Vascularity in the macula flava of human vocal fold as a stem cell niche.

OBJECTIVES: There is growing evidence that the maculae flavae of the human vocal fold are a stem cell niche, which is a microenvironment nurturing tissue stem cells. This study investigated the microenvironment, especially vascularity, in the maculae flavae of the human vocal fold. METHODS: Three normal human adult, three normal newborn vocal folds obtained from autopsy cases and three surgical specimens of glottic carcinoma were investigated using light and electron microscopy. For scanning electron microscopy, a chemical digestion method (modified sodium hydroxide maceration method) was used to observe the inner 3-dimensional structure of the macula flava. RESULTS: Capillaries ran around the anterior and posterior maculae flavae in adults and newborns. However, there was no vascularity in the maculae flavae of the vocal fold. The inner 3-dimensional electron microscopic structure of the macula flava showed there were no blood vessels in the maculae flavae of the vocal fold. Glottic carcinoma (squamous cell carcinoma) surrounded and was in contact with the macula flava, however, the carcinoma did not invade the macula flava indicating there was no vascular supply into the macula flava from the surrounding tissue. CONCLUSIONS: There was no vascularity in the anterior and posterior maculae flavae in the human adult and newborn vocal folds. The present study is consistent with the hypothesis that the hypoxic microenvironment in the maculae flavae of the adult and newborn vocal fold as a stem cell niche is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.

Evaluation of Diffusional Characteristics and Microstructure in Unilateral Vocal Fold Paralysis Using Diffusion Tensor Imaging.

OBJECTIVE: To investigate the value of diffusion tensor imaging (DTI) in the evaluation of vocal fold tissue microstructure after recurrent laryngeal nerve (RLN) injury. METHODS: Six canines were divided into 2 groups: a unilateral vocal fold paralysis group (n = 4) and a control group (n = 2). The RLN was cut in the unilateral vocal fold paralysis group, and no intervention was applied in the control group. After 4 months, the canines' larynges were removed and placed in a small animal magnetic resonance imaging (MRI) system (9.4T BioSpec MRI; Bruker, Germany). After scanning, the vocal folds were isolated, sectioned, and stained. The slides were then analyzed for the cross-sectional area and muscle fiber density through feature extraction technology. Pearson correlation analysis was performed on the DTI scan and histological section extraction results. RESULTS: In the vocal fold muscle layer, the fractional anisotropy (FA) of the unilateral RLN injury group was higher than that of the control group, and the Tensor Trace was lower than that of the control group. This difference was statistically significant, P < .05. In the lamina propria, the FA of the unilateral RLN injury group was lower than that of the control group, P > .05, and the Tensor Trace was lower than that of the control group, P < .05. The muscle fiber cross-sectional area of the RLN injury group was significantly smaller than the control group with statistical significance, P < .05, and the density of muscle fibers was lower, P < .05. The correlation coefficient between FA and the cross-sectional area was -0.838, P = .002, and .726; P = .017 between Tensor Trace and the cross-sectional area. CONCLUSION: Diffusion tensor imaging is an effective method to assess the changes in the microstructure of atrophic vocal fold muscle tissue after RLN injury.

Epithelium of the human vocal fold as a vibrating tissue.

OBJECTIVES: The human adult vocal fold mucosa, especially, superficial layer of the lamina propria (Reinke's space) has attracted notice as an important vibrating structure. However, fine structures of the stratified squamous epithelium of the human adult vocal fold, which is another histological component of the mucosa, remain enigmatic. METHODS: Three normal human adult vocal folds and epiglottises and three newborn vocal folds were investigated. Observations using transmission electron microscopy and light microscopy including immunohistochemistry were performed. RESULTS: The most obvious feature of the epithelium of the human adult vocal folds was that the intercellular spaces between adjacent epithelial cells were large (984 ± 186 nm) compared with the stratified squamous epithelium of the human adult epiglottis and the human newborn vocal fold. Even though intercellular spaces between adjacent epithelial cells of the human adult vocal fold were large, desmosomes at the junctions of two adjacent epithelial cells made firm intercellular adhesion. Tonofilaments composed of the bundles of intermediate filaments anchored to the desmosomes. Desmosomes formed a continuous cytoskeletal network throughout the epithelial cells and epithelium of the human adult vocal fold. In addition, a great deal of E-cadherin (adhesive glycoprotein) was present between epithelial cells especially the lower half of the stratified squamous epithelium of the human adult vocal fold. CONCLUSIONS: From the functional morphological point of view, stratified squamous epithelium of the human adult vocal fold seems to form a structural framework of tensile strength with pliability suitable structure for vibration.

