Cigarette smoke-induced changes in the murine vocal folds: a Raman spectroscopic observation.

Raman spectroscopic methods are being projected as novel tools to study the early invisible molecular level changes in a label-free manner. In the present study, we have used Raman spectroscopy to explore the earliest biochemical changes in murine vocal folds in response to time-bound cigarette smoke exposure. Mice were exposed to cigarette smoke for 2 or 4-weeks through a customized smoke inhalation system. The larynx was collected and initial evaluations using standard methods of analysis such as histopathology and immunofluorescence was performed. Concurrent unstained sections were used for Raman imaging. Two common pathological features of vocal fold disorders including alterations in collagen content and epithelial hypercellularity, or hyperplasia, were observed. The mean spectra, principal component analysis, and Raman mapping also revealed differences in the collagen content and hypercellularity in the smoke exposed tissues. The differences in 2-week exposed tissues were found to be more prominent as compared to 4-week. This was attributed to adaptive responses and the already reported biphasic effects, which suggest that collagen synthesis is significantly reduced at higher cigarette smoke concentrations. Overall findings of the study are supportive of the prospective application of Raman imaging in monitoring changes due to cigarette smoke in the vocal folds.

Deep brain stimulation for vocal tremor: a comprehensive, multidisciplinary methodology.

Tremulous voice is a characteristic feature of a multitude of movement disorders, but when it occurs in individuals diagnosed with essential tremor, it is referred to as essential vocal tremor (EVT). For individuals with EVT, their tremulous voice is associated with significant social embarrassment and in severe cases may result in the discontinuation of employment and hobbies. Management of EVT is extremely difficult, and current behavioral and medical interventions for vocal tremor result in suboptimal outcomes. Deep brain stimulation (DBS) has been proposed as a potential therapeutic avenue for EVT, but few studies can be identified that have systematically examined improvements in EVT following DBS. The authors describe a case of awake bilateral DBS targeting the ventral intermediate nucleus for a patient suffering from severe voice and arm tremor. They also present their comprehensive, multidisciplinary methodology for definitive treatment of EVT via DBS. To the authors' knowledge, this is the first time comprehensive intraoperative voice evaluation has been used to guide microelectrode/stimulator placement, as well as the first time that standard pre- and post-DBS assessments have been conducted, demonstrating the efficacy of this tailored DBS approach.

Method for Collecting Single Epithelial Cells from the Mouse Larynx.

OBJECTIVE: The larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non-laryngeal sites and non-epithelial laryngeal cells. METHODS: Mice were euthanized, and the larynx was carefully exposed and separated from non-laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan-Keratin, EpCAM, NGFR, p63, K5, ß-tubulin, MUC5AC) and non-epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single-cell RNA sequencing (scRNA-seq) was performed to confirm this method can capture diverse laryngeal cell types. RESULTS: We collected 6000-8000 cells from a single larynx and 35000-40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA-seq was successfully conducted and displayed nine unique cell clusters. CONCLUSION: We developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA-seq, protein analyses, and cell-culture-based experiments, following laryngeal injury. LEVEL OF EVIDENCE: NA Laryngoscope, 134:786-794, 2024.

Establishing a Mouse Model of Surgical Vocal Fold Injury.

OBJECTIVE: Animal models of vocal fold (VF) surgical injury and scar formation provide insight into the wound healing process. The purpose of this study was to establish an alternative model of surgical injury to the mouse VF using materials commonly available in most research laboratories or for purchase and to investigate wound healing of the epithelium (EP) and lamina propria (LP). METHODS: Mice were anesthetized by isoflurane gas delivery and positioned on a platform so that the larynx could be observed using a laryngoscope and dissection microscope. Unilateral VF injury was created using a wire brush. Mice were euthanized and the larynx evaluated 1-, 3-, 5-, 7-, 14-, and 28-days following injury. Histological and immunofluorescent analysis was used to evaluate thickness of the EP, LP area, proliferative (Ki67+) and basal cells (p63+) in the EP, and collagen III content in the LP. RESULTS: The depth of injury reached the superficial thyroarytenoid muscle on Day 1. The thickness of the EP of the injured VF was increased on Days 3 and 5, and the LP area was increased on Days 3, 5, and 7 as compared with the uninjured VF. Ki67+ and p63+ cells were increased on Day 3 and collagen III content was increased on Days 5 and 28 as compared with the uninjured VF. CONCLUSION: We successfully established an alternative method of creating unilateral VF injury in the mouse. This method will be useful for future research regarding VF surgical injury and wound healing. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

