On-tissue amidation of sialic acid with aniline for sensitive imaging of sialylated N-glycans from FFPE tissue sections via MALDI mass spectrometry.

Spatial visualization of glycans within clinical tissue samples is critical for discovery of disease-relevant glycan dysregulations. Herein, we develop an on-tissue derivatization strategy for sensitive spatial visualization of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections, based on amidation of sialic acid residues with aniline. The sialylated N-glycans were stabilized and given enhanced signal intensity owing to selective capping of a phenyl group to the sialic acid residue after aniline labeling. Proof-of-concept experiments, including determinations of sialylglycopeptide and N-glycans enzymatically released from glycoproteins, were performed. Further, mass spectrometry (MS) imaging of N-glycans on human laryngeal cancer FFPE tissue sections was conducted via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), based on our strategy for on-tissue amidation of sialylated N-glycans. We obtained higher sialylated N-glycan coverages for both the glycoproteins and cancer tissue samples, demonstrating that the detection sensitivity for sialylated N-glycans is notably improved by amidation derivatization. We also characterized N-glycan heterogeneity across the human laryngeal cancer tissue section, showing N-glycan dysregulation in the tumor region.

Quantification and molecular imaging of fatty acid isomers from complex biological samples by mass spectrometry.

Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend their biological functions in various physiological and pathological processes. Herein, we report a novel approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for localization of the C[double bond, length as m-dash]C bond in unsaturated FAs, which enables both quantification and spatial visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the C[double bond, length as m-dash]C positions were produced upon fragmentation of the FA epoxides derived from either in-solution or on-tissue PAA epoxidation of free FAs. The performance of the proposed approach was evaluated by analysis of FAs in human cell lines as well as mapping the FA isomers from cancer tissue samples with MALDI-TOF/TOF-MS. Merits of the newly developed method include high sensitivity, simplicity, high reaction efficiency, and capability of spatial characterization of FA isomers in tissue samples.

High-resolution magnetic resonance and mass spectrometry imaging of the human larynx.

High-resolution, noninvasive and nondestructive imaging of the subepithelial structures of the larynx would enhance microanatomic tissue assessment and clinical decision making; similarly, in situ molecular profiling of laryngeal tissue would enhance biomarker discovery and pathology readout. Towards these goals, we assessed the capabilities of high-resolution magnetic resonance imaging (MRI) and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) imaging of rarely reported paediatric and adult cadaveric larynges that contained pathologies. The donors were a 13-month-old male, a 10-year-old female with an infraglottic mucus retention cyst and a 74-year-old female with advanced polypoid degeneration and a mucus retention cyst. MR and molecular imaging data were corroborated using whole-organ histology. Our MR protocols imaged the larynges at 45-117 µm2 in-plane resolution and capably resolved microanatomic structures that have not been previously reported radiographically-such as the vocal fold superficial lamina propria, vocal ligament and macula flavae; age-related tissue features-such as intramuscular fat deposition and cartilage ossification; and the lesions. Diffusion tensor imaging characterised differences in water diffusivity, primary tissue fibre orientation, and fractional anisotropy between the intrinsic laryngeal muscles, mucosae and lesions. MALDI-MS imaging revealed peptide signatures and putative protein assignments for the polypoid degeneration lesion and the N-glycan constituents of one mucus retention cyst. These imaging approaches have immediate application in experimental research and, with ongoing technology development, potential for future clinical application.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis.

