Voice-Vibratory Assessment With Laryngeal Imaging (VALI) Form: Reliability of Rating Stroboscopy and High-speed Videoendoscopy.

OBJECTIVE: The purpose of the study was to evaluate the inter-judge and intra-judge reliability of raters using the Voice-Vibratory Assessment with Laryngeal Imaging (VALI) rating form that was developed for assessing videostroboscopy and high-speed videoendoscopic (HSV) recordings. SUBJECTS AND METHODS: Nine speech-language pathologists with an average of 12.8 years of experience with laryngeal imaging were trained to use the VALI form for rating 66 de-identified and randomized samples with voice disorders. Inter-judge reliability for parameters with scale data (amplitude, mucosal wave, nonvibratory portion, supraglottic activity, phase closure, symmetry, and regularity or periodicity) was assessed with intraclass correlations, and parameters with nominal data (glottal closure, vertical level, and free edge contour) were assessed with Fleiss' kappa. Intra-judge reliability was assessed using the Spearman rho statistic for scale data and percentage of concordant pairs for nominal data. RESULTS: Inter-judge reliability for parameters with scale data ranged from 0.57 to 0.96 for stroboscopy and from 0.81 to 0.94 for HSV. For nominal parameters, correlations ranged from 0.18 to 0.35 for stroboscopy and from 0.13 to 0.33 for HSV. Intra-judge reliability correlations for parameters with scale data ranged from 0.19 to 0.87 for stroboscopy and from 0.28 to 0.85 for HSV. For parameters with nominal data, percentage of concordance ranged from 44% to 78% for stroboscopy and from 52% to 89% for HSV. CONCLUSIONS: The VALI rating form and the training protocol is a first, a priori developed rating form that includes visual-perceptual ratings of both stroboscopy and HSV. The current form can be used to make reliable visual-perceptual judgments for selected features of vibratory motion from stroboscopy and HSV.

Histological Effect of Basic Fibroblast Growth Factor on Chronic Vocal Fold Scarring in a Rat Model.

OBJECTIVES: Vocal fold scarring is one of the most challenging laryngeal disorders to treat and there are currently no consistently effective treatments available. Our previous studies have shown the therapeutic potential of basic fibroblast growth factor (bFGF) for vocal fold scarring. However, the histological effects of bFGF on scarred vocal fold have not been elucidated. The aim of this study was to examine the histological effects of bFGF on chronic vocal fold scarring. METHODS: Sprague-Dawley rats were divided into phosphate buffered saline (sham) and bFGF groups. Unilateral vocal fold stripping was performed and the drug was injected into the scarred vocal fold for each group 2 months postoperatively. Injections were performed weekly for 4 weeks. Two months after the last injection, larynges were harvested and histologically analyzed. RESULTS: A significant increase of hyaluronic acid was observed in the vocal fold of the bFGF group compared with that of the sham group. However, there was no remarkable change in collagen expression nor in vocal fold contraction. CONCLUSION: Significant increase of hyaluronic acid by local bFGF injection was thought to contribute to the therapeutic effects on chronic vocal fold scarring.

Homeostasis of hyaluronic acid in normal and scarred vocal folds.

OBJECTIVES/HYPOTHESIS: Vocal fold scarring is one of the most challenging laryngeal disorders to treat. Hyaluronic acid (HA) is the main component of lamina propria, and it plays an important role in proper vocal fold vibration and is also thought to be important in fetal wound healing without scarring. Although several animal models of vocal fold scarring have been reported, little is known about the way in which HA is maintained in vocal folds. The purpose of this study was to clarify the homeostasis of HA by examining the expression of hyaluronan synthase (Has) and hyaluronidase (Hyal), which produce and digest HA, respectively. STUDY DESIGN: Experimental prospective animal study. METHODS: Vocal fold stripping was performed on 38 Sprague-Dawley rats. Vocal fold tissue was collected at five time points (3 days-2 months). Expression of HA was examined by immunohistochemistry, and messenger RNA (mRNA) expression of Has and Hyal was examined by real-time polymerase chain reaction and in-situ hybridization. RESULTS: In scarred vocal folds, expression of Has1 and Has2 increased at day 3 together with expression of HA and returned to normal at 2 weeks. At 2 months, Has3 and Hyal3 mRNA showed higher expressions than normal. CONCLUSIONS: Expression patterns of Has and Hyal genes differed between normal, acute-scarred, and chronic-scarred vocal folds, indicating the distinct roles of each enzyme in maintaining HA. Continuous upregulation of Has genes in the acute phase may be necessary to achieve scarless healing of vocal folds.

