An Integrated Experimental-Computational Study of Vocal Fold Vibration in Type I Thyroplasty.

Subject-specific computational modeling of vocal fold (VF) vibration was integrated with an ex vivo animal experiment of type 1 thyroplasty to study the effect of the implant on the vocal fold vibration. In the experiment, a rabbit larynx was used to simulate type 1 thyroplasty, where one side of the vocal fold was medialized with a trans-muscular suture while the other side was medialized with a silastic implant. Vocal fold vibration was then achieved by flowing air through the larynx and was filmed with a high-speed camera. The three-dimensional computational model was built upon the pre-operative scan of the laryngeal anatomy. This subject-specific model was used to simulate the vocal fold medialization and then the fluid-structure interaction (FSI) of the vocal fold. Model validation was done by comparing the vocal fold displacement with postoperative scan (for medialization), and by comparing the vibratory characteristics with the high-speed images (for vibration). These comparisons showed the computational model successfully captured the effect of the implant and thus has the potential for presurgical planning.

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.

Subject-Specific Modeling of Implant Placement for Type I Thyroplasty Surgery.

Type I thyroplasty is widely used to improve voice production in patients affected by unilateral vocal fold paralysis. Almost two-thirds of laryngologists report using Silastic® implants to medialize the vocal fold, with implant size, shape, and location determined experientially. However, post-surgical complications arising from this procedure (extrusion, migration, resizing) necessitate revision in 4.5-16% of patients. To improve initial surgical outcomes, we have developed a subject-specific modeling tool, PhonoSim, which uses model reconstruction from MRI scans to predict the optimal implantation location. Eleven vocal fold sample sides from eight larynges of New Zealand white rabbits were randomized to two groups: PhonoSim informed (n = 6), and control (no model guidance, n = 5). Larynges were scanned ex vivo in the abducted configuration using a vertical-bore 11.7 T microimaging system, and images were used for subject-specific modeling. The PhonoSim tool simulated vocal fold adduction for multiple implant location placements to evaluate vocal fold adduction at the medial surface. The best implant placement coordinates were output for the 6 samples in the PhonoSim group. Control placements were determined by the same surgeon based on anatomical landmarks. Post-surgical MRI scans were performed for all samples to evaluate medialization in implanted vocal folds. Results show that PhonoSim-guided implantation achieved higher vocal fold medialization relative to controls (28 to 55% vs. - 29 to 39% respectively, in the glottal area reduction), suggesting that this tool has the potential to improve outcomes and revision rates for type I thyroplasty.

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 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.

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.

Subject-Specific Computational Fluid-Structure Interaction Modeling of Rabbit Vocal Fold Vibration.

A full three-dimensional (3D) fluid-structure interaction (FSI) study of subject-specific vocal fold vibration is carried out based on the previously reconstructed vocal fold models of rabbit larynges. Our primary focuses are the vibration characteristics of the vocal fold, the unsteady 3D flow field, and comparison with a recently developed 1D glottal flow model that incorporates machine learning. The 3D FSI model applies strong coupling between the finite-element model for the vocal fold tissue and the incompressible Navier-Stokes equation for the flow. Five different samples of the rabbit larynx, reconstructed from the magnetic resonance imaging (MRI) scans after the in vivo phonation experiments, are used in the FSI simulation. These samples have distinct geometries and a different inlet pressure measured in the experiment. Furthermore, the material properties of the vocal fold tissue were determined previously for each individual sample. The results demonstrate that the vibration and the intraglottal pressure from the 3D flow simulation agree well with those from the 1D flow model based simulation. Further 3D analyses show that the inferior and supraglottal geometries play significant roles in the FSI process. Similarity of the flow pattern with the human vocal fold is discussed. This study supports the effective usage of rabbit larynges to understand human phonation and will help guide our future computational studies that address vocal fold disorders.

Exploring the presence of multiple abnormal non-motor features in patients with cervical dystonia.

This study's aim was to investigate prevalence of four non-motor symptoms in patients with cervical dystonia and healthy controls to explore whether the presence of multiple non-motor features is associated with cervical dystonia diagnosis. Fifteen patients with cervical dystonia and 15 healthy controls underwent non-invasive testing of spatial discrimination threshold, temporal discrimination threshold, vibration-induced illusion of movement, and kinesthesia. All spatial discrimination threshold, temporal discrimination threshold, and vibration-induced illusion of movement measures were converted to standardized Z scores with scores >2.0 considered abnormal. Any incorrect kinesthesia response was considered abnormal. Prevalence of each abnormal non-motor feature was compared between groups using a chi-squared test. A higher proportion of patients with cervical dystonia had abnormal spatial discrimination threshold (p = 0.01) and abnormal kinesthesia (p = 0.03) scores compared to healthy control subjects. There were no significant differences between the proportion of patients with cervical dystonia versus healthy controls for abnormal temporal discrimination threshold (p = 0.07) or abnormal vibration-induced illusion of movement (p = 0.14). Forty-seven percent of patients with cervical dystonia (7/15) demonstrated one abnormal non-motor feature, 20% (3/15) displayed two abnormal features, and 13% (2/15) displayed three abnormal features. Kinesthesia was the only non-motor feature identified as abnormal in the control group (20%, 3/15). All four tests demonstrated high specificity (80-100%) and low-moderate sensitivity (13-60%). These findings suggest that non-motor feature testing, specifically for spatial discrimination threshold and kinesthesia, could be a highly specific diagnostic tool to inform cervical dystonia diagnosis. Further investigation is needed to confirm these findings.

