Faculty Bibliography

INTRODUCTION/BACKGROUND:Glucocorticoids (GCs) are commonly prescribed for laryngeal indications due to their potent anti-inflammatory properties. However, GCs effect on vocal fold (VF) epithelial morphology and barrier function following injury is overlooked and may be key to efficacy. In this study, the effects of GCs on epithelial morphology and barrier function were quantified in injured VFs. We seek to increase our understanding of biochemical processes underlying GC mechanisms to refine therapeutic strategies. METHODS:Microflap injury was induced in 65 rabbits. Seven days after injury, animals received bilateral 20 μL intracordal injections of saline, dexamethasone, methylprednisolone, or triamcinolone (n = 15 per condition). Five rabbits in each condition were euthanized 1, 7, or 60 days following treatment. An additional five animals served as non-injured/untreated controls. To quantify transepithelial electrical resistance (TEER), 1 mm epithelial biopsies were placed in an Ussing chamber. The contralateral VF was processed for transmission electron microscopy and epithelial depth analysis. RESULTS:At 60 days, GC treatment maintained TEER levels similar to non-injured/untreated controls. However, triamcinolone reduced TEER compared with saline-treated conditions. Acutely, epithelial hyperplasia typically persisted in all injured VFs. At 60 days, only dexamethasone and triamcinolone increased epithelial depth in injured VFs; all GCs increased epithelial depth compared with non-injured/untreated controls. CONCLUSION/CONCLUSIONS:Acutely, GCs did not alter TEER. Additionally, GCs did not alter epithelial depth compared with saline treatment, indicating alignment with natural healing responses. At 60 days, GCs exhibited varying degrees of TEER restoration and epithelial hyperplasia, possibly due to distinct pharmacodynamic profiles. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2024.

OBJECTIVES/OBJECTIVE:Vocal fold scar remains a therapeutic challenge. Vocal fold fibroblasts (VFFs) secrete extracellular matrix (ECM), and transforming growth factor-beta 1 (TGF-β1)-mediated fibroblast to myofibroblast differentiation is central to the development of fibrosis. The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels, and activation of TRP ankyrin 1 (TRPA1) channel has been shown to have antifibrotic effects through TGF-β1/Smad signaling in various organs. This study aimed to elucidate expression of TRPA1 and the impact of TRPA1 activation on TGF-β1/Smad signaling in VFFs. METHODS: M) for 4 or 24 h. Trpa1, Smad3, Smad7, Col1a1, Acta2, and Has1 mRNA expression were quantified via qPCR. RESULTS:TRPA1 was expressed in cultured VFFs and the lamina propria. TGF-β1 administration significantly increased Trpa1 compared to control. AITC alone did not alter Smad3, Smad7, Acta2, or ECM related genes. However, the combination of AITC and TGF-β1 significantly increased Smad3 and decreased Smad7 and Acta2 compared to TGF-β1 alone; A-967079 significantly reduced this response. CONCLUSIONS:VFFs expressed TRPA1, and the activation of TRPA1 regulated TGF-β1/Smad signaling in VFFs. These findings provide preliminary insights into potential anti-fibrotic mechanisms of TRPA1 activation through TGF-β1/Smad signaling in VFFs. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2024.

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/METHODS:4 Laryngoscope, 134:4599-4603, 2024.

Endotracheal Tubes (ETTs) maintain and secure a patent airway; however, prolonged intubation often results in unintended injury to the mucosal epithelium and inflammatory sequelae which complicate recovery. ETT design and materials used have yet to adapt to address intubation associated complications. In this study, a composite coating of electrospun polycaprolactone (PCL) fibers embedded in a four-arm polyethylene glycol acrylate matrix (4APEGA) is developed to transform the ETT from a mechanical device to a dual-purpose device capable of delivering multiple therapeutics while preserving coating integrity. Further, the composite coating system (PCL-4APEGA) is capable of sustained delivery of dexamethasone from the PCL phase and small interfering RNA (siRNA) containing polyplexes from the 4APEGA phase. The siRNA is released rapidly and targets smad3 for immediate reduction in pro-fibrotic transforming growth factor-beta 1 (TGFϐ1) signaling in the upper airway mucosa as well as suppressing long-term sequelae in inflammation from prolonged intubation. A bioreactor was used to study mucosal adhesion to the composite PCL-4APEGA coated ETTs and investigate continued mucus secretory function in ex vivo epithelial samples. The addition of the 4APEGA coating and siRNA delivery to the dexamethasone delivery was then evaluated in a swine model of intubation injury and observed to restore mechanical function of the vocal folds and maintain epithelial thickness when observed over 14 days of intubation. This study demonstrated that increase in surface lubrication paired with surface stiffness reduction significantly decreased fibrotic behavior while reducing epithelial adhesion and abrasion.

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/METHODS:In vitro. METHODS:THP-1 monocyte-derived macrophages were stimulated with interferon-γ (IFN-γ), lipopolysaccharide (LPS), or transforming growth factor-β (TGF-β) 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/CONCLUSIONS: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/METHODS:N/A Laryngoscope, 2023.

OBJECTIVES/HYPOTHESIS/OBJECTIVE: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/METHODS:In vitro. METHODS: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/CONCLUSIONS: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/METHODS:N/A Laryngoscope, 2023.

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.

OBJECTIVES/HYPOTHESIS/OBJECTIVE: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/METHODS: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/CONCLUSIONS: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/METHODS:N/A Laryngoscope, 2022.

OBJECTIVES/OBJECTIVE:Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF-β1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis. METHODS: M) ± TGF-β1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real-time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre-injury day 5 to post-injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture. RESULTS: M) + TGF-β1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF-β1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment. CONCLUSIONS:TAM has antifibrotic potential via the regulation of TGF-β1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis. LEVEL OF EVIDENCE/METHODS:NA Laryngoscope, 2022.

OBJECTIVES/HYPOTHESIS/UNASSIGNED: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/UNASSIGNED: = 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/UNASSIGNED: = .004). CONCLUSIONS/UNASSIGNED: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.