Normal Vocal Fold Research Articles

Characterization of non-epithelial cells embedded within the vocal fold epithelial barrier

The vocal folds vibrate to produce voice, undergoing significant stress due to contact and shearing force. The epithelium operates as the primary protective layer of the tissue against stress and vibratory damage, as well as to provide a barrier against foreign organisms and toxins. Within the vocal fold epithelium, non-epithelial cells were identified that may interrupt the epithelium and compromise the epithelial barrier’s protective function. Human vocal fold samples with a variety of pathologies were compared to normal vocal folds. Analysis included the number of cells in the epithelium and epithelial thickness. Vocal fold sections from 10 human tissue samples were assessed via H&E staining and immunofluorescent co-labeling. Three cell populations (vimentin expressing, CD-45 expressing, and cells expressing both) were identified within the epithelium. Statistical analysis revealed that the abnormal samples had a significantly greater number of vimentin-positive cells/area within the epithelium compared to the normal samples. Additionally, normal tissue samples had a significantly greater epithelial depth, suggesting a more robust epithelial barrier compared to tissue with pathology. Knowledge of the function of these cells could lead to a better understanding of how the local immune environment near and within vocal fold epithelium changes in the presence of different pathologies.

Acoustic Character Governing Variation in Normal, Benign, and Malignant Voices.

Benign and malignant vocal fold lesions (VFLs) are growths that occur on the vocal folds. However, the treatments for these two types of lesions differ significantly. Therefore, it is imperative to use a multidisciplinary approach to properly recognize suspicious lesions. This study aimed to determine the important acoustic characteristics specific to benign and malignant VFLs. The acoustic model of voice quality was utilized to measure various acoustic parameters in 157 participants, including individuals with normal, benign, and malignant conditions. The study comprised 62 female and 95 male participants (43 ± 10 years). Voice samples were collected at the Shanghai Eye, Ear, Nose, and Throat Hospital of Fudan University between May 2020 and July 2021. The acoustic variables of the participants were analyzed using Principal Component Analysis (PCA) to present important acoustic characteristics that are specific to normal vocal folds, benign VFLs, and malignant VFLs. The similarities and differences in acoustic factors were also studied for benign conditions including Reinke's edema, polyps, cysts, and leukoplakia. Using the PCA method, the components that accounted for the variation in the data were identified, highlighting acoustic characteristics in the normal, benign, and malignant groups. The analysis indicated that coefficients of variation in root mean square energy were observed solely within the normal group. Coefficients of variation in pitch (F0) were found to be significant only in benign voices, while higher formant frequencies and their variability were identified as contributors to the acoustic variance within the malignant group. The presence of formant dispersion (FD) as a weighted factor in PCA was exclusively noted in individuals with Reinke's edema. The amplitude ratio between subharmonics and harmonics (SHR) and its coefficients of variation were evident exclusively in the polyps group. In the case of voices with cysts, both pitch (F0) and coefficients of variation for FD were observed to contribute to variations. Additionally, higher formant frequencies and their coefficients of variation played a role in the acoustic variance among voices of patients with leukoplakia. Experimental evidence demonstrates the utility of the PCA method in the identification of vibrational alterations in the acoustic characteristics of voice affected by lesions. Furthermore, the PCA analysis has highlighted underlying acoustic differences between various conditions such as Reinke's edema, polyps, cysts, and leukoplakia. These findings can be used in the future to develop an automated malignant voice analysis algorithm, which will facilitate timely intervention and management of vocal fold conditions.

Analysis of vocal fold movement characteristics in patients with laryngeal neurogenic injury

