Vocal Fold Epithelium Research Articles

Experimental study and theoretical simulation of stress relaxation behavior of vocal fold lamina propria tissue.

The viscoelastic properties of vocal fold lamina propria tissue play an important role in tissue modeling, as well as in clinical studies of the effects of carcinoma, scarring, atrophy, or edema on dysphonia. Although the impact of these properties has been well studied, quantification of viscoelastic behaviors such as creep and stress‐relaxation continue to be important in the research. The current study examined the stress‐relaxation curves of ten canine vocal fold cover samples by stretching the tissue to 5%, 10%, 15%, and 20% of the sample reference length. The force on the lamina propria tissue was then recorded for 5 mins, and graphed so the stress‐relaxation response could be seen. The curves obtained were what we hypothesized, with the lamina propria tissue exhibiting exponential relaxation initially, and very slow decay at the end of the trial. These results are consistent with our previous studies on stress‐relaxation predicted by the biphasic theory of vocal fold tissue [Zhang et al., J. Acoust. Soc. Am. 123, 1627–1636 (2008)]. Finite element simulation has also been given to investigate the stress distributions within the vocal fold epithelium and lamina propria tissues.

Pesquisa de estrógeno e progesterona no epitélio das pregas vocais de mulheres por imunohistoquímica

A laringe é extremamente sensível a mudanças endocrinológicas. A maioria das alterações da mucosa das pregas vocais é causada por modificações do conteúdo líquido das pregas vocais e das suas modificações epiteliais. O estrógeno e a progesterona interferem e modificam esse conteúdo líquido das pregas vocais. O objetivo deste trabalho é verificar a presença de receptores de estrógeno e progesterona no epitélio das pregas vocais de mulheres. MATERIAL E MÉTODO: Estudo de casos prospectivos. Foram realizados exames de imunohistoquímica para receptores de estrógeno e progesterona em 19 espécimes de epitélio de pregas vocais que não apresentavam quaisquer indícios de afecção, inclusive inflamatória. Foram descartados casos de pacientes com idade superior a 40 anos e inferior a 15 anos. RESULTADOS: Foram encontrados receptores para progesterona em 18 de 19 pacientes. Os receptores de progesterona estão localizados tanto no núcleo quanto no citoplasma e principalmente na camada basal. Não houve nenhum caso de receptores de estrógeno nas pregas vocais. CONCLUSÃO: O epitélio das pregas vocais apresenta receptores para progesterona, tanto no citoplasma quanto no núcleo. Não foram encontrados receptores para estrógeno no epitélio das pregas vocais estudadas.

Microendoscopy of Reinke's Space

Contemporary surgical treatment of the superficial layer of the lamina propria or Reinke's space is most commonly performed through an incision in the overlying vocal fold epithelium. This approach may disrupt normal tissue, induce scarring, and allow extrusion of implanted materials. Previously reported external approaches to Reinke's space required either a laryngofissure or a "minithyrotomy" for access. These surgical approaches were performed without direct imaging of Reinke's space. Instruments placed below the vocal fold epithelium via this external approach were visualized through the translucent vocal fold epithelium. We designed this study to identify the feasibility of limited-access surgery of the lamina propria using microendoscopes placed into Reinke's space through an external approach. A cadaveric human larynx was dissected, and microendoscopes were directly advanced into Reinke's space through a subepithelial puncture of the cricothyroid membrane, as well as lateral fenestration through the thyroid cartilage. Photodocumentation of the undersurface of vocal fold epithelium, the opposing surface of the vocal ligament, and the intervening Reinke's space was successfully accomplished. Advances in both microendoscopes and accompanying instrumentation permit access to the superficial layer of the lamina propria without disrupting the overlying epithelium. This approach to microendoscopy of Reinke's space may allow for more effective surgical treatment of cysts, chronic edema, vascular abnormalities, atrophy, scarring, and sulcus vocalis.