Cytoskeleton of cells in vocal fold macula flava unphonated for a long period.

Cells in the maculae flavae (MFe) are inferred to be involved in the metabolism of extracellular matrices of the human vocal fold mucosa. The latest research has supported the hypothesis that the tension caused by phonation (vocal fold vibration) regulates the behavior of these cells in the MFe of the human vocal fold. Tensile and compressive strains have direct effects on cell morphology and structure including changes in cytoskeletal structure and organization. Cytoskeletons are one of the structures which play a role as mechanoreceptors for the cells. The microstructure of the intermediate filaments and the expression of their proteins were investigated regarding the cells in the MFe of the human vocal fold unphonated over a decade. The adult vocal fold mucosa of a 64-year-old male with cerebral hemorrhage unphonated for 11 years was investigated by immunohistochemistry and electron microscopy. The intermediate filaments in the cytoplasm of the cells had become fewer in number. And the expression of their characteristic proteins (vimentin, desmin, GFAP) was also reduced. The results of this study are consistent with the hypothesis that mechanotransduction caused by vocal fold vibration could possibly be a factor in regulating the function and fate of the cells in the MFe.

Dilated Minute Chambers in Laryngeal Vocal Fold Polyps: Histopathological and Ultrastructural Features.

In Reinke's space of human vocal fold, type III collagen forms a three dimensional network and this contains numerous minute chambers in between these fibers. These compartments are occupied by glycosaminoglycans and glycoproteins. In laryngeal fold lesions, such as Reinke's edema and vocal fold polyps, proteoglycan (PG)/hyaluronic acid (HA) components of extracellular matrix increased. We investigated the size and quantity of the minute chambers within Reinke's space, filled with PG/HA with the aid of transmission electron microscopy. Eight vocal fold polyps and 10 mucosal biopsies (as control group) were all evaluated by light microscopy and electron microscopy. We detected that PG/HA in extracellular matrix had been increased in vocal fold lesions when compared with control group, by Alcian Blue-pH 2.5 stain. The mean volume of the chambers in Reinke's space of normal larynx was measured as 0.040233 µm2 whereas the mean volume of these chambers in vocal fold polyps was measured as 6.420221 µm2. The difference between the volumes of these chambers in vocal fold polyps and in control group was statistically significant (P = 0.001). Within these chambers PG/HA were found and PG/HA filling these chambers were increased in vocal fold polyps. We think proteoglycan and glycosaminoglycans, especially HA, play an important role in determining biochemical properties of vocal fold lesions.

Acute exposure to vibration is an apoptosis-inducing stimulus in the vocal fold epithelium.

Clinical voice disorders pose significant communication-related challenges to patients. The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (30, 60, 120min) or a control group (120min of vocal fold adduction and abduction). Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency. Laryngeal tissue specimens were evaluated for apoptosis and gene transcript and protein levels of TNF-α. Results revealed that terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was significantly higher after 120min of vibration compared to the control. Transmission electron microscopy (TEM) revealed no significant effect of time-dose on the mean area of epithelial cell nuclei. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Our findings suggest that apoptosis can be induced in the vocal fold epithelium after 120min of modal intensity phonation. In contrast, shorter durations of vibration exposure do not result in apoptosis signaling. However, morphological features of apoptosis are not observed using TEM. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases.

Scanning Electron Microscopy of the Presbylarynx.