Objective:To analyze the characteristics of vocal fold movement and glottic closure in patients with laryngeal neurogenic injury. Methods:A total of 185 patients with vocal fold paralysis diagnosed by laryngeal electromyography as neurogenic damage to cricothyroid muscle, thyreoarytenoid muscle and posterior cricoarytenoid muscle were enrolled, they were divided into unilateral vocal fold paralysis group and bilateral vocal fold paralysis group, respectively, and superior laryngeal paralysis group, recurrent laryngeal nerve paralysis group and vagal nerve paralysis group according to nerve injury. The characteristics of vocal fold movement and glottic closure were analyzed under strobe laryngoscope. The qualitative evaluation of vocal fold movement was fixed vocal fold, reduced vocal fold movement and normal vocal fold movement, and the qualitative evaluation of glottic closure was glottic closure and glottic imperfection. The results were analyzed statistically. Results:The proportion of normal, reduced and fixed vocal fold motion in bilateral vocal fold paralysis group was significantly different from that in unilateral vocal fold paralysis group（P<0.05）, the composition of normal and reduced vocal fold motion in bilateral vocal fold paralysis group（47.70%） was significantly greater than that in unilateral vocal fold paralysis group（12.27%）. There was no significant difference between the proportion of glottic closure and glottic imperfecta in bilateral vocal fold paralysis group and unilateral vocal fold paralysis group（P<0.05）. The proportion of decreased vocal fold motion in superior laryngeal nerve paralysis group（50.00%） was higher than that in recurrent laryngeal nerve paralysis group（9.32%） and vagal nerve paralysis group（9.00%）. The proportion of decreased and fixed vocal fold motion in superior laryngeal nerve paralysis group, recurrent laryngeal nerve paralysis group and vagal nerve paralysis group was statistically significant（P<0.05）.There was no significant difference in glottic closure among the three groups（P<0.05）. Conclusion:Vocal fold movement characteristics of patients with laryngeal neurogenic injury were mainly vocal fold fixation, or normal or weakened vocal fold movement. There may be missed diagnosis of unilateral vocal fold paralysis in clinical practice. In half of the patients with superior laryngeal nerve palsy, vocal fold movement is characterized by vocal fold fixation.

High-Resolution Profiling of Human Vocal Fold Cellular Landscapes With Single-Nuclei RNA Sequencing.

The function of the vocal folds (VFs) is determined by the phenotype, abundance, and distribution of differentiated cells within specific microenvironments. Identifying this histologic framework is crucial in understanding laryngeal disease. A paucity of studies investigating VF cellular heterogeneity has been undertaken. Here, we examined the cellular landscape of human VFs by utilizing single-nuclei RNA-sequencing. Normal true VF tissue was excised from five patients undergoing pitch elevation surgery. Tissue was snap frozen in liquid nitrogen and subjected to cellular digestion and nuclear extraction. Nuclei were processed for single-nucleus sequencing using the 10X Genomics Chromium platform. Sequencing reads were assembled using cellranger and analyzed with the scanpy package in python. RNA sequencing revealed 18 global cell clusters. While many were of epithelial origin, expected cell types, such as fibroblasts, immune cells, muscle cells, and endothelial cells were present. Subcluster analysis defined unique epithelial, immune, and fibroblast subpopulations. This study evaluated the cellular heterogeneity of normal human VFs by utilizing single-nuclei RNA-sequencing. With further confirmation through additional spatial sequencing and microscopic imaging, a novel cellular map of the VFs may provide insight into new cellular targets for VF disease. NA Laryngoscope, 134:3193-3200, 2024.

Empirical Distribution of Glottal Edges (EDGE): A Statistical Assessment of Vocal Fold Kinematics Using High-Speed Videoendoscopy.

Although laryngeal high-speed videoendoscopy (HSV) is crucial for studying vocal fold vibrations, its translation to clinical practice has been hindered by the large volume of data it produces and the difficulty in interpreting current analysis methods. Although image processing techniques have been developed to map spatial-temporal data into two-dimensional representations, they alter the geometrical construction of the glottis and do not provide standard quantitative features, thus challenging clinical interpretation. In response, we propose a new visualization and analysis framework for assessing the dynamics of vocal folds based on the empirical distribution of the glottal edge using HSV. This procedure analyzes vocal fold oscillations by preserving the shape of the glottis and quantifying the asymmetry between right and left vocal fold displacements along the anterior-posterior axis. This method was evaluated on four groups of participants: ten with normal voices, ten with vocal fold nodules, ten with muscle tension dysphonia, and two with unilateral vocal fold paralysis. The proposed method produces distinct representations for normal and pathological vocal fold vibratory behaviors and derived features based on amplitude and phase asymmetry metrics that show statistically significant differences between normal and pathological groups. Comparative analysis with state-of-the-art techniques indicates that our proposed method can complement the assessment of vocal fold vibration and enhance the clinical translation of HSV.

Multivariate Analysis of Vocal Fold Vibrations in Normal Speakers Using High-Speed Digital Imaging.

