Macrophages alter inflammatory and fibrotic gene expression in human vocal fold fibroblasts.

Macrophage phenotypes are simplistically classified as pro-inflammatory (M1) or anti-inflammatory/pro-fibrotic (M2). Phenotypically different macrophages are putatively involved in vocal fold (VF) fibrosis. The current study investigated interactions between macrophages and VF fibroblasts. THP-1 monocyte-derived macrophages were treated with interferon-gamma (IFN-γ), lipopolysaccharide (LPS)/IFN-γ, interleukin-10 (IL10), transforming growth factor-ß1 (TGF-ß), or interleukin-4 (IL4) for 24 h (M(IFN), M(IFN/LPS), M(IL10), M(TGF), and M(IL4), respectively; M(-) denotes untreated macrophages). Differentially activated macrophages and human VF fibroblasts were co-cultured ± direct contact. Expression of CXCL10, CCN2, ACTA2, FN1, TGM2, and LOX was quantified by real-time polymerase chain reaction. Type I collagen and smooth muscle actin (SMA) were observed by immunofluorescence. CXCL10 and PTGS2 were upregulated in fibroblasts indirectly co-cultured with M(IFN) and M(IFN/LPS). M(TGF) stimulated CCN2, ACTA2, and FN1 in fibroblasts. Enzymes involved in extracellular matrix crosslinking (TGM2, LOX) were increased in monocultured M(IL4) compared to M(-). Direct co-culture with all macrophages increased type I collagen and SMA in fibroblasts. Macrophage phenotypic shift was consistent with stimulation and had downstream differential effects on VF fibroblasts. Direct contact with macrophages, regardless of phenotype, stimulated a pro-fibrotic response in VF fibroblasts. Collectively, these data suggest meaningful interactions between macrophages and fibroblasts mediate fibrosis.

Custom Laser-Cut Silastic® Implants for Type I Thyroplasty in a Preclinical Model.

This study aimed to produce customized silicone elastomer implants of varied size and shape for optimization of type I thyroplasty procedures in a rabbit model. Computer-aided design models of different implant designs were designed and used to program laser cutting of a medical-grade Silastic® sheet. Laser-cut implants were produced rapidly and cost-efficiently. Surgical implantation demonstrated vocal fold medialization and phonation in 5 test subjects. This technique may provide a low-cost alternative or adjunct method to hand-carving or commercial implants.

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

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

The Impact of Vocal Tremor on Deglutition: A Pilot Study.

OBJECTIVE: Vocal tremor (VT) poses treatment challenges due to uncertain pathophysiology. VT is typically classified into two phenotypes: isolated vocal tremor (iVT) and essential tremor-related voice tremor (ETvt). The impact of phenotypes on upper aerodigestive tract physiology during swallowing remains unclear. Qualitative and quantitative measures were employed to characterize tremor phenotypes and investigate the effects on swallowing physiology. METHODS: Eleven ETvt participants (1 Male, 10 Female; x̄ age = 74) and 8 iVT participants (1 Male, 7 Female; x̄ age = 71) swallowed 20 mL boluses in cued and uncued conditions under standardized fluoroscopic visualization. Sustained/a/productions were captured to assess the rate and extent of fundamental frequency (F0) modulation. Penetration and Aspiration Scale (PAS) scores were obtained and swallowing biomechanics were captured using Swallowtail™ software. Participants also completed the Swallowing Quality of Life (SWAL-QOL) questionnaire. RESULTS: Hypopharyngeal transit was faster in both VT phenotypes compared with Swallowtail™ normative reference data. Total pharyngeal transit times, however, were only faster in patients with iVT, relative to reference data. No significant differences were observed on the SWAL-QOL or PAS between tremor phenotypes. SWAL-QOL scores revealed that these patients rarely reported dysphagia symptoms. CONCLUSIONS: Subtle differences in swallowing patterns were observed across VT phenotypes, possibly related to adaptive mechanisms resulting in quicker pharyngeal bolus transit. Most patients did not report swallowing issues or dysphagia symptoms. This study is foundational for larger studies on this challenging population. LEVEL OF EVIDENCE: 4 Laryngoscope, 2024.

A Novel Method for Thyroarytenoid Myofiber Culture.