Interspecies comparison of stellate cell-containing macula flavae and vitamin A storage in vocal fold mucosa.

The macula flavae (MF), populated by vitamin A-storing stellate cells (SCs), are believed to play a fundamental role in development, maintenance and repair of the vocal fold (VF) mucosa; however, to date, they have mostly been examined in observational human cadaver studies. Here, we conducted an interspecies comparison of MF and SC phenotype, as well as vitamin A quantification and localization, in human, pig, dog, rabbit and rat VF mucosae. MF containing vitamin A-positive SCs were only identified in human and rat specimens. Pig, dog and rabbit VF mucosae contained no discernable MF, but rather exhibited preferential vitamin A localization to mucous (pig), serous (dog) or mixed (rabbit) glands. This glandular vitamin A storage corresponded to exceedingly high concentrations of retinol in pig and dog mucosae, and retinyl ester in dog mucosa. These findings have significant implications for the presumed role of the MF and SCs in VF biology, the nature of vitamin A storage within the VF mucosa, and the selection of an appropriate animal model for future experimental studies.

Effects of aging on thyroarytenoid muscle regeneration.

OBJECTIVES/HYPOTHESIS: Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following injury are unclear. The purpose of this study was to investigate the regenerative properties of the thyroarytenoid (TA) muscle in an aging rat model. The hypothesis was that following myotoxic injury, old animals would exhibit a decrease in mitotic activities of muscle satellite cells when compared with younger rats, suggesting reduced regenerative potential in the aging rat TA muscle. STUDY DESIGN: Animal group comparison. METHODS: Regeneration responses following injury to the TA muscle were examined in 18 young adult, middle-aged, and old Fischer 344/Brown Norway rats. TA muscle fiber cross-sectional area (CSA), satellite cell mitosis (number/fiber), and regeneration index (CSA injured side/CSA noninjured side) were measured and compared across age groups. RESULTS: Young adult animals had a significantly higher regeneration index than the middle-aged and old groups. Within the lateral region of the TA muscle (LTA), the regeneration index was significantly higher in the young adult animals than in the middle-aged and old animals. The regeneration index of the medial TA was significantly higher than the LTA across all age groups. CONCLUSIONS: The regenerative capacity of the TA muscle is impaired with increasing age.

Nonlinear dynamic-based analysis of severe dysphonia in patients with vocal fold scar and sulcus vocalis.

OBJECTIVE: The primary goal of this study was to evaluate a nonlinear dynamic approach to the acoustic analysis of dysphonia associated with vocal fold scar and sulcus vocalis. STUDY DESIGN: Case-control study. METHODS: Acoustic voice samples from scar/sulcus patients and age-/sex-matched controls were analyzed using correlation dimension (D2) and phase plots, time-domain based perturbation indices (jitter, shimmer, signal-to-noise ratio [SNR]), and an auditory-perceptual rating scheme. Signal typing was performed to identify samples with bifurcations and aperiodicity. RESULTS: Type 2 and 3 acoustic signals were highly represented in the scar/sulcus patient group. When data were analyzed irrespective of signal type, all perceptual and acoustic indices successfully distinguished scar/sulcus patients from controls. Removal of type 2 and 3 signals eliminated the previously identified differences between experimental groups for all acoustic indices except D2. The strongest perceptual-acoustic correlation in our data set was observed for SNR and the weakest correlation was observed for D2. CONCLUSIONS: These findings suggest that D2 is inferior to time-domain based perturbation measures for the analysis of dysphonia associated with scar/sulcus; however, time-domain based algorithms are inherently susceptible to inflation under highly aperiodic (ie, type 2 and 3) signal conditions. Auditory-perceptual analysis, unhindered by signal aperiodicity, is therefore a robust strategy for distinguishing scar/sulcus patient voices from normal voices. Future acoustic analysis research in this area should consider alternative (e.g., frequency- and quefrency-domain based) measures alongside additional nonlinear approaches.