Immunohistochemical searching for estrogen and progesterone receptors in women vocal fold epithelia

SummaryLarynx is extremely sensitive to endocrinologic changes. Most vocal fold mucosa alterations are caused by changes in vocal fold liquid content and its epithelial changes. Estrogen and progesterone interfere and change this liquid content in the vocal folds. Our goal with the present paper is to study the presence of estrogen and progesterone receptors on vocal fold epithelium in 19 vocal fold epithelium specimens that did not present any indication of disease, especially inflammatory disease. We discarded those cases of patients above 40 years of age and those below 15.Resultswe found progesterone receptors in 18 of the 19 patients. The progesterone receptors are located both in the nucleus and the cytoplasm of cells, and mainly in the basal layer. There was no report of estrogen receptors present in the vocal folds.ConclusionVocal fold epithelium bears progesterone receptors, in the cytoplasm and in the nucleus. We did not find estrogen receptors in the epithelia of the vocal folds investigated.

Vocal fold elasticity of the Rocky Mountain elk (Cervus elaphus nelsoni) – producing high fundamental frequency vocalization with a very long vocal fold

The vocal folds of male Rocky Mountain elk (Cervus elaphus nelsoni) are about 3 cm long. If fundamental frequency were to be predicted by a simple vibrating string formula, as is often done for the human larynx, such long vocal folds would bear enormous stress to produce the species-specific mating call with an average fundamental frequency of 1 kHz. Predictions would be closer to 50 Hz. Vocal fold histology revealed the presence of a large vocal ligament between the vocal fold epithelium and the thyroarytenoid muscle. In tensile tests, the stress-strain response of vocal fold epithelium and the vocal ligament were determined. Elasticity of both tissue structures reached quantitative values similar to human tissue. It seems unlikely that the longitudinal stress in elk vocal folds can exceed that in human vocal folds by an order of magnitude to overcome the drop in fundamental frequency due to a 3:1 increase in vocal fold length. Alternative hypotheses of how the elk produces high fundamental frequency utterances, despite its very long vocal fold, include a reduced effective vocal fold length in vibration, either due to bending properties along the vocal fold, or by actively moving the boundary point with muscle stiffening. The relationships between an individual's average fundamental frequency, vocal fold length and body size are discussed.

Detecção de receptor de ácido hialurônico em prega vocal humana por método imunohistoquímico

O receptor do ácido Hialurônico é uma glicoproteína da membrana plasmática, sendo o principal o CD44, e está expresso em vários tipos de células onde possui a função de adesão celular. OBJETIVO: Estudar a possibilidade de empregar o método imunohistoquímico para identificar a distribuição dos receptores de ácido hialurônico ao longo da prega vocal humana. MATERIAL E MÉTODOS: Foram ressecadas as pregas vocais normais de um indivíduo de 23 anos, sexo masculino, cor negra. As lâminas foram analisadas por meio de estudo histomorfométrico, comparando-se a intensidade das cores nas camadas superficial, média e profunda da lâmina própria. Nas lâminas silanizadas foi utilizado método imunohistoquímico, sendo avaliadas através de microscopia óptica com aumento 40 vezes, obtendo coloração marrom onde houve a reação com receptor para ácido hialurônico. RESULTADOS: Os achados imunohistoquímicos mostraram presença de receptores para ácido hialurônico no epitélio de cobertura da prega vocal tendo maior concentração na região central da prega vocal. CONCLUSÃO: A técnica de imunohistoquímica, utilizada para avaliar a distribuição dos receptores para ácido hialurônico na pregas vocais humanas, mostrou sua disposição em epitélio da prega vocal e predomínio no terço médio, em relação às demais regiões na prega vocal estudada.

Detection of Hyaluronic Acid Receptor in Human Vocal Folds by immunohistochemistry

SummaryHyaluronic acid receptor is a glycoprotein of the plasmatic membrane, and the CD44 is its representative, expressed in many cell types where it has the task of cell adhesion.Aimthe goal of the present experimental study is to investigate the possibility of using immunohistochemistry to identify the distribution of hyaluronic acid along the vocal fold.Materials and MethodsWe resected the normal vocal folds from a normal 23 year-old male black individual. The slides were analyzed by means of a histomorphometric study, comparing the color intensity in the superficial, middle and deep layers of the lamina propria. In the silanized slides we used immunohistochemistry, and evaluated the slides under light microscopy with 40x magnification, and the color changed to brown when there was a reaction with the receptor for hyaluronic acid.ResultsImmunohistochemical findings showed the presence of hyaluronic acid receptors in the epithelium covering the vocal fold, being more concentrated in the central region of the vocal fold.Conclusionimmunohistochemistry, used to assess the distribution of hyaluronic acid receptors in the central portion of the vocal fold, proved it to be present in the vocal fold epithelium and it prevailed in its middle third.