OBJECTIVES/HYPOTHESIS: Myofiber culture has been employed to investigate muscle physiology in vitro and is well-established in the rodent hind limb. Thyroarytenoid (TA) myofiber culture has not been described, providing an opportunity to employ this method to investigate distinct TA myofiber functions. The purpose of this study was to assess the feasibility of a TA myofiber culture model. STUDY DESIGN: In vitro. METHODS: TA muscles from five Sprague Dawley rats were independently isolated and digested for 90 min. A smooth-tip, wide-bored pipette dissociated TA myofibers from cartilage, and the fibers were distributed on collagen-coated dishes and incubated at 37°C, 5% CO2 for 2 h. Myofiber specificity was determined via immunolabeling for desmin and myosin heavy chain (MHC). Myofibers viability was assessed over 7 days via esterase assay. Additional myofibers were immunolabeled for satellite cell marker Pax-7. Glucocorticoid (GC) receptor (GR) was immunolabeled following GC treatment. RESULTS: The harvest technique yielded ~120 myofibers per larynx. By day 7, ~60% of the fibers remained attached and were calcein AM-positive/ethidium homodimer-negative, indicating viability. Myofibers were positive for desmin and MHC, indicating muscle specificity. Cells surrounding myofibers were positive for Pax-7, indicating the presence of myogenic satellite cells. Myofibers also responded to GC treatment as determined by GR nuclear translocation. CONCLUSION: TA myofibers remained viable in culture for at least 7 days with a predictable response to exogenous stimuli. This technique provides novel investigative opportunities regarding TA structure and function. LEVEL OF EVIDENCE: N/A Laryngoscope, 133:3109-3115, 2023.

Epithelial response to vocal fold microflap injury in a preclinical model.

OBJECTIVES: Functional outcomes following microflap surgery for vocal fold pathology are favorable. Although the stratified squamous epithelium appears to heal rapidly, persistent physiologic tissue alterations are likely. We sought to elucidate key biochemical processes including recruitment of immune cells, regulation of cellular junction proteins, and long-term alterations to epithelial tissue permeability following microflap with an eye toward enhanced clinical outcomes. METHODS: Forty New Zealand rabbits were assigned to eight groups (n = 5/group): no-injury control or bilateral microflap with survival for 0 h, 12 h, 1 day, 3 days, 7 days, 30 days, and 60 days post-microflap. The epithelium was dissected from one vocal fold and transepithelial resistance was quantified. The contralateral fold was subjected to transmission electron microscopy. Images were evaluated by a blinded rater and paracellular space dilation was quantified using ImageJ. Immune cell infiltration was evaluated and recorded qualitatively. RESULTS: Increased innate immune response was observed 12 h as well as 7 and 30 days after microflap. At 60 days following injury, decreased epithelial resistance was observed. Paracellular spaces were dilated at all time-points following injury. CONCLUSIONS: The vocal fold epithelium was significantly altered at 60 days following microflap. The implications for this tissue phenotype are unclear. However, compromised epithelial barrier function is implicated in various diseases and may increase the risk of subsequent injury. LEVEL OF EVIDENCE: NA Laryngoscope, 133:350-356, 2023.

Glucocorticoid Dose Dependency on Gene Expression in Vocal Fold Fibroblasts and Macrophages.

OBJECTIVE: Glucocorticoids (GCs) modulate multiple cellular activities including inflammatory and fibrotic responses. Outcomes of GC treatment for laryngeal disease vary, affording opportunity to optimize treatment. In the current study, three clinically employed GCs were evaluated to identify optimal in vitro concentrations at which GCs mediate favorable anti-inflammatory and fibrotic effects in multiple cell types. We hypothesize a therapeutic window will emerge as a foundation for optimized therapeutic strategies for patients with laryngeal disease. STUDY DESIGN: In vitro. METHODS: Human vocal fold fibroblasts and human macrophages derived from THP-1 monocytes were treated with 0.03-1000 nM dexamethasone, 0.3-10,000 nM methylprednisolone, and 0.3-10,000 nM triamcinolone in combination with interferon-γ, tumor necrosis factor-α, or interleukin-4. Real-time polymerase chain reaction was performed to analyze inflammatory (CXCL10, CXCl11, PTGS2, TNF, IL1B) and fibrotic (CCN2, LOX, TGM2) genes, and TSC22D3, a target gene of GC signaling. EC50 and IC50 to alter inflammatory and fibrotic gene expression was calculated. RESULTS: Interferon-γ and tumor necrosis factor-α increased inflammatory gene expression in both cell types; this response was reduced by GCs. Interleukin-4 increased LOX and TGM2 expression in macrophages; this response was also reduced by GCs. GCs induced TSC22D3 and CCN2 expression independent of cytokine treatment. EC50 for each GC to upregulate CCN2 was higher than the IC50 to downregulate other genes. CONCLUSION: Lower concentrations of GCs repressed inflammatory gene expression and only moderately induced genes involved in fibrosis. These data warrant consideration as a foundation for optimized clinical care paradigms to reduce inflammation and mitigate fibrosis. LEVEL OF EVIDENCE: NA Laryngoscope, 133:1169-1175, 2023.