OBJECTIVE: To describe the findings on the presbylarynx under scanning electron microscopy. STUDY DESIGN: Cadaver study. SETTING: Universidade Estadual Paulista (Botucatu, São Paulo, Brazil). SUBJECTS AND METHODS: Sixteen vocal folds were removed during necropsies and distributed into 2 age groups: control (n = 8; aged 30-50 years) and elderly (n = 8; aged 75-92 years). The right vocal fold was dissected, fixed in glutaraldehyde 2.5%, and prepared for scanning electron microscopy. The thickness of the epithelium was measured using a scandium morphometric digital program. RESULTS: In the control group, the epithelium had 5 to 7 overlapped cell layers, rare desquamation cells, and little undulation with protruding intercellular junctions. The lamina propria showed a uniform network of collagen and elastic fibers in the superficial layer. A dense network of collagen was identified in the deeper layer. In the elderly group, the epithelium was atrophic (2-3 cells), with more desquamation cells and intercellular junctions delimited by deep sulci. The epithelial thickness was lower in elderly than in controls (mean [SD], 221.64 [145.90] µm vs 41.79 [21.40] µm, respectively). The lamina propria had a dense and irregular distribution of collagen and elastic fibers in the superficial layer. In the deep layers, the collagen fibers formed a true fibrotic and rigid skeleton. CONCLUSION: Scanning electron microscopy identified several changes in the elderly larynx, differentiating it from the controls. These alterations are probably related to the aging process of the vocal folds. However, the exact interpretation of these findings requires additional studies, even to the molecular level, having the fibroblasts as targets.

In vivo investigation of acidified pepsin exposure to porcine vocal fold epithelia.

OBJECTIVES/HYPOTHESIS: The study objective was to investigate epithelial changes in response to direct, repeated, acidified pepsin exposures in an in vivo porcine model. We hypothesized that 12 acidified pepsin applications to simulate reflux would elicit a vocal fold response characterized by inflammation, epithelial proliferation, and increased intercellular space, as well as changes in the gene expression of epithelial junctional proteins, ion transporter proteins, and proinflammatory cytokines. STUDY DESIGN: Prospective, in vivo study. METHODS: Pigs received acidified pepsin (pH = 4) or saline (sham) applied directly to vocal folds. Larynges were collected following three exposures per week for 4 weeks. Vocal fold tissue morphology, collagen, and elastin were evaluated histologically. Gene expression of E-cadherin, zona occludens-1, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, interleukin-1ß, tumor necrosis factor-α, and interferon-γ were measured. Ultrastructural alterations, epithelial intercellular space diameter, and microridge height were measured using transmission electron microscopy. RESULTS: There were no significant differences in histology, gene transcripts, epithelial ultrastructure, intercellular space, and microridge height after acidified pepsin exposure. CONCLUSIONS: Twelve simulated reflux challenges were insufficient to elicit epithelial changes, demonstrating the resistance of healthy vocal folds to direct, repeated acidified pepsin exposures. These data increase our understanding of healthy vocal fold defenses and lay the groundwork for a prospective, uninjured, nonsurgical, laryngopharyngeal reflux model where pigs can be exposed directly to acidified pepsin.

Investigation of nanostructural changes following acute injury using atomic force microscopy in rabbit vocal folds.

There continues to be a paucity of data regarding the nanostructural changes of vocal fold (VF) collagen after injury. The aim of this study is to investigate the nanostructural and morphological changes in the rabbit VF lamina propria following acute injury using atomic force microscopy (AFM). Unilateral VF injury was performed on 9 New Zealand breeder rabbits. Sacrifice and laryngeal harvest were performed at three time points: 1 day, 3 days, and 7 days after injury. Histology and immunohistochemistry data were collected to confirm extracellular matrix (ECM) changes in rabbit VF. The progressive changes in thickness and D-spacing of VF collagen fibrils were investigated over a 7-day postinjury period using AFM. At post-injury day 1, a fibrin clot and inflammatory cell infiltration were observed at the injured VF. The inflammatory score at postinjury day 1 was highest in injured VF tissue, with a significant decrease at postinjury day 7. The immunoreactivity of inflammatory proteins (COX-2, TNF-α) was observed in VF up to day 7 after injury. AFM investigation showed clustered and disorganized collagen fibrils at the nanoscale resolution at post-injury day 7. Collagen fibrils in injured VF at postinjury day 7 were significantly thicker than control and postinjury days 1 and 3 (P < 0.001). D-spacing of collagen at postinjury day 7 was not studied due to loss of distinct edges resulting from immature collagen deposition. AFM investigation of VF could add valuable information to understanding micromechanical changes in VF scar tissue.