Evidence for cAMP‐mediated transepithelial ion flux in porcine vocal fold epithelia

Vocal fold epithelia are exposed to osmotic challenge and inflammatory perturbations at the glottal interface. Emerging data on transepithelial vectorial ion fluxes and associated passive water movement suggest potential mechanisms by which vocal folds may defend against osmotic and chemical challenge. However, the ion transport mechanisms underlying vocal fold defense to inflammatory perturbations are not understood. In vocal folds from an Ossabaw swine model, we used electrophysiological techniques to confirm the presence of the epithelial Na+channel, ENaC, and the Cl−channel CFTR. As cAMP plays an important role in signal transduction and may serve as a second messenger for a variety of inflammatory mediators and cytokines, we examined whether porcine vocal fold epithelia are sensitive to the cAMP activator, forskolin. 30 minutes of exposure to forskolin (5mM) significantly increased ion transport, the characteristics of which are consistent with Cl− secretion as compared to sham controls (N = 10, p < 0.05) and concurrently significantly increased intracellular cAMP (N = 4, p < 0.05). These findings provide evidence for the presence of cAMP mediated ion flux in porcine vocal fold epithelia. Vocal fold inflammation is a leading cause of laryngeal disease and understanding the mechanisms involved in the inflammatory process will lead to improved treatment options for this common communication disorder.

Vitamin a Deficiency Causes Metaplasia in Vocal Fold Epithelium: A Rat Study

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. 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. 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. 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.

Safe, complete resection of epiglottic cysts with phono-ultra-microsurgical technique

Generally, epiglottic cysts have been treated by the puncture technique or surgical resection. However, these techniques can result in accidental injury to the cyst wall. As a result, the contents of the cyst may spread throughout the larynx, trachea and pharyngeal cavity. The ideal treatment would involve complete resection without cyst perforation. We use a completely different approach for the resection of epiglottic cysts, with successful results, which we term the advanced phono-ultra-microsurgical technique. One difference between this method and previous techniques of surgical epiglottic cyst resection is that the cyst is sealed off before resection in order to prevent rupture and spread of its contents. To enable this, we have developed a special device to facilitate suturing of the vocal fold epithelium, utilising a knot-pusher. This suturing technique allows complete removal of epiglottic cysts. Our technique allows optimal prevention of cyst rupture and spread of contents.

Vocal Fold Epithelial Response to Luminal Osmotic Perturbation

Dry-air challenges increase the osmolarity of fluid lining the luminal surface of the proximal airway. The homeostasis of surface fluid is thought to be essential for voice production and laryngeal defense. Therefore, the authors hypothesized that viable vocal fold epithelium would generate a water flux to reduce an osmotic challenge (150 mOsm mannitol) on the lumen. Bidirectional transepithelial water fluxes were measured in vocal folds exposed to physiologically realistic luminal osmotic perturbations in vitro. Thirty-six native ovine vocal folds were exposed to either luminal hyperosmotic or isosmotic perturbations. Vocal fold viability and water fluxes toward the lumen and into the mucosa were measured at prechallenge baseline and for 30 min after challenge. Vocal fold electrophysiological viability was maintained for the duration of osmotic perturbation. Luminal osmotic exposure increased luminally directed transepithelial water fluxes in 60% of vocal folds. This increase was electrically silent, of short duration, and would not negate the osmotic gradient. Ovine vocal fold epithelia detect osmotic perturbations to the luminal surface in vitro. This ability to detect and respond to changes in surface composition may be important in homeostatic regulation of vocal fold surface fluid during osmotic perturbations in respiration and phonation.