Effects of Short-term Electronic(e)-Cigarette Aerosol Exposure in the Mouse Larynx.

OBJECTIVES: The effects of electronic cigarettes (e-cigarettes) on the larynx are relatively unknown. This study examined the short-term effects of e-cigarette inhalation on cellular and inflammatory responses within the mouse laryngeal glottic and subglottic regions after exposure to pod-based devices (JUUL). METHODS: Male C57BL6/J mice (8-9 weeks) were assigned to control (n = 9), JUUL flavors Mint (JMi; n = 10) or Mango (JMa; n = 10). JUUL mice were exposed to 2 h/day for 1, 5, and 10 days using the inExpose inhalation system. Control mice were in room air. Vocal fold (VF) epithelial thickness, cell proliferation, subglandular area and composition, inflammatory cell infiltration, and surface topography were evaluated in the harvested larynges. Mouse body weight and urinary nicotine biomarkers were also measured. Chemical analysis of JUUL aerosols was conducted using selective ion flow tube mass spectrometry. RESULTS: JUUL-exposed mice had reduced body weight after day 5. Urinary nicotine biomarker levels indicated successful JUUL exposure and metabolism. Quantitative analysis of JUUL aerosol indicated that chemical constituents differ between JMi and JMa flavors. VF epithelial thickness, cellular proliferation, glandular area, and surface topography remained unchanged after JUUL exposures. Acidic mucus content increased after 1 day of JMi exposure. VF macrophage and T-cell levels slightly increased after 10 days of JMi exposures. CONCLUSIONS: Short-term e-cigarette exposures cause minimal flavor- and region-specific cellular and inflammatory changes in the mouse larynx. This work provides a foundation for long-term studies to determine if these responses are altered with multiple e-cigarette components and concentrations. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1316-1326, 2024.

Impact of Electronic Cigarettes on the Upper Aerodigestive Tract: A Comprehensive Review for Otolaryngology Providers.

Objective: The use and effects of electronic (e)-cigarettes (e-cigs) are particularly relevant for otolaryngology providers as tobacco plays a major role in benign and malignant diseases of the upper aerodigestive tract. This review aims to (1) summarize the recent policies regarding e-cigs and important patterns of use and (2) serve as a comprehensive resource for clinical providers on the known biologic and clinical effects of e-cigs on the upper aerodigestive tract. Data Sources: PubMed/MEDLINE. Review Methods: We conducted a narrative review on (1) general information on e-cig use and informative findings in the lower respiratory system and a comprehensive review on (2) the effects of e-cigs on cell and animal models and the clinical implications of these products on human health as is relevant to otolaryngology. Conclusions: Although e-cigs are likely less harmful than conventional cigarettes, preliminary research on e-cigs suggest several deleterious effects including in the upper aerodigestive tract. Due to this, there has been increased interest in restricting e-cig usage, particularly among the adolescent population, and caution in recommending e-cigs to current smokers. Implications for Practice: Chronic e-cig use is likely to have clinical implications. It is critical for otolaryngology providers to be aware of the rapidly changing regulations and use patterns regarding e-cigs and how e-cigs influence human health, particularly with regards to the upper aerodigestive tract, to accurately council patients regarding potential risks and benefits of use.