[The electron microscopic study of tumour-like structures on the vocal cords].

We have undertaken the electron microscopic investigation into peculiarities of six tumour-like structures on the vocal cords. The study has demonstrated changes in the number and distribution patterns of intercellular junctions, keratin and tonofilament contents in epithelial cells, basal membrane structure, and composition of the basic substance in lamina propria. All the examined tumour-like structures contained bacteria an two of them had viral particles in vacuoles of fibroblasts. Moreover, the bacteria were found on the surface of epithelium, between epithelial cells and in the basic substance in lamina propria. Cytoplasm of epithelial cells and fibroblasts not infrequently contained bacteria in the phase of division.

Reduction of thermocoagulative injury via use of a picosecond infrared laser (PIRL) in laryngeal tissues.

The carbon dioxide (CO2) laser is routinely used in glottic microsurgery for the treatment of benign and malignant disease, despite significant collateral thermal damage secondary to photothermal vaporization without thermal confinement. Subsequent tissue response to thermal injury involves excess collagen deposition resulting in scarring and functional impairment. To minimize collateral thermal injury, short-pulse laser systems such as the microsecond pulsed erbium:yttrium-aluminium-garnet (Er:YAG) laser and picosecond infrared laser (PIRL) have been developed. This study compares incisions made in ex vivo human laryngeal tissues by CO2 and Er:YAG lasers versus PIRL using light microscopy, environmental scanning electron microscopy (ESEM), and infrared thermography (IRT). In comparison to the CO2 and Er:YAG lasers, PIRL incisions showed significantly decreased mean epithelial (59.70 µm) and subepithelial (22.15 µm) damage zones (p < 0.05). Cutting gaps were significantly narrower for PIRL (133.70 µm) compared to Er:YAG and CO2 lasers (p < 0.05), which were more than 5 times larger. ESEM revealed intact collagen fibers along PIRL cutting edges without obvious carbonization, in comparison to diffuse carbonization and tissue melting seen for CO2 and Er:YAG laser incisions. IRT demonstrated median temperature rise of 4.1 K in PIRL vocal fold incisions, significantly less than for Er:YAG laser cuts (171.85 K; p < 0.001). This study has shown increased cutting precision and reduced lateral thermal damage zones for PIRL ablation in comparison to conventional CO2 and Er:YAG lasers in human glottis and supraglottic tissues.

Effects of phonation time and magnitude dose on vocal fold epithelial genes, barrier integrity, and function.

OBJECTIVES/HYPOTHESIS: To investigate the effects of increasing time and magnitude doses of vibration exposure on transcription of the vocal fold's junctional proteins, structural alterations, and functional tissue outcomes. STUDY DESIGN: Animal study. METHODS: 100 New Zealand White breeder rabbits were studied. Dependent variables were measured in response to increasing time doses (30, 60, or 120 minutes) and magnitude doses (control, modal intensity, and raised intensity) of vibration exposure. Messenger RNA expression of occludin, zonula occluden-1 (ZO-1), E-cadherin, ß-catenin, interleukin 1ß, cyclooxygenase-2, transforming growth factor ß-1, and fibronectin were measured. Tissue structural alterations were assessed using transmission electron microscopy (TEM). Transepithelial resistance was used to measure functional tissue outcomes. RESULTS: Occludin gene expression was downregulated in vocal folds exposed to 120-minute time doses of raised-intensity phonation, relative to control, and modal-intensity phonation. ZO-1 gene expression was upregulated following a 120-minute time dose of modal-intensity phonation, compared to control, and downregulated after a 120-minute time dose of raised-intensity phonation, compared to modal-intensity phonation. E-cadherin gene expression was downregulated after a 120-minute time dose of raised-intensity phonation, compared to control and modal-intensity phonation. TEM revealed extensive desquamation of the stratified squamous epithelial cells with increasing time and magnitude doses of vibration exposure. A general observation of lower transepithelial resistance measures was made in tissues exposed to raised-intensity phonation compared to all other groups. CONCLUSIONS: This study provides evidence of vocal fold tissue responses to varying time and magnitude doses of vibration exposure. LEVEL OF EVIDENCE: NA.