Vocal fold vibratory characteristics in normal female speakers from high-speed digital imaging.

The purpose of the study was to investigate relationships between vocal fold vibrations and voice quality. Laryngeal images obtained from high-speed digital imaging (HSDI) were examined for their open-closed timing characteristics and perturbation values. A customized software delineated the glottal edges and used the Hilbert transform-based method of analysis to provide objective quantification of glottal perturbation. Overlay tracings of the transformed glottal cycles provided visual patterns on the overall vibratory dynamics. In this paper, we described the use of this method in looking at vibratory characteristics of a group of young female speakers (N=23). We found that, females with no voice complaints and who had been perceived to have normal voices were not a homogeneous group in terms of their glottal vibratory patterns during phonation. Their vibratory patterns showed characteristics similar to exemplar voices targeted to be clear (50%), pressed (27%), breathy (15%), or a mixed quality (8%). Perturbation range in terms of cycle-to-cycle frequency and amplitude was small and did not discriminate patterns. All these patterns yielded perceptually normal voices suggesting that in normal young speakers, the level of perturbation may be more important to the judgment than the actual pattern of closure.

Prospective multi-arm evaluation of surgical treatments for vocal fold scar and pathologic sulcus vocalis.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to compare the clinical effectiveness of type I thyroplasty, injection laryngoplasty, and graft implantation for the treatment of vocal fold scar and pathologic sulcus vocalis. STUDY DESIGN: Prospective, multi-arm, quasi-experimental research design. METHODS: Twenty-eight patients with newly diagnosed vocal fold scar and/or pathologic sulcus vocalis were assigned to one of three treatment modalities: type I thyroplasty (n = 9), injection laryngoplasty (n = 9), and graft implantation (n = 10). Psychosocial, auditory-perceptual, acoustic, aerodynamic, and videostroboscopic data were collected pretreatment and at 1, 6, 12, and 18 months posttreatment. RESULTS: Type I thyroplasty and graft implantation both resulted in reduced voice handicap with no concomitant improvement in auditory-perceptual, acoustic, aerodynamic, or vocal fold physiologic performance. Injection laryngoplasty resulted in no improvement on any vocal function index. Patients who underwent graft implantation exhibited the slowest improvement trajectory across the 18-month follow-up period. CONCLUSIONS: A persistent challenge in this area is that no single treatment modality is successful for the majority of patients, and there is no evidence-based decision algorithm for matching a given treatment to a given patient. Progress therefore requires the identification and categorization of predictive clinical features that can drive evidence-based treatment assignment.

Differential vibratory characteristics of adductor spasmodic dysphonia and muscle tension dysphonia on high-speed digital imaging.

OBJECTIVES: The purpose of this study was to quantify disorder-specific signature kinematic disturbances of vibratory motion in adductor spasmodic dysphonia (AdSD) and muscle tension dysphonia (MTD), in voice disturbances of a severe nature, with the use of high-speed digital imaging (HSDI). A secondary hypothesis of the study was to investigate the sensitivity and specificity of the signature kinematic features obtained from HSDI, in differentiating between AdSD and MTD. METHODS: We used vibratory features from automated extraction of vocal fold motion waveforms and glottal cycle montage analysis from HSDI for differential kinematic profiling of AdSD and MTD. RESULTS: Novel features of motion irregularities and micromotions (as small as 27 ms) were greater in number for AdSD, whereas reduced motion irregularities, absence of oscillatory breaks, absence of micromotions, and increased hyperfunction characterized the MTD group. Oscillatory breaks (as small as 8 ms), although present only in the AdSD group, were not statistically significant because of their reduced number of occurrences compared to the other features. Further montage analysis of successive glottal cycles of oscillatory breaks in the AdSD group revealed 3 different kinematic patterns within the AdSD group, indicative of likely AdSD with: 1) possible predominant thyroarytenoid muscle involvement, 2) possible predominant cricothyroid muscle involvement, and 3) possible combined involvements of the thyroarytenoid and lateral cricoarytenoid muscles. Four consistent but unique kinematic patterns were identified within the MTD group: 1) diplophonia, 2) vocal fry, 3) breathy phonation, and 4) pressed phonation. Sensitivity and specificity analysis revealed that only motion irregularity was a significant predictor of the presence of AdSD. CONCLUSIONS: Fine kinematic analysis from HSDI can be used to aid detailed clinical profiling of the source characteristics of AdSD and MTD.