Effects of Electronic (E)-cigarette Vapor and Cigarette Smoke in Cultured Vocal Fold Fibroblasts.

OBJECTIVE: The public use of electronic-cigarettes (e-cigs) is rapidly growing. When heated, e-cigs produce a vapor that is inhaled. The vocal folds are among the first tissues exposed to this insult. However, the impact of e-cigs on vocal fold health is almost entirely unknown. Our objective was to evaluate the effects of e-cig vapor on cultured human vocal fold fibroblasts (hVFFs), the primary cell type of the lamina propria. We compared the cellular effects of e-cig vapor without and with nicotine and conventional cigarette smoke. STUDY DESIGN: In vitro. METHODS: E-cig vapor extract (EVE) and cigarette smoke extract (CSE) were created by bubbling vapor and smoke, respectively, into the cell culture medium. hVFFs were exposed to EVE without or with nicotine or CSE for 24 hours. Untreated cells were used as a control group. Cells were harvested, and cytotoxicity, extracellular matrix and inflammatory gene expression, and DNA damage were assessed. RESULTS: Undiluted EVE without and with nicotine reduced the viability of hVFFs to a cytotoxic level. CSE reduced hVFFs viability to a greater extent than EVE and induced DNA damage as measured by DNA double-strand breaks. No changes in gene expression were observed following EVE or CSE exposure. CONCLUSION: EVE induces cytotoxicity in hVFFs. However, cellular responses were greater following exposure to CSE, suggesting cigarette smoke may induce more harm, at least in the short term. Findings from this investigation improve our understanding of responses of hVFFs to e-cigs and form the basis for an in vitro methodology to study the vocal fold responses to these products. LEVEL OF EVIDENCE: NA Laryngoscope, 133:139-146, 2023.

Diagnosis and Management of Vocal Complications after Chondrolaryngoplasty.

OBJECTIVE: Transfeminine patients (transwomen/feminine nonbinary folks assigned male at birth) can undergo chondrolaryngoplasty ("tracheal shave") to feminize their neck appearance. While isolated cases of vocal complications have been reported following the procedure, aggregated outcomes have not been quantitatively studied. We present acoustic and stroboscopic data to describe a patient cohort with vocal complications after chondrolaryngoplasty and discuss reparative surgical technique. METHODS: Subjective and objective data, including videostroboscopy, were collected from patients with voice complaints after chondrolaryngoplasty. Dislocated anterior commissures were reconstructed with feminization laryngoplasty. Postoperative voice data were recorded and statistically compared to preoperative data using paired t-tests. RESULTS: On consecutive chart review, of the 94 transfeminine women with prior outside history of chondrolaryngoplasty, 27 (29%) reported chronic postoperative hoarseness, deepened pitch, or loss of upper register. On endoscopy, short, lax vocal folds with persistent anterior glottic gap and phase asymmetry were commonly noted; anterior commissure dislocation was confirmed in-office by using needle localization through absent thyroid cartilage. After open resuspension of the anterior commissure with feminization laryngoplasty, post-repair modal-speaking, minimum, and maximum fundamental frequencies (F0) increased on average by 7, 8, and 5 semitones, respectively (p < 0.01), when compared to pre-repair values. On average, perioperative maximum phonation time did not change significantly (p = 0.15). Average self-assessment of vocal femininity increased by 48% (p < 0.01). CONCLUSION: Anterior commissure dislocation should be suspected with signs of vocal impairment after chondrolaryngoplasty. Following proper diagnosis, resuspension of the anterior commissure via feminization laryngoplasty approach can be an effective reparative technique. LEVEL OF EVIDENCE: This work represents a 2011 OCEBM Level 4 evidence as a case series Laryngoscope, 133:2301-2307, 2023.

Social Perception of External Laryngeal Anatomy Related to Gender Expression in a Web-based Survey.