Cytoskeleton of newborn vocal fold stellate cells.

OBJECTIVES/HYPOTHESIS: Vocal fold stellate cells (VFSCs) in the human maculae flavae located at both ends of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Tension caused by phonation (vocal fold vibration) likely regulates the behavior of the VFSCs in the human maculae flava. Tensile and compressive strains have direct effects on cell morphology and structure, including changes in cytoskeletal structure and organization. Cytoskeletons play a role as mechanoreceptors for the cells. The microstructure of the intermediate filaments and the expression of their characteristic proteins were investigated regarding the human newborn VFSCs. STUDY DESIGN: Histopathologic analysis of the human newborn vocal fold. METHODS: Three newborn vocal fold mucosae were investigated by immunohistochemistry and electron microscopy. RESULTS: The intermediate filaments in the cytoplasm of the newborn VFSCs were few in number. However, their characteristic proteins (vimentin, desmin, GFAP [Glial fibrillary acidic protein], cytokeratin) had already expressed. CONCLUSION: The function and fate of VFSCs are regulated by various microenvironmental factors. Not only chemical factors but also mechanical factors could also modulate VFSC behaviors. The cytoskeletal structure of the newborn VFSCs is under development. And the newborn VFSCs have not acquired mechanical regulation. LEVEL OF EVIDENCE: N/A.

Quantification of acute vocal fold epithelial surface damage with increasing time and magnitude doses of vibration exposure.

Because the vocal folds undergo repeated trauma during continuous cycles of vibration, the epithelium is routinely susceptible to damage during phonation. Excessive and prolonged vibration exposure is considered a significant predisposing factor in the development of vocal fold pathology. The purpose of the present study was to quantify the extent of epithelial surface damage following increased time and magnitude doses of vibration exposure using an in vivo rabbit phonation model. Forty-five New Zealand white breeder rabbits were randomized to nine groups and received varying phonation time-doses (30, 60, or 120 minutes) and magnitude-doses (control, modal intensity phonation, or raised intensity phonation) of vibration exposure. Scanning electron microscopy and transmission electron microscopy was used to quantify the degree of epithelial surface damage. Results revealed a significant reduction in microprojection density, microprojection height, and depth of the epithelial surface with increasing time and phonation magnitudes doses, signifying increased epithelial surface damage risk with excessive and prolonged vibration exposure. Destruction to the epithelial cell surface may provide significant insight into the disruption of cell function following prolonged vibration exposure. One important goal achieved in the present study was the quantification of epithelial surface damage using objective imaging criteria. These data provide an important foundation for future studies of long-term tissue recovery from excessive and prolonged vibration exposure.

Microstructural characterization of vocal folds toward a strain-energy model of collagen remodeling.

Collagen fibrils are believed to control the immediate deformation of soft tissues under mechanical load. Most extracellular matrix proteins remain intact during frozen sectioning, which allows them to be scanned using atomic force microscopy (AFM). Collagen fibrils are distinguishable because of their periodic roughness wavelength. In the present study, the shape and organization of collagen fibrils in dissected porcine vocal folds were quantified using nonlinear laser scanning microscopy data at the micrometer scale and AFM data at the nanometer scale. Rope-shaped collagen fibrils were observed. The geometric characteristics for the fibrils were fed into a hyperelastic model to predict the biomechanical response of the tissue. The model simulates the micrometer-scale unlocking behavior of collagen bundles when extended from their unloaded configuration. Force spectroscopy using AFM was used to estimate the stiffness of collagen fibrils (1±0.5MPa). The presence of rope-shaped fibrils is postulated to change the slope of the force-deflection response near the onset of nonlinearity. The proposed model could ultimately be used to evaluate changes in elasticity of soft tissues that result from the collagen remodeling.

