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.

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.

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.

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.

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.

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.

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.

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.

Roles of vitamin A and macula flava in maintaining vocal folds.

OBJECTIVES: Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. METHODS: Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. RESULTS: Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor a in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. CONCLUSIONS: These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage.

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.

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.

Attitudes of children with dysphonia.

SUMMARY: Because voice disorders in childhood may have a negative impact on communicative effectiveness, social development, and self-esteem, the objective was to determine the impact of voice disorders on lives of children from the perspective of chronically dysphonic children and their parents. This study consisted of focused interviews with chronically dysphonic children and their caregivers. Focused interviews were conducted with 10 children in each of the following age groups: Toddler (2-4 years old), Young Child (5-7 years old), School-Aged Child (8-12 years old), and Adolescent (13-18 years old). Interview questions were formulated to elicit attitudes in the following conceptual domains: emotional, social/functional, and physical. Interviews were transcribed and subjected to systematic qualitative analyses that identified common themes within each age group for each conceptual domain. For Toddlers, interviews relied heavily on parents and the biggest concerns were found in the physical and functional domains. Young Children expressed that their biggest issues related to voice were physical ("run out of air," "sometimes voice does not work"). Ninety percent of Young Children were repeatedly asked to use a quieter voice. Emotional factors and physical factors were prominent in the interviews of School-Aged Children and Adolescents. Children and Adolescents often felt that their dysphonic voice received undue attention and also limited their participation in important events. Anger, sadness, and frustration were also expressed. Chronic dysphonia negatively affects the lives of children. This work will serve as the basis for development of a valid, reliable, and age-appropriate measure of voice-related quality of life in children.

Immuno-scanning electron microscopy of collagen types I and III in human vocal fold lamina propria.

OBJECTIVES: This study was undertaken to identify the types of collagen fibrils in the extracellular matrix of the human vocal fold lamina propria. METHODS: Human vocal folds were obtained from 3 autopsy cases less than 65 years of age. The vocal fold specimens were labeled by primary antibodies of anti-type I and anti-type III collagens, and then by secondary antibody conjugated with 15 nm colloidal gold. The specimens were observed with a scanning electron microscope. Secondary electron imaging and backscatter electron imaging of high-resolution field emission scanning electron microscopy were used to detect gold particles indicating immunolabeling. RESULTS: Type III collagen-labeling gold particles were abundant on the fibrils constructing collagenous fibers, whereas type I collagen-labeling gold particles were sparsely present on fibrils in collagenous fibers. A few reticular fibers were labeled by both collagen type I and collagen type III. CONCLUSIONS: The results suggest that collagen type I coexists with collagen type III in fibrils of both collagenous fibers and reticular fibers.

Voice handicap evaluation of patients with pathologic sulcus vocalis.

OBJECTIVES: The purpose of this study was to characterize the psychosocial impact of dysphonia on patients with pathologic sulcus vocalis by use of the Voice Handicap Index (VHI). METHODS: The VHI was administered to 15 patients (11 women and 4 men) with pathologic sulcus vocalis. The VHI subscale and total scores were compared with previously published data from individuals with no history of dysphonia and from patients with vocal fold scar. Additional comparisons were performed for patients with unilateral sulcus versus bilateral sulci, type II sulcus versus type III sulcus, and sulcus with concomitant vocal fold scar versus sulcus without concomitant scar. RESULTS: The VHI scores for patients with pathologic sulcus vocalis were significantly greater than those for individuals with no history of dysphonia and for patients with vocal fold scar. In addition, significantly greater VHI scores were observed for patients with sulcus vocalis with concomitant scar versus those with sulcus alone. CONCLUSIONS: These data suggest that pathologic sulcus vocalis can be a severely handicapping condition, particularly in the presence of concomitant scar.

Prenatal vitamin A deficiency causes laryngeal malformation in rats.