Immunolocalization of Aquaporins in Vocal Fold Epithelia

To investigate the presence of aquaporin (AQP) water channels 1, 2, and 3 in stratified squamous vocal fold epithelium. Immunolocalization analysis of excised ovine vocal fold epithelia. Sheep. Ovine vocal fold epithelia were prepared for immunoelectron microscopy using primary antibodies directed against AQP-1, AQP-2, and AQP-3. Photographic profiles of epithelium exposed to each antibody were used to calculate the immunogold labeling density of the plasma membrane and cytoplasm. Density of immunolabeling was compared across 3 regions that represent cell layers closest to the glottal lumen for the plasma membrane and cytoplasm, respectively. Labeling densities of AQP-1 and AQP-2 were significantly greater for the plasma membrane region of the luminal cells than for deeper cell layers. Cytoplasmic labeling and labeling of circular structures was greatest for cell layers 2 through 5 beneath the vocal fold surface compared with the surface cell layer. Immunogold labeling of AQP-3, an aquaglyceroporin, in vocal fold epithelium was inconclusive. Aquaporins 1 and 2, associated with the plasma membrane region of ovine vocal fold epithelial cells, demonstrate the presence of an intrinsic mechanism to permit transcellular water flux in response to osmotic gradients.

Vocal Folds Detect Ionic Perturbations on the Luminal Surface: An In Vitro Investigation

The homeostasis of fluid bathing the luminal surface of the vocal folds is important for phonation and laryngeal defense. Dehydration of the respiratory tract during mouth breathing can perturb the concentration of sodium and chloride ions in surface fluid. Exposure to dry air also increases the osmolarity of airway surface fluid. We hypothesized that viable vocal fold epithelium would detect changes in the ionic and osmotic composition of fluid on the luminal surface. Therefore, we examined bioelectric responses of vocal folds exposed to physiologically real, luminal ionic and osmotic challenges in vitro. The study used randomized factorial design with experimental and sham control groups. Fifty native ovine vocal folds were exposed to five challenges (ionic, osmotic, combined ionic-osmotic, and sham) on the luminal surface. Bioelectric measures of potential difference (PD), short-circuit current (I(SC)), and tissue resistance were assessed at prechallenge baseline, during challenge, and after removal of challenge. Ionic and combined ionic-osmotic challenges reduced PD and I(SC) (P<0.01). These reductions depended on the nature of the ionic challenge, were observed within 10 minutes, lasted for the duration of exposure, and were reversible after removal of the challenge. Conversely, sham or osmotic challenge did not alter bioelectric parameters over time (P>0.05). Viable ovine vocal fold epithelia detect ionic perturbations to the luminal surface. This sensitivity to luminal ionic challenge may be necessary to maintain the homeostasis of surface fluid.

Efficacy of Treating Children With Anterior Commissure and True Vocal Fold Respiratory Papilloma With the 585-nm Pulsed-Dye Laser

To report preliminary results regarding the safety and efficacy of the 585-nm pulsed-dye laser (PDL) for the treatment of juvenile-onset recurrent respiratory papillomatosis (JORRP) in the pediatric population. Prospective longitudinal cohort study. Two pediatric otolaryngology referral centers. Twenty-three pediatric patients ranging in age from 6 months to 17 years. The 585-nm PDL was used for at least 1 treatment on each of these patients to treat JORRP of the true vocal folds or anterior commissure. Complications from the use of the 585-nm PDL in the treatment of JORRP. There was no evidence of anterior commissure webbing or true vocal fold scarring in this group of 23 patients followed up for 3 months to 1 year. The 585-nm PDL seems to be a safe instrument for treatment of JORRP. There is the potential that improved voice outcomes may be apparent when compared with traditional therapies because the vocal fold epithelium seems to be unharmed when treated with this method. Furthermore, the lack of epithelial damage incurred by the 585-nm PDL should enable more aggressive surgical excision of anterior commissure disease. Further prospective longitudinal studies examining voice outcomes are needed.