Numerical and experimental investigations on vocal fold approximation in healthy and simulated unilateral vocal fold paralysis.

We have developed a novel surgical/computational model for the investigation of unilateral vocal fold paralysis (UVFP) which will be used to inform future in silico approaches to improve surgical outcomes in type I thyroplasty. Healthy phonation (HP) was achieved using cricothyroid suture approximation on both sides of the larynx to generate symmetrical vocal fold closure. Following high-speed videoendoscopy (HSV) capture, sutures on the right side of the larynx were removed, partially releasing tension unilaterally and generating asymmetric vocal fold closure characteristic of UVFP (sUVFP condition). HSV revealed symmetric vibration in HP, while in sUVFP the sutured side demonstrated a higher frequency (10 - 11%). For the computational model, ex vivo magnetic resonance imaging (MRI) scans were captured at three configurations: non-approximated (NA), HP, and sUVFP. A finite-element method (FEM) model was built, in which cartilage displacements from the MRI images were used to prescribe the adduction and the vocal fold deformation was simulated before the eigenmode calculation. The results showed that the frequency comparison between the two sides were consistent with observations from HSV. This alignment between the surgical and computational models supports the future application of these methods for the investigation of treatment for UVFP.

Protein Substrate Alters Cell Physiology in Primary Culture of Vocal Fold Epithelial Cells.

The basement membrane interacts directly with the vocal fold epithelium. Signaling between the basement membrane and the epithelium modulates gene regulation, differentiation, and proliferation. The purpose of this study was to identify an appropriate simple single-protein substrate for growth of rabbit vocal fold epithelial cells. Vocal folds from 3 New Zealand white rabbits (Oryctolagus cuniculus) were treated to isolate epithelial cells, and cells were seeded onto cell culture inserts coated with collagen I, collagen IV, laminin, or fibronectin. Transepithelial electrical resistance (TEER) was measured, and phase contrast microscopy, PanCK, CK14, and E-cadherin immunofluorescence were utilized to assess for epithelial cell-type characteristics. Further investigation via immunofluorescence labeling was conducted to assess proliferation (Ki67) and differentiation (Vimentin). There was a significant main effect of substrate on TEER, with collagen IV eliciting the highest, and laminin the lowest resistance. Assessment of relative TEER across cell lines identified a larger range of TEER in collagen I and laminin. Phase contrast imaging identified altered morphology in the laminin condition, but cell layer depth did not appear to be related to TEER, differentiation, or morphology. Ki67 staining additionally showed no significant difference in proliferation. All conditions had confluent epithelial cells and dispersed mesenchymal cells, with increased mesenchymal cell numbers over time; however, a higher proportion of mesenchymal cells was observed in the laminin condition. The results suggest collagen IV is a preferable basement membrane substrate for in vitro vocal fold epithelial primary cell culture, providing consistent TEER and characteristic cell morphology, and that laminin is an unsuitable substrate for vocal fold epithelial cells and may promote mesenchymal cell proliferation.

Medialization Laryngoplasty: A Review for Speech-Language Pathologists.

Purpose The purpose of this study is to familiarize speech-language-pathologists with the current state of the science regarding medialization laryngoplasty in the treatment of voice disorders, with emphasis on current evidence-based practice, voice outcomes, and future directions for research. Method A literature review was performed in PubMed and Embase using the keywords vocal fold/cord and laryngoplasty, thyroplasty, augmentation, or laryngeal framework. Articles published between 2010 and 2020 were reviewed for data about clinical applications, technical approach, voice-related outcomes, and basic science or clinical innovations with the potential to improve patient care. A synthesis of data was performed from articles meeting the outlined search criteria. Conclusions As key members in the multidisciplinary care of voice disorders, speech-language pathologists need to be informed of current research in medialization laryngoplasty, a procedure commonly used for patients with glottic insufficiency. Advances in anesthetic technique, office-based procedures, and the development of materials with increased bio-tolerability over the past decade have led to innovations in treatment and improved patient outcomes. Recent applications of computational and bioengineering approaches have the potential to provide new directions in the refinement of currently available techniques and the improvement of patient-based treatment outcomes.

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.

Prevalence of Comorbid Spasticity and Urinary Incontinence in Residents of a Long-Term Care Facility.

The current study evaluated the prevalence of comorbid spasticity and urinary incontinence (UI) in a long-term care facility. Medical history, presence of UI, and activities of daily living (ADL) dependency were obtained from medical records and Minimum Data Set 3.0. Quality of life was assessed with the EuroQoL-5D-5L (EQ-5D). Comorbid spasticity and UI presented in 29% of participants (14 of 49). Participants with spasticity and UI had higher ADL dependency and lower EQ-5D than participants without both conditions (4.9, 95% confidence interval [CI] [1.6, 80.], p = 0.003; -0.17, 95% CI [-0.33, 0.00], p = 0.044; respectively). More than one half of participants with lower limb spasticity had severe UI, compared to only 10% without lower limb spasticity (relative risk = 5.5; 95% CI [1.9, 15.9]; p = 0.006). Comorbid spasticity and UI may be common in the long-term care setting and negatively associated with ADL and quality of life. Further investigation is needed to confirm these findings. [Journal of Gerontological Nursing, 46(10), 35-42.].

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.