Picosecond infrared laser (PIRL): an ideal phonomicrosurgical laser?

A comparison of tissue cutting effects in excised cadaver human vocal folds after incisions with three different instruments [scalpel, CO2 laser and the picosecond infrared laser-(PIRL)] was performed. In total, 15 larynges were taken from human cadavers shortly after death. After deep freezing and thawing for the experiment, the vocal folds suspended in the hemilarynx were incised. Histology and environmental scanning electron microscopy (ESEM) analyses were performed. Damage zones after cold instrument cuts ranged from 51 to 135 µm, as compared to 9-28 µm after cutting with the PIRL. It was shown that PIRL incision had smaller zones of tissue coagulation and tissue destruction, when compared with scalpel and CO2 laser cuts. The PIRL technology provides an (almost) atraumatic laser, which offers a quantum jump towards realistic 'micro'-phonosurgery on a factual cellular dimension, almost entirely avoiding coagulation, carbonization, or other ways of major tissue destruction in the vicinity of the intervention area. Although not available for clinical use yet, the new technique appears promising for future clinical applications, so that technical and methodological characteristics as well as tissue experiments seem worthwhile to be communicated at this stage of development.

Histopathologic investigations of the unphonated human child vocal fold mucosa.

OBJECTIVES: Vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) located at both ends of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices. MFe are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate VFSCs to accelerate production of extracellular matrices. Human child vocal fold mucosae unphonated since birth were investigated histologically. STUDY DESIGN: Histologic analysis of human child vocal fold mucosa. METHODS: Vocal fold mucosae, which have remained unphonated since birth, of two children (7 and 12 years old) with cerebral palsy were investigated by light and electron microscopy and compared with normal subjects. RESULTS: Vocal fold mucosae and MFe were hypoplastic and rudimentary and did not have a vocal ligament, Reinke's space, or the layered structure. The lamina propria appeared as a uniform structure. Some VFSCs in the MFe showed degeneration and not many vesicles were present at the periphery of the cytoplasm. The VFSCs synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. The VFSCs appeared to have decreased activity. CONCLUSION: Vocal fold vibration (phonation) after birth is an important factor in the growth and development of the human vocal fold mucosa.

Morphological properties of collagen fibers in porcine lamina propria.

OBJECTIVES: Collagen influences the biomechanical properties of vocal folds. Altered collagen morphology has been implicated in dysphonia associated with aging and scarring. Documenting the morphological properties of native collagen in healthy vocal folds is essential to understand the structural and functional alterations to collagen with aging and disease. Our primary objective was to quantify the morphological properties of collagen in the vocal fold lamina propria. Our secondary exploratory objective was to investigate the effects of pepsin exposure on the morphological properties of collagen in the lamina propria. STUDY DESIGN: Experimental, in vitro study with porcine model. METHODS: Lamina propria was dissected from 26 vocal folds and imaged with atomic force microscopy (AFM). Morphological data on d-periodicity, diameter, and roughness of collagen fibers were obtained. To investigate the effects of pepsin exposure on collagen morphology, vocal fold surface was exposed to pepsin or sham challenge before lamina propria dissection and AFM imaging. RESULTS: The d-periodicity, diameter, and roughness values for native vocal fold collagen are consistent with literature reports of collagen fibers in other body tissues. Pepsin exposure on vocal fold surface did not appear to change the morphological properties of collagen fibers in the lamina propria. CONCLUSIONS: Quantitative data on collagen morphology were obtained at nanoscale resolution. Documenting collagen morphology in healthy vocal folds is critical for understanding the physiological changes to collagen with aging and scarring and for designing biomaterials that match the native topography of lamina propria.