OBJECTIVE: Idiopathic subglottic stenosis (iSGS) is a chronic inflammatory condition that causes dyspnea and affects middle-aged women of White race and non-Latino or Hispanic ethnicity. To better characterize its phenotype and pathogenesis, we assessed the proteomic and genomic methylation signatures of subglottic tissue collected from iSGS patients compared to controls. STUDY DESIGN: Molecular analysis of clinical biospecimens. METHODS: We collected subglottic tissue biopsies from 12 patients during direct laryngoscopy, immediately prior to surgical treatment of iSGS; as well as from 4 age-, sex-, and race/ethnicity-matched control patients undergoing other direct laryngoscopic procedures. We isolated protein and genomic DNA, acquired proteomic data using label-free quantitative mass spectrometry techniques, and acquired genome-wide methylation data using bisulfite conversion and a microarray platform. We compared molecular profiles across the iSGS and control groups, and with respect to clinical course in the iSGS group. Eight of the 12 iSGS patients underwent subsequent blood collection and plasma isolation for further assessment. RESULTS: Proteomic analysis revealed 42 differentially abundant proteins in the iSGS biopsies compared to controls, inferring enrichment of biological pathways associated with early wound healing, innate immunity, matrix remodeling, and metabolism. Proteome-based hierarchical clustering organized patients into two iSGS and one control subgroups. Methylation analysis revealed five hypermethylated genes in the iSGS biopsies compared to controls, including the biotin recycling enzyme biotinidase (BTD). Follow-up analysis showed elevated plasma BTD activity in iSGS patients compared to both controls and published normative data. CONCLUSION: iSGS exhibits distinct proteomic and genomic methylation signatures. These signatures expand current understanding of the iSGS phenotype, support the possibility of disease subgroups, and should inform the direction of future experimental studies. LEVEL OF EVIDENCE: Not applicable Laryngoscope, 131:E540-E546, 2021.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Differentiated fibrocytes assume a functional mesenchymal phenotype with regenerative potential.

Fibrocytes (FCs) are hematopoietic lineage cells that migrate to sites of injury, transition to a mesenchymal phenotype, and help to mediate wound repair. Despite their relevance to human fibrotic disorders, there are few data characterizing basic FC biology. Herein, using proteomic, bioenergetic, and bioengineering techniques, we conducted deep phenotypic characterization of differentiating and mature FCs. Differentiation was associated with metabolic reprogramming that favored oxidative phosphorylation. Mature FCs had distinct proteomes compared to classic mesenchymal cells, formed functional stromae that supported epithelial maturation during in vitro organotypic culture, and exhibited in vivo survival and self-tolerance as connective tissue isografts. In an in vitro scratch assay, FCs promoted fibroblast migration and wound closure by paracrine signaling via the chemokine CXCL8 (interleukin-8). These findings characterize important aspects of FC differentiation and show that, in addition to their role in wound healing, FCs hold potential as an easily isolated autologous cell source for regenerative medicine.

High- and ultrahigh-field magnetic resonance imaging of naïve, injured and scarred vocal fold mucosae in rats.

Subepithelial changes to the vocal fold mucosa, such as fibrosis, are difficult to identify using visual assessment of the tissue surface. Moreover, without suspicion of neoplasm, mucosal biopsy is not a viable clinical option, as it carries its own risk of iatrogenic injury and scar formation. Given these challenges, we assessed the ability of high- (4.7 T) and ultrahigh-field (9.4 T) magnetic resonance imaging to resolve key vocal fold subepithelial tissue structures in the rat, an important and widely used preclinical model in vocal fold biology. We conducted serial in vivo and ex vivo imaging, evaluated an array of acquisition sequences and contrast agents, and successfully resolved key anatomic features of naïve, acutely injured, and chronically scarred vocal fold mucosae on the ex vivo scans. Naïve lamina propria was hyperintense on T1-weighted imaging with gadobenate dimeglumine contrast enhancement, whereas chronic scar was characterized by reduced lamina propria T1 signal intensity and mucosal volume. Acutely injured mucosa was hypointense on T2-weighted imaging; lesion volume steadily increased, peaked at 5 days post-injury, and then decreased - consistent with the physiology of acute, followed by subacute, hemorrhage and associated changes in the magnetic state of hemoglobin and its degradation products. Intravenous administration of superparamagnetic iron oxide conferred no T2 contrast enhancement during the acute injury period. These findings confirm that magnetic resonance imaging can resolve anatomic substructures within naïve vocal fold mucosa, qualitative and quantitative features of acute injury, and the presence of chronic scar.

Practice variations in voice treatment selection following vocal fold mucosal resection.