OBJECTIVES: Our previous research demonstrated that vitamin A might be related to vocal fold development. The purpose of this study was to determine whether vitamin A deficiency affects prenatal laryngeal development in rats. METHODS: Two considerations were necessary in designing a study using a rat model: for embryonic survival, vitamin A is necessary through day 10 of gestation, and laryngeal formation occurs primarily after day 11. Thus, we created a rat model that developed vitamin A deficiency after embryonic day 11. Ten pregnant rats (5 vitamin A-deficient rats and 5 control rats) were studied. Embryos were collected at embryonic day 18.5 and analyzed histologically. RESULTS: Eighteen percent of the vitamin A-deficient embryos were alive and demonstrated laryngotracheal cartilage malformation, incomplete separation of the glottis, and/or laryngoesophageal clefts. CONCLUSIONS: These results document the important role played by vitamin A in laryngeal development.

Symptoms of extraesophageal reflux in a community-dwelling sample.

OBJECTIVES: Extraesophageal reflux disease (EERD) is linked with serious and varied health concerns. The authors' goal was to determine the prevalence of EERD and the effect on health-related quality of life in adults within a large community-based sample. STUDY DESIGN: Survey study conducted in a community setting. METHODS: In the Madison, Wisconsin area, 1845 adult subjects were surveyed. Main outcome measures were frequently of GERD and laryngeal symptoms, the Medical Outcomes Trust Short Form 12 (SF-12), Voice Handicap Index (VHI), and Quality of Life in Reflux and Dyspepsia (QOLRAD) questionnaires. RESULTS: More than 66% of subjects reported either GERD or laryngeal symptoms, and 26% reported both GERD and laryngeal symptoms (which reflect symptoms commonly reported for EERD). In addition, 39% of our sample took medicine for heartburn. Forty-four percent of subjects with both GERD and laryngeal symptoms reported occasional breathing difficulties, and 38% of these subjects reported a voice disorder. Significant reductions in perceived quality of general health, digestive health, and voice-related quality of life were found in subjects with both GERD and laryngeal symptoms, in comparison with subjects manifesting each symptom group alone, or in subjects with no symptoms. CONCLUSIONS: The presence of GERD and laryngeal symptoms, and their concurrence, was identified in a substantial number of subjects. The GERD and laryngeal symptoms surveyed represent those most commonly reported for EERD. Because EERD has been linked with tissue damage and reductions in health-related quality of life, it is important to identify these persons and provide treatment before progression of the condition.

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.

Collagen subtypes in human vocal folds.

OBJECTIVES: The collagen subtypes in human vocal folds are of particular interest, because each collagen subtype has different features that make it uniquely suited for performing specific tissue tasks and each collagen subtype can affect the tissue properties of the vocal fold lamina propria. METHODS: Human vocal folds from 5 autopsy cases (less than 65 years old) were examined by immunohistochemistry for collagen types I, III, IV, and V and elastin. RESULTS: Collagen type III was distributed throughout the whole lamina propria. Type I was found just beneath the basal membrane, in the deep layer of the lamina propria and in the anterior and posterior maculae flavae. Types IV and V were present in the epithelial and endothelial basal membrane. Three-dimensional images from thick specimens reconstructed with confocal microscopy showed 2 distinct patterns: type III fibers were wavy, collagenous fibers, as previously observed in the vocal folds, and type I fibers were thinner than type III fibers. These results suggest that type III fibers help maintain the lamina propria structure and that type I fibers provide the tensile strength required around the basal membrane and vocal ligament to maintain the vocal fold shape while withstanding vibratory forces.

Histological study of acute vocal fold injury in a rat model.

OBJECTIVES: We used an acute vocal fold injury in a rat model to characterize vocal fold wound healing by studying the expression pattern of the extracellular matrix components in the vocal fold lamina propria. METHODS: Vocal fold stripping was performed unilaterally in 27 Sprague-Dawley rats. The vocal folds were harvested at 5 time points (1, 3, 5, 7, and 14 days) and histologically analyzed by Alcian blue stain, trichrome stain, and immunofluorescence with antibodies to collagen type I, collagen type III, and fibronectin. RESULTS: Re-epithelialization occurred by day 3 and was complete by day 14. Granulation tissue was formed by day 3. Hyaluronic acid and collagen type I appeared in injured vocal folds by day 3, peaked at day 5, and thereafter decreased. Collagen type III and fibronectin appeared by day 1 and continued to be intense at all time points after day 3. CONCLUSIONS: These results suggest that the expression of these extracellular matrix components peaks in the period around days 3 to 5, and that the characteristics of wound healing in the vocal fold are similar to those in the skin in the early phases, but differ during the subsequent remodeling phase.