Dose-Dependent Glucocorticoid Regulation of Transcription Factors in Vocal Fold Fibroblasts and Macrophages.

OBJECTIVE: Variable outcomes of glucocorticoid (GC) therapy for laryngeal disease are putatively due to diverse interactions of the GC receptor (GR) with cell signaling pathways, limited consideration regarding concentration-dependent effects, and inconsistent selection of GCs. In the current study, we evaluated the concentration-dependent effects of three frequently administered GCs on transcription factors with an emphasis on the phosphorylation of GR at Ser203 and Ser211 regulating the nuclear translocation of GR. This study provides foundational data regarding the diverse functions of GCs to optimize therapeutic approaches. STUDY DESIGN: In vitro. METHODS: Human vocal fold fibroblasts and THP1-derived macrophages were treated with different concentrations of dexamethasone, methylprednisolone, and triamcinolone in combination with IFN-γ, TNF-α, or IL4. Phosphorylated STAT1, NF-κB family molecules, and phosphorylated STAT6 were analyzed by Western blotting. Ser211-phosphorylated GR (S211-pGR) levels relative to GAPDH and Ser203-phosphorylated GR (S203-pGR) were also analyzed. RESULTS: GCs differentially altered phosphorylated STAT1 and NF-κB family molecules in different cell types under IFN-γ and TNF-α stimuli. GCs did not alter phosphorylated STAT6 in IL4-treated macrophages. The three GCs were nearly equivalent. A lower concentration of dexamethasone increased S211-pGR/GAPDH ratios relative to increased S211-pGR/S203-pGR ratios regardless of cell type and treatment. CONCLUSION: The three GCs employed in two cell lines had nearly equivalent effects on transcription factor regulation. Relatively high levels of Ser203-phosphorylation at low GC concentrations may be related to concentration-dependent differential effects of GCs in the two cell lines. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2704-2711, 2023.

Concentration Effects of Methylprednisolone in Human Vocal Fold Fibroblast-Macrophage Co-Culture.

OBJECTIVE: The diversity of glucocorticoid (GC) properties may underlie variability of clinical efficacy for vocal fold (VF) disease. Optimized therapeutic approaches must account for tissue complexity as well as interactions between cell types. We previously reported that reduced GC concentrations inhibited inflammation without eliciting fibrosis in mono-cultured VF fibroblasts and macrophages. These data suggested that a refined approach to GC concentration may improve outcomes. In the current study, co-culture of VF fibroblasts and macrophages was employed to investigate the effects of different concentrations of methylprednisolone on fibrotic and inflammatory response genes in VF fibroblasts to optimize management paradigms. STUDY DESIGN: In vitro. METHODS: THP-1 monocyte-derived macrophages were stimulated with interferon-γ (IFN-γ), lipopolysaccharide (LPS), or transforming growth factor-ß (TGF-ß) to induce inflammatory (M(IFN/LPS)) and fibrotic (M(TGF)) phenotypes. Macrophages were then co-cultured with a human VF fibroblast cell line using a 0.4 µm pore membrane with or without 0.1-3000 nM methylprednisolone. Inflammatory (CXCL10, TNF, and PTGS2) and fibrotic (ACTA2, CCN2, and COL1A1) gene expression was quantified in fibroblasts. RESULTS: Incubating VF fibroblasts with M(IFN/LPS) macrophages increased expression of TNF and PTGS2, and this effect was inhibited by methylprednisolone. Incubation of VF fibroblasts with M(TGF) macrophages increased expression of ACTA2, CCN2, and COL1A1, and this effect was enhanced by methylprednisolone. The concentration of methylprednisolone required to downregulate inflammatory genes (TNF and PTGS2) was lower than that to upregulate fibrotic genes (ACTA2, CCN2, and COL1A1). CONCLUSION: Reduced concentration of methylprednisolone effectively suppressed inflammatory genes without enhancing fibrotic genes, suggesting that a refined approach to GC concentration may improve clinical outcomes. LEVEL OF EVIDENCE: N/A Laryngoscope, 133:3116-3122, 2023.