Die Kehlkopfkonfiguration beim Flüstern

The purpose of the study was to assess glottal and supraglottal configurations during whispering in comparison to habitual phonation and to visually estimate to what extent force is exerted onto the vocal fold epithelium during whispering. Is whispering a good compromise between preservation of epithelium and permission to communicate orally, e. g. postoperatively? We investigated on 100 patients with organic or non-organic voice disorders. Patients with vocal fold immobility and patients with organic lesions large enough to impede glottal closure were excluded. Videolaryngoscopy and stroboscopy was performed via flexible endoscopy. During fiberoscopy the patients were asked (i) to phonate and then whisper a series of vowels in various loudness levels and (ii) to count to 10 voiced and whispered in various loudness levels. The extent of vocal fold adduction and supraglottal configuration were assessed and whispering compared with voiced phonation. Interindividual differences in glottal and supraglottal configuration during whispering were seen. The two different and most frequently seen glottal area patterns were determined by the position of the vocal processes - either adducted or abducted. Adduction resulted in sole cartilaginous triangle formation or 'kissing processes', abduction in ligamentous and cartilaginous triangle. Another surprising result was that with increasing loudness either a narrowing or widening of glottal and supraglottal structures was visible. There are different patterns of glottal and supraglottal configuration during whispering. Although the vocal folds can show partial or total contact over the membranous parts, the lack of additional vibration of the vocal folds suggests that epithelial forces are smaller than with additional vibratory shear stress. Thus, whispering may be considered and allowed for postoperative communication purposes, when patients follow instructions for low-effort whispering.

Efeitos da inalação passiva da fumaça de cigarro sobre as pregas vocais de ratos

Poucos estudos demonstram as reações patológicas devidas à inalação de fumaça pelas vias aéreas de ratos. OBJETIVO: Estudar e analisar os possíveis efeitos histopatologicos produzidos pela inalação crônica de fumaça de cigarro nas pregas vocais de ratos. DESENHO DE ESTUDO: Experimental. MATERIAL E MÉTODOS: 36 ratos masculinos (Rattus norvergicus Wistar), de 60 dias de idade, foram mantidos em gaiolas e expostos à inalação da fumaça produzida por 10 cigarros, 3 vezes ao dia, 7 dias por semana, para períodos de 25, 50 e 75 dias, e os controles respectivos. Os animais foram sacrificados e as suas laringes dissecadas e submetidas a análise histológica com coloração de Hematoxilina e Eosina e analisados através de microscopia simples. RESULTADOS: Os ratos expostos ao cigarro exibiram menor (p &lt;0,05) massa corporal que o grupo controle. Havia hiperplasia e metaplasia escamosa na extremidade livre da prega vocal e hiperplasia escamosa na porção mediana da prega vocal em todos os 3 períodos de estudo. Além disso, o grupo de 50 dias revelou metaplasia com queratinização nesta área. Não foram observadas alterações morfológicas em outras áreas da laringe e reação inflamatória da lâmina propria. CONCLUSÃO: Foi concluído que a inalação passiva de fumaça de cigarro rende mudanças morfológicas importantes no epitélio da prega vocal que podem, eventualmente, progredir para neoplasia.

Regulation of aquaporin 1 in epithelia of vocal fold and trachea

We sought to identify and regulate a pathway for transcellular water flux in native epithelia of the ovine vocal fold and trachea. Immunogold localization visualized by transmission electron microscopy was used to identify presence of AQP1 and AQP2 in ovine vocal fold (N=2) and tracheal (N=2) epithelia. Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to examine regulation of AQP1 and AQP2 mRNA in ovine vocal fold (N=34) and tracheal (N=24) epithelia by vasopressin (4 hours, luminal exposure). In the vocal fold, plasma membrane labeling density of AQP1 and AQP2 was greater for squamous cells at the lumen than for deeper cells (AQP1 p< 0.02, AQP2 p<0.0001). Labeling of trachea was associated with the luminal plasma membrane of pseudostratified ciliated epithelial cells. In vocal fold epithelia only, vasopressin (but not sham control) significantly reduced CT values for AQP1 (p<0.01). No difference was seen in CT values of AQP2 in vocal fold or trachea following vasopressin exposure. Vasopressin-dependent increase of AQP1 mRNA in stratified squamous epithelium of the vocal fold suggests a hormonal mechanism for upregulation of water permeability in luminal vocal fold cells, possibly distinct from other regions of the airway. Supported by NIDCD #F31DC5733 (DL) and #K23DC00168 (KF).