OBJECTIVES/HYPOTHESIS: To characterize initial voice treatment selection following vocal fold mucosal resection in a Medicare population. STUDY DESIGN: Retrospective analysis of a large, nationally representative Medicare claims database. METHODS: Patients with > 12 months of continuous Medicare coverage who underwent a leukoplakia- or cancer-related vocal fold mucosal resection (index) procedure during calendar years 2004 to 2009 were studied. The primary outcome of interest was receipt of initial voice treatment (thyroplasty, vocal fold injection, or speech therapy) following the index procedure. We evaluated the cumulative incidence of each postindex treatment type, treating the other treatment types as competing risks, and further evaluated postindex treatment utilization using the proportional hazards model for the subdistribution of a competing risk. Patient age, sex, and Medicaid eligibility were used as predictors. RESULTS: A total of 2,041 patients underwent 2,427 index procedures during the study period. In 14% of cases, an initial voice treatment event was identified. Women were significantly less likely to receive surgical or behavioral treatment compared to men. From age 65 to 75 years, the likelihood of undergoing surgical treatment increased significantly with each 5-year age increase; after age 75 years, the likelihood of undergoing either surgical or behavioral treatment decreased significantly every 5 years. Patients with low socioeconomic status were significantly less likely to undergo speech therapy. CONCLUSION: The majority of Medicare patients do not undergo voice treatment following vocal fold mucosal resection. Further, the treatments analyzed here appear disproportionally utilized based on patient sex, age, and socioeconomic status. Additional research is needed to determine whether these observations reflect clinically explainable differences or disparities in care. LEVEL OF EVIDENCE: 2c. Laryngoscope, 126:2505-2512, 2016.

Bioengineered vocal fold mucosa for voice restoration.

Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration and physiologically capable of maintaining a barrier against the airway lumen. We isolated primary human VF fibroblasts and epithelial cells and cocultured them under organotypic conditions. The resulting engineered mucosae showed morphologic features of native tissue, proteome-level evidence of mucosal morphogenesis and emerging extracellular matrix complexity, and rudimentary barrier function in vitro. When grafted into canine larynges ex vivo, the mucosae generated vibratory behavior and acoustic output that were indistinguishable from those of native VF tissue. When grafted into humanized mice in vivo, the mucosae survived and were well tolerated by the human adaptive immune system. This tissue engineering approach has the potential to restore voice function in patients with otherwise untreatable VF mucosal disease.

Clinical features and surgical outcomes following closed reduction of arytenoid dislocation.

IMPORTANCE: Arytenoid dislocation is a rare condition characterized by vocal fold immobility and is easily mistaken as recurrent laryngeal nerve paralysis. OBJECTIVE: To describe the presenting features, multimodal diagnostic evaluation, and surgical outcomes following closed reduction (CR) of arytenoid dislocation. DESIGN, SETTING, AND PARTICIPANTS: Prospective case series at a single academic medical center. Evaluation and treatment data were obtained from 22 consecutive patients with arytenoid dislocation over a 7-year period. INTERVENTIONS: Patients underwent direct laryngoscopy and CR of the dislocated arytenoid, with adjunct injection laryngoplasty or botulinum toxin administration in select cases. MAIN OUTCOMES AND MEASURES: Initial diagnosis was confirmed using flexible laryngeal endoscopy with stroboscopy, computed tomography, electromyography, and interoperative palpation. Arytenoid motion (primary outcome measure) and vocal function data (secondary outcome measures) were collected before treatment and up to 6 months after treatment. RESULTS: Key history features included emergent intubation, elective intubation, and external laryngeal trauma. Sixteen patients (73%) had anterior and 6 patients (27%) posterior dislocation. One patient experienced spontaneous recovery. Following CR, with or without adjunct therapy, 18 of the remaining patients (86%) exhibited arytenoid motion recovery with concomitant voice improvement. Recovery was sustained at 6 months after CR. Closed reduction performed within 21 days of the presumed dislocation event was associated with a superior arytenoid motion recovery rate. CONCLUSIONS AND RELEVANCE: These data represent the largest clinical series on arytenoid dislocation with complete vocal function data and follow-up at 6 months after CR. These findings also corroborate existing evidence for early surgical intervention.

