged vibration exposure. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: The vocal fold epithelium provides a barrier to the entry of inhaled and systemic challenges. However, the location of the epithelium makes it vulnerable to damage. Past research suggests, but does not directly demonstrate, that exposure to gastric reflux adversely affects the function of the epithelial barrier. Understanding the nature of reflux-induced epithelial barrier dysfunction is necessary to better recognize the mechanisms for vocal fold susceptibility to this disease. Therefore, we examined the effects of physiologically relevant reflux challenges on vocal fold transepithelial resistance and gross epithelial and subepithelial appearance. Study Design: Ex vivo, mixed design with between-group and repeated-measures analyses. Methods: Healthy, native porcine vocal folds (N = 52) were exposed to physiologically relevant acidic pepsin, acid-only, or pepsin-only challenges and examined with electrophysiology and light microscopy. For all challenges, vocal folds exposed to a neutral pH served as control. Results: Acidic pepsin and acid-only challenges, but not pepsin-only or control challenges significantly reduced transepithelial resistance within 30 minutes. Reductions in transepithelial resistance were irreversible. Challenge exposure produced minimal gross changes in vocal fold epithelial or subepithelial appearance as evidenced by light microscopy. Conclusions: These findings demonstrate that acidic environments characteristic of gastric reflux compromise epithelial barrier function without gross structural changes. In healthy, native vocal folds, reductions in transepithelial resistance could reflect reflux-related epithelial disruption. These results might guide the development of pharmacologic and therapeutic recommendations for patients with reflux, such as continued acid-suppression therapy and patient antireflux behavioral education. Laryngoscope, 2010 [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: Inhaled air must be adequately humidified to prevent vocal fold drying, which is detrimental to phonation. The water content of inspired air is reduced by parameters, such as increased breathing rate and oral route. Accelerated oral breathing challenges induce airway dehydration and are posited to affect airway function. The primary objective of this study was to investigate whether accelerated oral breathing challenges are detrimental to phonation. The secondary objective of this study was to determine whether individuals at increased risk for developing voice problems (i.e., smokers) have greater adverse phonatory effects after accelerated breathing challenge than nonsmoking controls. Study Design: Prospective study with between-subjects, repeated-measures design. Methods: Female smokers (n = 12) and nonsmoking controls (n = 12) participated in this experimental study over 2 days that differed in ambient humidity. Phonation threshold pressures (PTP) were collected prior to and following short-term accelerated and habitual breathing challenges. Respiratory measures were collected during the challenges. Results: Short-term accelerated breathing challenges significantly increased PTP. This increase in PTP with accelerated breathing was transient and not significantly influenced by breathing route, ambient humidity, or smoking status. Likewise, respiratory measures were not affected by breathing route, ambient humidity, or smoking status. Conclusions: During daily activities, such as exercise, individuals may engage in accelerated breathing for prolonged durations. This study demonstrates that even extremely short durations of accelerated breathing may affect phonation. [ABSTRACT FROM AUTHOR]

Objectives: Inhaled medications prescribed for the hypersensitive airway typically combine corticosteroids and long-acting β2 adrenergic agonists (LABAs). The phonatory side effects of these combination treatments are widely recognized. However, there is limited understanding of the physiological changes induced by these medications that underlie the phonatory side effects. The objective of this study was to investigate the distinct effects of corticosteroids and LABAs on vocal fold mucosal physiology. Understanding the physiological changes to the vocal folds after corticosteroid and LABA treatments is necessary to prevent the prevalent vocal decrement associated with these medications. Study Design: Experimental in vitro design with treatment and control groups. Methods: Native porcine vocal fold mucosae (N = 38) were exposed to corticosteroid or LABA treatments. Ion transport was measured continuously at baseline and after treatment. To quantify the nature of ion transport, vocal folds were also treated with chloride and sodium channel inhibitors. Results: Corticosteroid treatment did not alter ion transport. Conversely, exposure to LABAs significantly increased ion transport. This increase in ion transport was transient, observed immediately after treatment in all tissue and associated with increased chloride secretion. Conclusions: The distinct effects of corticosteroids and LABAs on vocal fold physiology have not been examined to date. This study demonstrates that short-term treatment with LABAs, but not corticosteroids, significantly increases ion transport. These findings suggest that one underlying physiological mechanism for phonatory changes associated with inhaled treatments may be related to acute alterations in vocal fold ion transport and surface hydration. Laryngoscope, 2009 [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: Cyclic adenosine monophosphate (cAMP) is an important biological molecule that regulates ion transport and inflammatory responses in epithelial tissue. The present study examined whether the adenylyl cyclase activator, forskolin, would increase cAMP concentration in porcine vocal fold mucosa and whether the effects of increased cAMP would be manifested as a functional increase in transepithelial ion transport. Additionally, changes in cAMP concentrations following exposure to an inflammatory mediator, tumor necrosis factor-α (TNFα) were investigated. Study Design: In vitro experimental design with matched treatment and control groups. Methods: Porcine vocal fold mucosae (N = 30) and tracheal mucosae (N = 20) were exposed to forskolin, TNFα, or vehicle (dimethyl sulfoxide) treatment. cAMP concentrations were determined with enzyme-linked immunosorbent assay. Ion transport was measured using electrophysiological techniques. Results: Thirty minute exposure to forskolin significantly increased cAMP concentration and ion transport in porcine vocal fold and tracheal mucosae. However, 30-minute and 2-hour exposure to TNFα did not significantly alter cAMP concentration. Conclusions: We demonstrate that forskolin-sensitive adenylyl cyclase is present in vocal fold mucosa, and further, that the product, cAMP increases vocal fold ion transport. The results presented here contribute to our understanding of the intracellular mechanisms underlying vocal fold ion transport. As ion transport is important for maintaining superficial vocal fold hydration, data demonstrating forskolin-stimulated ion transport in vocal fold mucosa suggest opportunities for developing pharmacological treatments that increase surface hydration. [ABSTRACT FROM AUTHOR]