Boot camp: a novel intensive approach to voice therapy.

It is well known in the disciplines of neurobiology, exercise physiology, motor learning, and psychotherapy that desirable learning and behavior changes occur primarily from practice that involves high-intensity overload, variability, and specificity of training. We propose a novel treatment approach called intensive short-term voice therapy that uses these practice parameters for recalcitrant dysphonia. Intensive short-term voice therapy involves multiple sessions with a variety of clinicians, incorporating multiple simultaneous therapeutic approaches. The intensive short-term voice therapy approach is characterized by voice therapy for 1-4 successive days each with an average of 5 hours of therapy and five clinicians. This form of intensive voice therapy provides rigorous practice, involving not only overload but also opportunities for specificity and individuality thereby facilitating better transfer of learned skills. This article discusses the conceptual, theoretical, and practical foundations of this novel therapy approach.

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.

Reactive response of fibrocytes to vocal fold mucosal injury in rat.

Fibrocytes hold a prominent role in inflammatory and tissue repair processes in various organ systems. In this study, we identified and quantified a reactive fibrocyte population in the vocal fold mucosa postinjury using immunohistochemistry and stereological analysis. These cells, which expressed CD11b on their surface and prolyl-4-hydroxylase ß (P4H-ß) intracellularly, were largely restricted to the lamina propria, and were morphologically and immunochemically distinguishable from newly recruited epithelial cells. We validated our immunohistochemistry findings using flow cytometry, and additionally characterized a reactive fibrocyte population in circulating peripheral blood using a novel detection panel (CD16(-) CD11b(+) P4H-ß(+) ). Fibrocyte recruitment peaked at 3 days postinjury in peripheral blood, and 5 days postinjury in the vocal fold mucosa. These findings suggest that circulating fibrocytes are recruited to sites of tissue injury in the vocal fold mucosa, and may play an important role in vocal fold tissue repair. The results of this study are consistent with published data from other organ systems and strongly suggest the importance of fibrocytes as therapeutic targets. Our newly reported antigen panel facilitating the direct characterization of fibrocytes via flow cytometry is a useful tool with the potential to facilitate improved study of this cell population.

Morphological and extracellular matrix changes following vocal fold injury in mice.

Mouse experimental models are commonly utilized tools in biomedical research but remain underrepresented in vocal fold biology, presumably due to the small size of the larynx and limited description of the anatomical, cellular and extracellular composition of the vocal folds. In this study, we provide a whole-mount serial section-based histological description of vocal fold morphology of wild-type FVB strain mice, alongside a histological and immunohistochemical (IHC)-based quantitative analysis of extracellular matrix (ECM) alteration 1, 7, 14, 28, 42 and 56 days following unilateral vocal fold injury. IHC was specific for procollagen type I, collagen type I, collagen type III, collagen type IV, elastin, decorin, fibronectin and hyaluronic acid binding protein 2. The histological description confirmed the presence of a laryngeal alar structural complex in the mouse, which appears to be a morphological feature unique to rodents. The lamina propria appeared uniform without evidence of a distinct layer structure as has been reported in larger animals and humans. Time-dependent alterations in vocal fold morphology, ECM organization and ECM protein/glycoconjugate abundance were observed in injured vocal folds compared to control. The presence of a mature scar was observed between 28 and 42 days postinjury. Morphological and ECM changes following vocal fold injury in the mouse were generally consistent with those reported in other animal models, particularly the rat, although wound repair in the mouse appears to occur at a faster rate.

Alteration in cellular morphology, density and distribution in rat vocal fold mucosa following injury.