Little is known about the normal variations in vocal fold vibrations. We conducted a prospective study on normal subjects using high-speed digital imaging (HSDI) to elucidate key parameters regarding age/gender-related normal variations. Forty-six healthy adult volunteers were divided into young (aged ≤35 years) male, young female, elderly (aged ≥65 years) male, and elderly female subgroups. HSDI data of sustained phonation of /i/ at a comfortable pitch and loudness were obtained, and vibratory parameters were calculated using the visual-perceptual rating, laryngotopography, digital kymography, and glottal area waveform. Multivariate analysis was then performed on these parameters to clarify the subgroup-specific key parameters. Four key parameters were identified from a total of 83: one from visual perceptual rating and three from laryngotopography. Subgroup analyses showed that posterior-to-anterior longitudinal phase difference (PD) and high fundamental frequency (F0) were specific to young female participants. A low F0 was specific to young male participants. Large anterior-to-posterior longitudinal PD and its left-right difference were specific to elderly male participants. There were no key parameters for elderly female participants. Methods that can assess F0 and longitudinal PD, such as visual-perceptual rating and laryngotopography, were effective in the evaluation of normal vocal fold vibrations and their variations.

Glycolytic Metabolism of the Tissue Stem Cells in the Maculae Flavae of the Human Vocal Fold

Metabolic programs in the stem cells are essential for maintaining homeostasis and protecting against stem cell aging. There is growing evidence that the tissue stem cells reside in the anterior and posterior maculae flavae of the human vocal fold mucosa. Our previous studies observed that the glycolysis of the cell in the human maculae flavae seems to rely more on anaerobic glycolysis for energy supply in comparison with oxidative phosphorylation. However, previous studies showed only the metabolic enzymes of glycolysis and functional morphology of the mitochondria, therefore, it has not yet been determined whether anaerobic glycolysis actually took place. The purpose of this study is to investigate the glycolytic metabolites of the cells in the maculae flavae of the human vocal fold in vitro. Four normal human vocal folds were used. After extraction of the anterior maculae flavae, cells in the maculae flavae were cultured and proliferated. Glucose transporter-1 was assessed using immunocytochemistry and metabolites of glycolysis (lactate and NADPH) were measured. The cells in the maculae flavae expressed glucose transporter-1 in the cytoplasm and the cell membranes. In addition, the cultured cells produced lactate (metabolites of anaerobic glycolysis) and NADPH (metabolites of the pentose phosphate pathway). The cells in the maculae flavae of the human vocal folds were found to undergo anaerobic glycolysis via the pentose phosphate pathway. This suggests that the cells in the maculae flavae of the human vocal fold have a metabolism that favors the maintenance of stemness and undifferentiated states.

Pathogenesis of Reinke's Edema of the Vocal Fold.

The most frequent etiologic factor of Reinke's edema (RE) is considered to be smoking. However, the mechanism for the onset and development of the disease remains unclear. Hypoxia-inducible factor-1α (HIF-1α) is an oxygen-dependent transcriptional activator which plays crucial roles in angiogenesis in hypoxic microenvironments. HIF-1α induces the expression of vascular endothelial growth factor (VEGF) which involves angiogenesis and enhances vascular permeability. This study investigated the roles of HIF-1α in the pathogenesis of RE. Surgical specimens of RE from patients who underwent endolaryngeal microsurgery were used. Normal vocal folds were used as a control group. Expression of HIF-1α and VEGF was analyzed by immunohistochemistry. Three-dimensional fine structures of the vessels in RE were investigated using correlative light and electron microscopy (CLEM) technique. HIF-1α and VEGF were broadly expressed in the stromal, inflammatory, and endothelial cells in the lamina propria of the vocal fold of RE. The expression of HIF-1α and VEGF of RE were significantly higher than in the lamina propria of the normal vocal fold mucosa. CLEM showed vascularization and telangiectasia and there were many dilated capillaries with thin endothelium not covered with pericytes indicating the vessels were fragile. Transcription factor HIF-1α and induced VEGF likely play roles in the pathogenesis of RE. And increased vascular permeability with fragile vessels in angiogenesis is likely to be an etiology of RE. Transcription factor HIF-1α and induced VEGF are potential therapeutic targets for RE. NA Laryngoscope, 134:1785-1791, 2024.

Ventricular fold oscillations lower the vocal pitch in rhesus macaques.

We carried out ex vivo and in vivo experiments to explore the functional role of the ventricular folds in sound production in macaques. In the ex vivo experiments, 29 recordings out of 67 showed that the ventricular folds co-oscillated with the vocal folds. Transitions from normal vocal fold oscillations to vocal-ventricular fold co-oscillations as well as chaotic irregular oscillations were also observed. The in vivo experiments indicated that the vocal-ventricular fold co-oscillations were also observed in two macaque individuals. In both ex vivo and in vivo experiments, the vocal-ventricular fold co-oscillations significantly lowered the fundamental frequency. A mathematical model revealed that the lowering of the fundamental frequency was caused by a low oscillation frequency inherent in the ventricular folds, which entrained the vocal folds to their low-frequency oscillations. From a physiological standpoint, the macaques may utilize the ventricular fold oscillations more frequently than humans. The advantages as well as disadvantages of using the ventricular folds as an additional vocal repertory are discussed.