Acute In Vitro and In Vivo Effects of Dexamethasone in the Vocal Folds: a Pilot Study.

OBJECTIVES/HYPOTHESIS: Glucocorticoids (GC)s are commonly employed to treat vocal fold (VF) pathologies. However, VF atrophy has been associated with intracordal GC injections. Dexamethasone-induced skeletal muscle atrophy is well-documented in other tissues and believed to be mediated by increased muscle proteolysis via upregulation of Muscle Ring Finger (MuRF)-1 and Atrogin-1. Mechanisms of dexamethasone-mediated VF atrophy have not been described. This pilot study employed in vitro and in vivo models to investigate the effects of dexamethasone on VF epithelium, thyroarytenoid (TA) muscle, and TA-derived myoblasts. We hypothesized that dexamethasone will increase atrophy-associated gene expression in TA muscle and myoblasts and decrease TA muscle fiber size and epithelial thickness. STUDY DESIGN: In vitro, pre-clinical. METHODS: TA myoblasts were isolated from a female Sprague-Dawley rat and treated with 1 µM dexamethasone for 24-h. In vivo, 15 New Zealand white rabbits were randomly assigned to three treatment groups: (1) bilateral intracordal injection of 40 µL dexamethasone (10 mg/ml; n = 5), (2) volume-matched saline (n = 5), and (3) untreated controls (n = 5). Larynges were harvested 7-days post-injection. Across in vivo and in vitro experimentation, MuRF-1 and Atrogin-1 mRNA expression were measured via RT-qPCR. TA muscle fiber cross-sectional area (CSA) and epithelial thickness were also quantified in vivo. RESULTS: Dexamethasone increased MuRF-1 gene expression in TA myoblasts. Dexamethasone injection, however, did not alter atrophy-associated gene expression, TA CSA, or epithelial thickness in vivo. CONCLUSION: Dexamethasone increased atrogene expression in TA myoblasts, providing foundational insight into GC induced atrophic gene transcription. Repeated dexamethasone injections may be required to elicit atrophy in vivo. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2264-2270, 2023.

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

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

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

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

The Patient Experience: The Relationship Between Vocal Handicap, Congruency, Perceived Present Control, and Mood Across Four Voice Disorders.

The potential for negative sequalae in psychosocial well-being presents clinical importance to the assessment of voice disorders. Despite the impairment voice disorders cause in the psychosocial domain, the clinical assessment of these disorders relies heavily on visual perceptual judgments of the larynx, audio-perceptual, as well as acoustic and aerodynamic measures. While these measures aid in accurate diagnosis and are necessary for standard of care, they present little insight into the patient experience of having a voice disorder. DESIGN: Retrospective between-subject, non-experimental design. METHODS: Data from 335 patients from the University of Pittsburgh Voice Center were collected from scores of the Voice Handicap Index-10 (VHI-10) and two recent questionnaires, the Voice Present Perceived Control scale (VPPC), and the Vocal Congruency Scale (VCS). Examining how these voice-specific scales related to three mental health screeners for stress (Perceived Stress Scale-4), anxiety (Generalized Anxiety Disorder-7) and depression (Patient Health Questionnaire-9) were also examined. Patient diagnoses included primary muscle tension dysphonia (pMTD), unilateral vocal fold paralysis (UVFP), vocal fold atrophy, and mid membranous vocal fold lesions. RESULTS: There were significant differences in scores from the voice-specific scales between diagnostic groups with UVFP being the highest (worst) in VHI-10 and UVFP being the lowest (worst) in VCS compared to healthy controls. There was no significant difference in VPPC scores between diagnostic groups. Results showed statistically significant inverse relationships between the VHI-10 and the VPPC and between the VHI-10 and VCS for all diagnostic groups. A significant direct relationship was found between the VPPC and the VCS for patients diagnosed with MTD, UVFP and Lesions. In sum, patients with UVFP presented with the most frequent and sometimes strongest relationships between voice and mental health measures. DISCUSSION: This study marks an initial investigation into the nuanced patient experience of having a voice disorder. Three theoretically unrelated voice constructs: handicap, perceived control, and sense of self, were measured via self-report. Results from this study describe the patient experience correlating to these constructs with weak correlations to stress, anxiety, and depression. Findings also clearly suggest that patient experience varies among diagnostic groups, as well as varying constructs. Measures of multiple constructs of patient perception provide valuable insight into a patient's experience of their voice disorder, guidance on the direction of voice treatment, and justification for such treatments.

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

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