Correlation between Reflux and multichannel intraluminal impedance pH monitoring in untreated volunteers.

OBJECTIVES/HYPOTHESIS: Although probable causative agents have been identified (e.g., refluxate components, tobacco smoke), the definitive mechanism for inflammation-related laryngeal mucosal damage remains elusive. Multichannel intraluminal impedance combined with pH monitoring (MII/pH) has emerged as a sensitive tool for diagnosis and characterization of gastroesophageal reflux disease with laryngopharyngeal manifestations. To determine the relationship between laryngeal signs and MII/pH, we examined correlations between Reflux Finding Score (RFS) ratings of videostroboscopic laryngeal examinations and findings from MII/pH. STUDY DESIGN: Correlational study. METHODS: Healthy, untreated volunteers (n = 142) underwent reflux diagnosis using data acquired from MII/pH testing. Eight trained clinicians performed RFS ratings of corresponding laryngeal examinations. Averaged RFS ratings were compared to MII/pH data using Pearson correlation coefficients. The relationship between RFS and MII/pH findings and demographic/clinical information (age, sex, smoking status, reflux) was assessed using general linear modeling. Rater reliability was evaluated. RESULTS: Posterior commissure hypertrophy was negatively correlated with minutes of nonacid refluxate (R = -0.21, P = .0115). General linear modeling revealed that 28% to 40% of the variance in ratings of ventricular obliteration, erythema/hyperemia, vocal fold edema, diffuse laryngeal edema, posterior commissure hypertrophy, and granulation/granuloma could be explained by main and interaction effects of age, sex, smoking status, and reflux. Intra- and inter-rater reliability for RFS were poor-fair. CONCLUSIONS: These results support the theory that the RFS is not specific for reflux in healthy, untreated volunteers, suggesting there may be alternate explanations for inflammatory clinical signs commonly ascribed to reflux in this population.

TGF-ß3 modulates the inflammatory environment and reduces scar formation following vocal fold mucosal injury in rats.

Transforming growth factor (TGF)-ß1 and TGF-ß3 have been reported to exert differential effects on wound healing, and possibly even account for tissue-specific differences in scar formation. Scarring is particularly detrimental in the vocal fold mucosa (VFM), where destruction of the native extracellular matrix causes irreparable biomechanical changes and voice impairment. Here, in a series of in vitro and in vivo experiments, we identified differences in TGF-ß1 and TGF-ß3 transcription and immunolocalization to various cell subpopulations in naïve and injured rat VFM, compared with oral mucosa (which undergoes rapid healing with minimal scar) and skin (which typically heals with scar). Treatment of cultured human vocal fold fibroblasts with TGF-ß3 resulted in less potent induction of profibrotic gene transcription, extracellular matrix synthesis and fibroblast-myofibroblast differentiation, compared with treatment with TGF-ß1 and TGF-ß2. Finally, delivery of exogenous TGF-ß3 to rat VFM during the acute injury phase modulated the early inflammatory environment and reduced eventual scar formation. These experiments show that the TGF-ß isoforms have distinct roles in VFM maintenance and repair, and that TGF-ß3 redirects wound healing to improve VFM scar outcomes in vivo.

Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa.

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.

E-cadherin and transglutaminase-1 epithelial barrier restoration precedes type IV collagen basement membrane reconstruction following vocal fold mucosal injury.

The vocal fold epithelium is critical to upper airway immunologic defense and water/ion transport; therefore, any form of physical trauma or insult increases the vulnerability of this structure to functional impairment and pathogen invasion/infection. In this study, we examined the reestablishment of epithelial and basement membrane barrier structures in a well-established rat model of vocal fold mucosal injury. We observed active cell recruitment culminating in peak hyperplasia at 3 days postinjury, the establishment of robust E-cadherin+ and transglutaminase-1+ biochemical barrier signals along the epithelial surface by 3 days postinjury, and the persistent absence of a type IV collagen+ basement membrane at 7 days postinjury. The distinct spatial and temporal immunoactivity of these molecules is consistent with a programmed repair process driving the restoration of vocal fold mucosal integrity and permeability. These data may inform future efforts to optimize functional mucosal recovery postinjury and avoid undesirable events such as barrier compromise or epithelial metaplasia.