OBJECTIVE(S): To quantify the effect of laryngeal prominence size on socially perceived attributes relating to gender expression. Chondrolaryngoplasty ("tracheal shave") is a common procedure performed for transgender women to feminize neck appearance. The extent of thyroid cartilage resection needed to convey socially-perceived feminine gender expression without destabilizing the voice is incompletely understood. METHODS: Cross-sectional evaluation of a randomized allocation of images of varying laryngeal prominence to a non-repeated, random sample from November 2021 to December 2021. Photos of laryngeal prominence were isolated against a constant neck baseline with lateral, oblique, and frontal views. The images were embedded into a web-based survey with visual analog scales to capture perceived scaled gender expression (masculinity, femininity) and social traits (e.g., attractiveness, friendliness, leadership). We performed bivariate and multivariate analyses relating the laryngeal prominence to perceived gender expression and social traits. RESULTS: The analytic sample included 1,026 respondents. Laryngeal grades similar to the demonstrated "grade M" in this study and smaller demonstrated similar perceptions of increased femininity and decreased masculinity. Grades larger than M demonstrate significantly increased perceived masculinity and significantly decreased perceived femininity. The lateral and oblique views of the neck appear to be the most gender-informative. CONCLUSION: This crowd-sourced analysis of external laryngeal anatomy by a large population of observers provides clear, reproducible insights into social perceptions of gender identity and specifically femininity. These data will meaningfully inform patient counseling and surgical planning for gender-affirming interventions by establishing normative data representing the general public's perceptions. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2292-2300, 2023.

End-to-end deep learning classification of vocal pathology using stacked vowels.

Objectives: Advances in artificial intelligence (AI) technology have increased the feasibility of classifying voice disorders using voice recordings as a screening tool. This work develops upon previous models that take in single vowel recordings by analyzing multiple vowel recordings simultaneously to enhance prediction of vocal pathology. Methods: Voice samples from the Saarbruecken Voice Database, including three sustained vowels (/a/, /i/, /u/) from 687 healthy human participants and 334 dysphonic patients, were used to train 1-dimensional convolutional neural network models for multiclass classification of healthy, hyperfunctional dysphonia, and laryngitis voice recordings. Three models were trained: (1) a baseline model that analyzed individual vowels in isolation, (2) a stacked vowel model that analyzed three vowels (/a/, /i/, /u/) in the neutral pitch simultaneously, and (3) a stacked pitch model that analyzed the /a/ vowel in three pitches (low, neutral, and high) simultaneously. Results: For multiclass classification of healthy, hyperfunctional dysphonia, and laryngitis voice recordings, the stacked vowel model demonstrated higher performance compared with the baseline and stacked pitch models (F1 score 0.81 vs. 0.77 and 0.78, respectively). Specifically, the stacked vowel model achieved higher performance for class-specific classification of hyperfunctional dysphonia voice samples compared with the baseline and stacked pitch models (F1 score 0.56 vs. 0.49 and 0.50, respectively). Conclusions: This study demonstrates the feasibility and potential of analyzing multiple sustained vowel recordings simultaneously to improve AI-driven screening and classification of vocal pathology. The stacked vowel model architecture in particular offers promise to enhance such an approach. Lay Summary: AI analysis of multiple vowel recordings can improve classification of voice pathologies compared with models using a single sustained vowel and offer a strategy to enhance AI-driven screening of voice disorders. Level of Evidence: 3.

Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke.