Effects of passive smoke inhalation on the vocal cords of rats

SummaryFew studies have demonstrated the pathologic reactions yielded by smoke inhalation on the airway in rats. Aim: The aim of this study was to analyze the possible histopathological effects produced by chronic cigarette smoke inhalation on the vocal folds of rats. Study design: Experimental. Material and Method: 36 male rats (Rattus norvergicus Wistar strain), aged 60 days, were kept in cages and exposed to inhalation of the smoke produced by 10 cigarettes lit 3 times a day, 7 days a week, for periods of 25, 50 and 75 days, and their respective controls. Thereafter the animals were killed and their larynxes were dissected and submitted to histological processing for achievement of histological sections, which were stained with Hematoxylin and Eosin and analyzed by light microscopy. Results: The rats exposed to smoke displayed smaller (p< 0,05) body mass than the control group. There was hyperplasia and squamous metaplasia in the free edge of the vocal fold and squamous hyperplasia on the middle portion of the vocal fold in all 3 study periods. Moreover, the 50-day group revealed keratinizing metaplasia in this area. Morphological alterations in other areas of the larynx and inflammatory reaction of the lamina propria were also not observed. Conclusion: It was concluded that the passive inhalation of cigarette smoke yields important morphological changes in the vocal fold epithelium, which may progress to neoplasia.

Contribution of the Paracellular Pathway to the Electrophysiological Asymmetry of Vocal Fold Epithelium

Wet stratified squamous epithelium of the vocal fold possesses electrophysiological asymmetry (lumen-negative) to support ionically-driven vectorial water fluxes. We studied the role of paracellular pathway in the maintenance of this bioelectrical asymmetry by demonstrating the tight junction (TJ) - related barrier function of vocal fold epithelia. Cryosections of fresh ovine vocal fold mucosae (N=5) were used for the immunostaining of occludin and ZO-1 proteins, markers of the TJ. An Ussing chamber system, and voltage/current clamp were used to measure the transepithelial electrical resistance (TER) with and without luminal histamine (N=46). Permeability of paracellular pathway to the protein tracer, horseradish peroxidase (HRP), was measured spectrophotometrically (N=35), and penetration was confirmed by detection of HRP activity within the epithelium. We detected positive immunoreactions of occludin and ZO-1 proteins with distinct patterns of distribution in all epithelia. Baseline TER of 410±173.9 Ω·cm2 was reduced by 94% (p<0.001) with high-dose histamine (2 hr, 0.1M) but not low dose (10μM, 1mM) or sham control. Histamine (but not sham) increased permeability (1mM, p<0.044, n=12; 0.1M, p<0.001, n=11) to HRP in a dose- and time-dependent manner. These results suggest that TJ-related barrier function in the paracellular pathway contributes to maintenance of the electrophysiological gradient in this epithelium. Interference with the TJ-related barrier function may provide a novel direction for the understanding of physiological as well as pathological mechanisms of vocal fold hydration. NIDCD K23DC00168 (KF)

Making a diagnosis of different lesions of vocal fold mucosa by contact endoscopy: The first usage in our clinical practice

During laryngomicroscopy, the superficial layers of vocal fold epithelium can be examined in vivo and in situ by contact endoscopy. Methylene blue is applied initially to stain the epihelial cells of the vocal folds. When in contact with mucosal tissue, this endoscope provides 60 and 150 times magnification and clear visualization of cellular patterns of the superficial epithelial layers. For the first time in our laryngological clinical practice, we confirmed a number of previosly established parametars such as regularity and arrangement of the epithelium, nucleus contour, and nucleus-cytoplasm ratio, what all enable recognition and easy evaluation of different clinical conditions such as chronic laryngitis, Reinke's edema, papiloma dysplasia or vocal fold carcinoma. The advantage of contact endoscopy in vivo and in situ allows for detailed scan and mapping of all cell changes of the whole mucosa surface. All these features definitely classify the contact endoscopy into additional diagnostic methods in laryngology.