Chronic rhinosinusitis with nasal polyps: a proteomic analysis.

OBJECTIVES: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a severe subtype of chronic rhinosinusitis that can affect patients despite medical and surgical interventions. The purpose of this study was to utilize the techniques of proteomics to investigate differences in protein abundance within the sinonasal mucosa of patients with CRSwNP compared to healthy controls. METHODS: In a case-control study at a tertiary-care academic medical center, sinonasal mucosa was harvested from 3 patients with CRSwNP and 3 control patients undergoing transsphenoidal excision of pituitary tumors. Two-dimensional gel electrophoresis was used to identify proteins with elevated or reduced abundance in CRSwNP patients compared to controls. The proteins showing the greatest abundance differences were characterized by mass spectrometry. RESULTS: More than 300 differentially abundant proteins (p < or = 0.05) were identified. Many of these protein species were involved in the host inflammatory response. Proteins up-regulated in CRSwNP patients included eosinophil lysophospholipase by a ratio (R) of 18.13, RHO-GDP dissociation inhibitor 2 (R = 2.80), and apolipoprotein A-1 (R = 1.73). Down-regulated proteins in CRSwNP patients included catalase (R = -5.87), annexin A1 (R = -6.27), and keratin II-8 (R = -6.73). A detailed analysis of additional protein species is outlined. CONCLUSIONS: The proteomic approach allows detection of significant differences in protein abundance in CRSwNP and provides unique insight into the pathophysiology of this common disease.

Characterization of vocal fold scar formation, prophylaxis, and treatment using animal models.

PURPOSE OF REVIEW: To review recent literature on animal models used to study the pathogenesis, detection, prevention, and treatment of vocal fold scarring. Animal work is critical to studying vocal fold scarring because it is the only way to conduct systematic research on the biomechanical properties of the layered structure of the vocal fold lamina propria, and therefore develop reliable prevention and treatment strategies for this complex clinical problem. RECENT FINDINGS: During the period of review, critical anatomic, physiologic, and wound healing characteristics, which may serve as the bases for selection of a certain species to help answer a specific question, have been described in mouse, rat, rabbit, ferret, and canine models. A number of different strategies for prophylaxis and chronic scar treatment in animals show promise for clinical application. The pathways of scar formation and methods for quantifying treatment-induced change have become better defined. SUMMARY: Recent animal vocal fold scarring studies have enriched and confirmed earlier work indicating that restoring pliability to the scarred vocal fold mucosa is challenging but achievable. Differences between animal models and differences in outcome measurements across studies necessitate considering each study individually to obtain guidance for future research. With increased standardization of measurement techniques it may be possible to make more inter-study comparisons.

Implantation of atelocollagen sheet for vocal fold scar.

PURPOSE OF REVIEW: This article reviews recent advances in scaffold-based interventions for the treatment of vocal fold scarring, with a particular emphasis on atelocollagen sheet implantation in the vocal fold lamina propria. RECENT FINDINGS: Scaffold-based therapies have demonstrated therapeutic promise in both preclinical and early clinical studies. Recent research has begun to shed light on the interactions between scaffold material properties, encapsulated and infiltrating cells, stimulatory molecules such as growth factors, and external regulatory variables such as stress, strain, and vibration. The atelocollagen sheet, a cross-linked collagen material with abundant micropores, has an established clinical track record as a scaffold for dermal and epidermal repair and exhibited potential therapeutic benefit in a recent study of patients with vocal fold scarring and sulcus vocalis. SUMMARY: Scaffolding is one of the useful tools in tissue engineering and atelocollagen sheet implantation has been shown to be effective in vocal fold regeneration. However, many of the scaffold materials under investigation still await clinical translation and those that have been investigated in human patients (such as the atelocollagen sheet) require additional research in appropriately powered placebo-controlled studies.

Pulsed dye laser-induced inflammatory response and extracellular matrix turnover in rat vocal folds and vocal fold fibroblasts.