Cigarette smoking is a major risk factor for laryngeal diseases. Despite well-documented cigarette smoke (CS) induced laryngeal histopathological changes, the underlying immunopathological mechanisms remain largely unexplored. The goal of this study was to evaluate inflammatory and immune cell responses in a CS-exposed larynx. Specifically, we used a 4-week subacute whole-body CS inhalation mouse model to assess these responses in the laryngeal mucosa upon exposure to low (LD; 1 h/day) and high dose (HD; 4 h/day) CS. Laryngeal tissues were harvested and evaluated using a 254-plex NanoString inflammation panel and neutrophil/macrophage/T-cell immunohistochemistry (IHC). NanoString global and differential gene expression analysis revealed a unique expression profile only in the HD group, with 26 significant differentially expressed genes (DEGs). StringDB KEGG pathway enrichment analysis revealed the involvement of these DEGs with pro-inflammatory pathways including TNF/TNFα and IL-17. Furthermore, inflammatory responses remained inhibited in conjunction with predicted activated states of anti-inflammatory regulators like PPARγ and NFE2L2 upon Ingenuity Pathway Analysis (IPA). Subglottic T-cell levels remained significantly inhibited as corroborated by IPA predictions. Overall, our key findings are consistent with HD exposures being anti-inflammatory and immunosuppressive. Furthermore, the identification of important regulatory genes and enriched pathways may help improve clinical interventions for CS-induced laryngeal diseases.

Feminization Laryngoplasty: 17-Year Review on Long-Term Outcomes, Safety, and Technique.

OBJECTIVES: Transfeminine patients can experience significant gender dysphoria in vocal communication. Feminization laryngoplasty (FL) is a gender-affirming surgery developed to elevate speaking vocal range, as well as alter vocal resonance and laryngeal cosmesis. The purpose here was to appraise FL's long-term voice outcomes across a 17-year review period. STUDY DESIGN: Level III, retrospective study and description of technique. SETTING: A single-institution transfeminine voice clinic. METHODS: Voice data (speaking fundamental frequency [F0], lowest F0, highest F0, F0 range in both Hertz and semitones, and maximum phonation time [MPT]) were collected and assessed. Self-assessment of voice femininity and complications were documented. RESULTS: The 162 patients, all transfeminine women, had a mean age of 40 years with 36-month mean follow-up. There were significant increases in mean speaking F0 (Δ = 50 ± 30 Hz, Δ = 6 ± 3 semitones; P < .001) and mean change in lowest F0 (Δ = 58 ± 31 Hz, Δ = 8 ± 4 semitones; P < .001). There was no significant difference in mean change in highest F0 or MPT. There was significant improvement (Δ = 60% ± 39%; P < .001) in perceptual self-assessment of vocal femininity. There was a 1.2% rate of major postoperative complications requiring inpatient admission or operative intervention. There were no differences in vocal outcomes between those patients who had less than 1-year follow-up and those who had 5-year follow-up. CONCLUSION: FL in this cohort was a safe and effective technique for increasing mean speaking F0, mean lowest F0, and voice gender perception over a prolonged follow-up period. These findings add to the possible treatments aimed at addressing the morbid dysphoria related to voice and communication for our transfeminine patients.

The Impact of COVID-19 on Speech-Language Pathologists Engaged in Clinical Practices With Elevated Coronavirus Transmission Risk.

Purpose This study assessed and described potential clinical practice changes secondary to COVID-19 that emerged as an early response to the pandemic for speech-language pathologists (SLPs) engaged in voice, alaryngeal, and swallowing activities that may increase the risk of virus transmission. Method SLPs from the United States and Canada (n = 665) who were engaged in clinical activities that might elevate the risk of COVID-19 exposure completed an online survey regarding their clinical practices. Topics assessed included potential clinical service modifications, COVID-19 testing and health, and potential financial impacts in the early time period of the pandemic. Results The percentage of SLPs completing the most endoscopic procedures prepandemic (≥ 10/week) was reduced from 39% of respondents to 3% due to the pandemic. Those who completed the most tracheoesophageal puncture voice prosthesis changes (≥ 5/week) reported a reduction in frequency from 24% to 6%. Twenty-five percent of SLPs reported that they were tested for COVID-19, and 6% reported a positive result. Descriptive statistics suggest that COVID-19 testing rates of SLPs, the percentage of SLPs experiencing a financial impact, and the percentage who were furloughed varied across SLP work setting. Conclusions These findings provide the first data characterizing the impact on COVID-19 on clinical practice for SLPs engaged in procedures such as endoscopy and laryngectomy care. The results indicate that, as frontline workers, SLPs were directly impacted in their practice patterns, personal health, safety, and financial security, and that these reported impacts occurred differently across SLP work settings.