The vocal fold mucosa plays an important role in voice production. Its cellular composition and density frequently change under various pathological conditions, often contributing to altered extracellular matrix production, tissue viscoelasticity, and voice quality. In this study, cellular changes in the rat mucosa following a unilateral stripping injury were investigated and analyzed semi-quantitatively. Distinctive and sequential changes in cellular morphology, composition, and density were observed in the mucosa post-injury. Cellular recruitment was a major event during the early stage of injury and reached its peak level by day 5 post-injury. Several types of cells, including neutrophil-like cells, epithelial cells, and fibroblast-like cells, were sequentially recruited. The sequential emergence of reactive cell populations following injury and subsequent reconstruction of the mucosa suggests their involvement in vocal fold tissue repair and scar formation processes.

A rat excised larynx model of vocal fold scar.

PURPOSE: To develop and evaluate a rat excised larynx model for the measurement of acoustic, aerodynamic, and vocal fold vibratory changes resulting from vocal fold scar. METHOD: Twenty-four 4-month-old male Sprague-Dawley rats were assigned to 1 of 4 experimental groups: chronic vocal fold scar, chronic vocal fold scar treated with 100-ng basic fibroblast growth factor (bFGF), chronic vocal fold scar treated with saline (sham treatment), and unscarred untreated control. Following tissue harvest, histological and immunohistochemical data were collected to confirm extracellular matrix alteration in the chronic scar group; acoustic, aerodynamic, and high-speed digital imaging data were collected using an excised larynx setup in all groups. Phonation threshold pressure (P(th)), glottal resistance (R(g)), glottal efficiency (E(g)), vibratory amplitude, and vibratory area were used as dependent variables. RESULTS: Chronically scarred vocal folds were characterized by elevated collagen Types I and III and reduced hyaluronic acid abundance. Phonation was achieved, and data were collected from all control and bFGF-treated larynges; however, phonation was not achieved with 3 of 6 chronically scarred and 1 of 6 saline-treated larynges. Compared with control, the chronic scar group was characterized by elevated P(th), reduced E(g), and intralarynx vibratory amplitude and area asymmetry. The bFGF group was characterized by P(th) below control-group levels, E(g) comparable with control, and vocal fold vibratory amplitude and area symmetry comparable with control. The sham group was characterized by P(th) comparable with control, E(g) superior to control, and vocal fold vibratory amplitude and area symmetry comparable with control. CONCLUSIONS: The excised larynx model reported here demonstrated robust deterioration across phonatory indices under the scar condition and sensitivity to treatment-induced change under the bFGF condition. The improvement observed under the sham condition may reflect unanticipated therapeutic benefit or artifact. This model holds promise as a tool for the functional characterization of biomechanical tissue changes resulting from vocal fold scar and the evaluation of experimental therapies.

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.

Changes in cytokine signaling and extracellular matrix production induced by inflammatory factors in cultured vocal fold fibroblasts.

OBJECTIVES: Modulating cytokine signaling in vocal fold fibroblasts after injury may influence extracellular matrix (ECM) production and eventual fibrotic outcome. To evaluate previously established in vivo cytokine and ECM gene expression hypotheses, we examined in vitro vocal fold fibroblast responses to exogenous inflammatory factor stimulation. METHODS: Rat vocal fold fibroblast lines derived from explants were separately treated with interleukin-13 (IL-13), interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta subtype 1 (TGF-beta1), or prostaglandin E2 (PGE2). We examined the in vitro messenger RNA expression profiles of IL-1beta, IFN-gamma, TNF-alpha, TGF-beta1, and cyclooxygenase 2 (COX-2), as well as those of hyaluronic acid synthase (HAS) 1, HAS-2, procollagen subtype 1, and procollagen subtype 3, at 1,4, 8, 16, 24, and 72 hours after treatment, and compared them to those of untreated fibroblasts and in vivo data, using real-time reverse transcription-polymerase chain reaction. RESULTS: IL-1beta and TNF-alpha induced each other and synergistically increased HAS-1 and HAS-2 expression. PGE2 also up-regulated HAS-1 and HAS-2 expression. IFN-gamma, IL-1beta, TNF-alpha, and TGF-beta1 up-regulated HAS expression alongside either transient up-regulation of, or no change in, procollagen 1 and 3 expression. Most treatments appeared to suppress procollagen expression, possibly through HAS up-regulation. All inflammatory factors attenuated TGF-beta1 expression. CONCLUSIONS: These results confirm several in vivo trends, identify potential cytokine pathways and therapeutic candidates, and suggest the utility of this in vitro setup for future studies.