BACKGROUND AND OBJECTIVES: Disruption of the vocal fold extracellular matrix (ECM) can induce a profound and refractory dysphonia. Pulsed dye laser (PDL) irradiation has shown early promise as a treatment modality for disordered ECM in patients with chronic vocal fold scar; however, there are limited data addressing the mechanism by which this laser energy might induce cellular and extracellular changes in vocal fold tissues. In this study, we examined the inflammatory and ECM modulating effects of PDL irradiation on normal vocal fold tissues and cultured vocal fold fibroblasts (VFFs). STUDY DESIGN/MATERIALS AND METHODS: We evaluated the effects of 585 nm PDL irradiation on inflammatory cytokine and collagen/collagenase gene transcription in normal rat vocal folds in vivo (3-168 hours following delivery of approximately 39.46 J/cm(2) fluence) and VFFs in vitro (3-72 hours following delivery of 4.82 or 9.64 J/cm(2) fluence). We also examined morphological vocal fold tissue changes 3 hours, 1 week, and 1 month post-irradiation. RESULTS: PDL irradiation altered inflammatory cytokine and procollagen/collagenase expression at the transcript level, both in vitro and in vivo. Additionally, PDL irradiation induced an inflammatory repair process in vivo that was completed by 1 month with preservation of normal tissue morphology. CONCLUSIONS: PDL irradiation can modulate ECM turnover in phenotypically normal vocal folds. Additional work is required to determine if these findings extend to disordered ECM, such as is seen in vocal fold scar. Lasers Surg. Med. 41:585-594, 2009. (c) 2009 Wiley-Liss, Inc.

Surgical method to create vocal fold injuries in mice.

OBJECTIVES: The goal of this study was to develop a surgical method for the creation of vocal fold injuries in mice, as a precursor to the use of genetically engineered mouse models in the study of vocal fold wound healing and scar formation. METHODS: Seven FVB strain mice were used in this study. A laryngoscope and 3 micro-instruments were designed and fabricated to facilitate endoscopic vocal fold visualization and the creation of vocal fold surgical injuries. The larynges were harvested 1 and 7 days after surgery, and the vocal fold injury sites were evaluated by routine hematoxylin and eosin staining. Additional immunohistochemical analysis of collagen type I and elastin distribution in the lamina propria was performed for an uninjured control larynx. RESULTS: Endoscopic visualization and vocal fold stripping resulting in thyroarytenoid muscle exposure were successful in all animals. Histologic and immunohistochemical analyses revealed a simple lamina propria structure with relatively even collagen type I and elastin distribution in the control vocal fold, obliteration of vocal fold mucosa 1 day after surgery, and complete reepithelialization by 7 days. CONCLUSIONS: These results demonstrate the feasibility of creating reproducible vocal fold injuries via an endoscopic approach in mice. The observation that the mouse lamina propria may have a relatively simple histologic structure indicates that additional characterization should be performed and caution used in translating findings between this and other model systems.

Viscoelastic measurements of vocal folds using the linear skin rheometer.

As the number of interventions for vocal fold scar grows and with the advancement of mathematical modeling, greater accuracy and precision in the measurement of vocal fold pliability will become essential. Although indirect pliability measures have been used successfully, direct measurement of tissue pliability is essential. Indirect measurement with parallel plate technology has limitations; it requires the tissue to be removed from the surrounding framework, allows no site specificity, and offers no future for in vivo use in animals or humans. We tested the linear skin rheometer (LSR) in the evaluation of vocal fold pliability. We measured site-specific rheology of vocal folds thereby creating "pliability maps" in human, dog, and rat cadaveric larynges under conditions of altered stiffness; the canine vocal folds possessed sulci, the rat vocal fold was stiff secondary to controlled biopsy, and the human vocal fold was injected with trichloroacetic acid. Histology was performed to confirm the site and type of canine sulci. We found that the LSR reliably detected stiffness in the vocal folds of all species and created "pliability maps" consistent with previous data and clinical observations. The LSR should prove useful in the evaluation of vocal fold pliability for ex vivo and ultimately for in vivo applications.