Mainstream Cigarette Smoke Impacts the Mouse Vocal Fold Epithelium and Mucus Barrier.

OBJECTIVES/HYPOTHESIS: Cigarette smoke (CS) is a primary risk factor for the development of numerous benign and malignant laryngeal diseases. The epithelium and mucus lining the vocal folds (VF) are the first barriers against CS. The primary objective of this study was to investigate epithelial and mucus barrier changes in the mouse laryngeal mucosa upon exposure to subacute CS. The secondary objective was to compare mucus barrier changes in mice and human smokers and nonsmokers. Study Design Animal model. METHODS: Mice were exposed to CS for 4 weeks for 4 hours (N = 12, high dose [HD]) or 1 hour (N = 12, low dose [LD]) per day. Air-exposed mice were used as a control group (N = 10). Larynges were harvested and VF epithelial barrier integrity was evaluated including cellular proliferation and expression of cell junctions. We also investigated mucus production by examining mucus cell area and mucin expression in mice and human smokers and nonsmokers. RESULTS: HD CS increased VF epithelial cellular proliferation but did not alter the expression of cell junctions. HD CS also induced hypertrophy of the mucus-producing submucosal glands. However, only LD CS increased MUC5AC gene expression. MUC5AC staining appeared elevated in laryngeal specimens from smokers, but this was not significant as compared to nonsmokers. CONCLUSIONS: These findings help us identify potential adaptive mechanisms to CS exposure as well as set the foundation for further study of key aspects of epithelial and mucus barrier integrity that may be implicated in laryngeal disease development following prolonged smoking. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2530-2539, 2021.

Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx.

The larynx is an essential organ in the respiratory tract and necessary for airway protection, respiration, and phonation. Cigarette smoking is a significant risk factor associated with benign and malignant laryngeal diseases. Despite this association, the underlying mechanisms by which cigarette smoke (CS) drives disease development are not well elucidated. In the current study, we developed a short-term murine whole body inhalation model to evaluate the first CS-induced cellular responses in the glottic [i.e. vocal fold (VF)] and subglottic regions of the larynx. Specifically, we investigated epithelial cell proliferation, cell death, surface topography, and mucus production, at various time points (1 day, 5 days, 10 days) after ∼ 2 h exposure to 3R4F cigarettes (Delivered dose: 5.6968 mg/kg per cigarette) and following cessation for 5 days after a 5 day CS exposure (CSE). CSE elevated levels of BrdU labeled proliferative cells and p63 labeled epithelial basal cells on day 1 in the VF. CSE increased proliferative cells in the subglottis at days 5, 10 and following cessation in the subglottis. Cleaved caspase-3 apoptotic activity was absent in VF at all time points and increased at day 1 in the subglottis. Evaluation of the VF surface by scanning electron microscopy (SEM) revealed significant epithelial microprojection damage at day 10 and early signs of necrosis at days 5 and 10 post-CSE. SEM visualizations additionally indicated the presence of deformed cilia at days 5 and 10 after CSE and post-cessation in the respiratory epithelium lined subglottis. In terms of mucin content, the impact of short-term CSE was observed only at day 10, with decreasing acidic mucin levels and increasing neutral mucin levels. Overall, these findings reveal regional differences in murine laryngeal cellular responses following short-term CSE and provide insight into potential mechanisms underlying CS-induced laryngeal disease development.

Vocal Hygiene in Collegiate Singers-Does Formal Training Relate to Practices?