Proteomic changes in rat thyroarytenoid muscle induced by botulinum neurotoxin injection.

Botulinum neurotoxin (BoNT) injection into the thyroarytenoid (TA) muscle is a commonly performed medical intervention for adductor spasmodic dysphonia. The mechanism of action of BoNT at the neuromuscular junction is well understood, however, aside from reports focused on myosin heavy chain isoform abundance, there is a paucity of data addressing the effects of therapeutic BoNT injection on the TA muscle proteome. In this study, 12 adult Sprague Dawley rats underwent unilateral TA muscle BoNT serotype A injection followed by tissue harvest at 72 h, 7 days, 14 days, and 56 days postinjection. Three additional rats were reserved as controls. Proteomic analysis was performed using 2-D SDS-PAGE followed by MALDI-MS. Vocal fold movement was significantly reduced by 72 h, with complete return of function by 56 days. Twenty-five protein spots demonstrated significant protein abundance changes following BoNT injection, and were associated with alterations in energy metabolism, muscle contractile function, cellular stress response, transcription, translation, and cell proliferation. A number of protein abundance changes persisted beyond the return of gross physiologic TA function. These findings represent the first report of BoNT-induced changes in any skeletal muscle proteome, and reinforce the utility of applying proteomic tools to the study of system-wide biological processes in normal and perturbed TA muscle function.

Inflammatory factor profiles one hour following vocal fold injury.

OBJECTIVES: Inflammatory factors are key mediators of wound healing processes following injury, and their modulation may improve healing outcomes. The objective of this study was to characterize in vivo inflammatory factor and extracellular matrix (ECM) messenger RNA (mRNA) expression levels 1 hour after vocal fold injury. METHODS: Five Sprague-Dawley rats were subjected to bilateral vocal fold injury, 5 rats were reserved as uninjured controls, and 1 rat was subjected to unilateral vocal fold injury and reserved for histology. Tissue was harvested 1 hour after injury. Real-time reverse transcription-polymerase chain reaction was performed to examine the mRNA expression profiles of inflammatory factors nuclear factor kappa beta (NF-kappabeta), interferon gamma (IFN-gamma), cyclooxygenase 2 (COX-2), transforming growth factor beta isoform 1 (TGF-beta1), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 beta (IL-1beta), as well as ECM genes hyaluronic acid synthase (HAS) 1, HAS-2, procollagen 1, procollagen 3, and elastin, in the injured samples compared with the uninjured controls. RESULTS: Injury resulted in subepithelial bleeding throughout the vocal fold. The COX-2, TNF-alpha, IL-1beta, and HAS-1 mRNA expression levels were significantly up-regulated 1 hour after injury compared with the uninjured controls. CONCLUSIONS: Inflammatory factor and ECM gene expression changes occur in vocal fold wound sites as early as 1 hour after injury. These results should inform future efforts to attenuate vocal fold scarring via the modulation of inflammatory factors.

Vitamin A deficiency causes metaplasia in vocal fold epithelium: a rat study.

OBJECTIVES: The roles of vitamin A in the vocal fold epithelium are not well documented, although vitamin A has been used as a conservative treatment for laryngeal leukoplakia. The purpose of this study was to analyze the roles of vitamin A in vocal fold epithelial differentiation. METHODS: Vitamin A-deficient (VAD) rats were generated, and the abnormality of their vocal fold epithelium was examined by hematoxylin and eosin staining and immunohistochemical analysis for keratin 10 and transglutaminase (TGase) 1. RESULTS: The VAD experimental rats exhibited orthokeratosis of the vocal fold epithelium. Keratin 10 and TGase 1 were up-regulated in the epithelium of the VAD rats. CONCLUSIONS: It is suggested that vitamin A suppresses TGase 1 expression in normal vocal folds to inhibit keratinization, and that the TGase 1 up-regulation caused by vitamin A deficiency may be related to the formation of metaplasia in the laryngeal epithelium.