BACKGROUND: Vocal hygiene is vitally important for protecting vocal quality and promoting vocal longevity. It includes adequate hydration, avoidance of environmental irritants, and refraining from phonotraumatic behaviors such as screaming, excessive talking, and throat clearing. Formal singing training in addition to enhancing singing techniques, is also assumed to promote good vocal hygiene. Consequently, we investigated whether collegiate singers with formal training are more likely to practice good vocal hygiene habits than untrained collegiate singers. METHODS: We completed a prospective cohort study, where collegiate singers at Stanford University were voluntarily enrolled in an annual vocal health clinic. Full laryngeal exams were performed and demographic information, formal vocal training status, and information on vocal hygiene habits were obtained through questionnaires. The results were then analyzed by grouping singers as "untrained" if less than 1 year of formal training, and "trained" if greater than a year. RESULTS: A total of 82 singers were included for analysis, with 49 (59.8%) having greater than one year of formal training. Trained singers were more likely to employ a primary musical singing style of classical or operatic singing compared to untrained singers who were more likely to utilize "R&B" (P < 0.001). No significant differences were found between groups for hydration practices (P = 0.20), caffeine consumption (P = 0.73), warm up practices (P = 0.08), and phonotraumatic behaviors. Alcohol consumption, smoking tobacco, marijuana, e-cigarette usage was similar between groups. Overall, 11% of our sample reported intermittent less than daily marijuana use, with one singer reporting daily consumption. None of the singers in this sample used e-cigarettes. These rates are noticeably lower than the agematched United States population. Perceptions of cigarettes, marijuana, electronic cigarettes, alcohol, and caffeine were also the same between groups, with the vast majority of singers perceiving these agents as harmful to the voice (64%-100% depending on the agent). No laryngeal pathology was identified during laryngeal examination of any singers. CONCLUSIONS: Vocal hygiene habits are similar between trained and untrained collegiate singers. This likely relates to a desire for vocal longevity irrespective of prior training. This may dispel the long-held assumption that formal training was necessary to develop good vocal habits. We do however, present a very homogenous, highly educated sample of collegiate singers from Stanford University, having significantly less at-risk behavior than their aged-matched counterparts in the general US population. A lack of laryngeal pathology despite extensive vocal demands may also be reflective of good vocal hygiene habits.

Clinical Efficacy and Dosing of Vibrotactile Coordinated Reset Stimulation in Motor and Non-motor Symptoms of Parkinson's Disease: A Study Protocol.

Enhanced neuronal synchronization of the subthalamic nucleus (STN) is commonly found in PD patients and corresponds to decreased motor ability. Coordinated reset (CR) was developed to decouple synchronized states causing long lasting desynchronization of neural networks. Vibrotactile CR stimulation (vCR) was developed as non-invasive therapeutic that delivers gentle vibrations to the fingertips. A previous study has shown that vCR can desynchronize abnormal brain rhythms within the sensorimotor cortex of PD patients, corresponding to sustained motor relief after 3 months of daily treatment. To further develop vCR, we created a protocol that has two phases. Study 1, a double blinded randomized sham-controlled study, is designed to address motor and non-motor symptoms, sensorimotor integration, and potential calibration methods. Study 2 examines dosing effects of vCR using a remote study design. In Study 1, we will perform a 7-month double-blind sham-controlled study including 30 PD patients randomly placed into an active vCR or inactive (sham) vCR condition. Patients will receive stimulation for 4 h a day in 2-h blocks for 6 months followed by a 1-month pause in stimulation to assess long lasting effects. Our primary outcome measure is the Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part III off medication after 6 months of treatment. Secondary measures include a freezing of gait (FOG) questionnaire, objective motor evaluations, sensorimotor electroencephalography (EEG) results, a vibratory temporal discrimination task (VTDT), non-motor symptom evaluations/tests such as sleep, smell, speech, quality of life measurements and Levodopa Equivalent Daily Dose (LEDD). Patients will be evaluated at baseline, 3, 6, and 7 months. In the second, unblinded study phase (Study 2), all patients will be given the option to receive active vCR stimulation at a reduced dose for an additional 6 months remotely. The remote MDS-UPDRS part III off medication will be our primary outcome measure. Secondary measures include sleep, quality of life, objective motor evaluations, FOG and LEDD. Patients will be evaluated in the same time periods as the first study. Results from this study will provide clinical efficacy of vCR and help validate our investigational vibrotactile device for the purpose of obtaining FDA clearance. Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT04877015.

Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers.

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-ß1 levels between smokers and non-smokers.

A Preliminary Study of Vocal Health Among Collegiate A Cappella Singers.

OBJECTIVE: Collegiate a cappella groups have grown significantly in popularity and prominence; however, there have been few studies that evaluate the vocal health of this subgroup of young singers. The objective of this preliminary study was to conduct a multiparametric evaluation of the vocal health characteristics of a sample of collegiate a cappella singers. We further tested whether differences in vocal health assessments exist between a cappella singers with and without vocal training and trained collegiate singers who do not participate in a cappella groups. STUDY DESIGN: Point prevalence study. METHODS: Forty-one collegiate singers participated in this study. Participants were divided into the following three groups: trained singers (TS), trained a cappella singers (ATS), and untrained a cappella singers (AUS). Participants were administered a set of surveys to assess self-perception of singing voice health and perceived access and attitudes toward voice-related health care. Acoustic and laryngoscopic assessments of participant's speaking and singing voice was performed and validated vocal health questionnaires administered as a means to objectively evaluate for the presence of voice problems. RESULTS: Overall, 87.5% of the ATS and 60% of the AUS groups reported experiencing problems with their singing voice. However, no vocal abnormalities were detected during laryngoscopic and acoustic assessments. Furthermore, minimal differences between any of the measured vocal health parameters were observed between the TS, ATS, and AUS groups. CONCLUSION: Collectively, a high percentage of collegiate a cappella singers with and without vocal training report singing voice problems. However, our sample of a cappella singers did not have increased singing voice problems as compared to vocally trained collegiate singers not in a cappella groups. We did find that a cappella singers may be more inclined to seek information about maintaining a healthy singing voice from their fellow musicians as opposed to singing teachers or other voice health professionals. Singing teachers, otolaryngologists, and speech-language pathologists may need to play a more active role in educating a cappella singers regarding maintaining good vocal health.

Increased Expression of Estrogen Receptor Beta in Idiopathic Progressive Subglottic Stenosis.

BACKGROUND/OBJECTIVES: Idiopathic progressive subglottic stenosis (IPSS) predominantly affects females in perimenopause. It has, therefore, been hypothesized that estrogen is involved in its pathogenesis. There are two main types of estrogen receptors: ER-α and ER-ß. Abnormal variants of ER-ß have previously been shown to be associated with poor wound healing. Estrogen receptors have recently been identified in subglottic tissue samples, with elevated levels of ER-α and progesterone receptors, and no expression of ER-ß, in stenotic specimens reported in one study. The objective of this study was to confirm the presence of estrogen receptors in the subglottis and investigate levels of expression and types of estrogen receptors in normal and stenotic subglottic tissue. METHODS: Subglottic tissue was obtained from three female and one male cadaver without laryngotracheal pathology to serve as controls. Subglottic tissue specimens from five female patients with IPSS were also analysed. Immunofluorescence stains for ER-α and ER-ß were performed on specimens. Staining patterns were compared qualitatively and semi-qualitatively between control and IPSS specimens. RESULTS: Immunofluorescence stains demonstrated the presence of both ER-α and ER-ß in subglottic tissue. IPSS specimens demonstrated significantly greater staining intensity of ER-α in the epithelium and ER-ß in glands and ducts compared to controls. CONCLUSIONS: This study confirms the presence of estrogen receptors in the subglottis. Increased expression of ER-α in the epithelium and ER-ß in glands and ducts in IPSS compared to controls may help to explain the predisposition to stenosis in these individuals. LEVEL OF EVIDENCE: 3b Laryngoscope, 130:2186-2191, 2020.