A self-fused hydrogel for the treatment of glottic insufficiency through outstanding durability, extracellular matrix-inducing bioactivity and function preservation.

Injection laryngoplasty with biomaterials is an effective technique to treat glottic insufficiency. However, the inadequate durability, deficient pro-secretion of extracellular matrix (ECM) and poor functional preservation of current biomaterials have yielded an unsatisfactory therapeutic effect. Herein, a self-fusing bioactive hydrogel comprising modified carboxymethyl chitosan and sodium alginate is developed through a dual-crosslinking mechanism (photo-triggered and dynamic covalent bonds). Owing to its characteristic networks, the synergistic effect of the hydrogel for vocal folds (VFs) vibration and phonation is adequately demonstrated. Notably, owing to its inherent bioactivity of polysaccharides, the hydrogel could significantly enhance the secretion of major components (type I/III collagen and elastin) in the lamina propria of the VFs both in vivo and in vitro. In a rabbit model for glottic insufficiency, the optimized hydrogel (C1A1) has demonstrated a durability far superior to that of the commercially made hyaluronic acid (HA) Gel. More importantly, owing to the ECM-inducing bioactivity, the physiological functions of the VFs treated with the C1A1 hydrogel also outperformed that of the HA Gel, and were similar to those of the normal VFs. Taken together, through a simple-yet-effective strategy, the novel hydrogel has demonstrated outstanding durability, ECM-inducing bioactivity and physiological function preservation, therefore has an appealing clinical value for treating glottic insufficiency.

Support of deep learning to classify vocal fold images in flexible laryngoscopy

PurposeTo collect a dataset with adequate laryngoscopy images and identify the appearance of vocal folds and their lesions in flexible laryngoscopy images by objective deep learning models. MethodsWe adopted a number of novel deep learning models to train and classify 4549 flexible laryngoscopy images as no vocal fold, normal vocal folds, and abnormal vocal folds. This could help these models recognize vocal folds and their lesions within these images. Ultimately, we made a comparison between the results of the state-of-the-art deep learning models, and another comparison of the results between the computer-aided classification system and ENT doctors. ResultsThis study exhibited the performance of the deep learning models by evaluating laryngoscopy images collected from 876 patients. The efficiency of the Xception model was higher and steadier than almost the rest of the models. The accuracy of no vocal fold, normal vocal folds, and vocal fold abnormalities on this model were 98.90 %, 97.36 %, and 96.26 %, respectively. Compared to our ENT doctors, the Xception model produced better results than a junior doctor and was near an expert. ConclusionOur results show that current deep learning models can classify vocal fold images well and effectively assist physicians in vocal fold identification and classification of normal or abnormal vocal folds.

Simulation en dermatologie 1 : consultation d’annonce d’un mélanome

To collect a dataset with adequate laryngoscopy images and identify the appearance of vocal folds and their lesions in flexible laryngoscopy images by objective deep learning models.We adopted a number of novel deep learning models to train and classify 4549 flexible laryngoscopy images as no vocal fold, normal vocal folds, and abnormal vocal folds. This could help these models recognize vocal folds and their lesions within these images. Ultimately, we made a comparison between the results of the state-of-the-art deep learning models, and another comparison of the results between the computer-aided classification system and ENT doctors.This study exhibited the performance of the deep learning models by evaluating laryngoscopy images collected from 876 patients. The efficiency of the Xception model was higher and steadier than almost the rest of the models. The accuracy of no vocal fold, normal vocal folds, and vocal fold abnormalities on this model were 98.90 %, 97.36 %, and 96.26 %, respectively. Compared to our ENT doctors, the Xception model produced better results than a junior doctor and was near an expert.Our results show that current deep learning models can classify vocal fold images well and effectively assist physicians in vocal fold identification and classification of normal or abnormal vocal folds.

Comparative Results of Vocal Fold Injury According to the Fiber-Based Laser in a Rabbit Vocal Fold Model

We assessed the safety of flexible fiber-based lasers including the potassium-titanyl-phosphate (KTP), CO2, and Holmium lasers using a rabbit vocal fold model and provided the safety evidence of laser before human clinical trial. In all, 120 male New Zealand white rabbits were used. Each laser was used to induce acute and chronic vocal fold injuries in 40 rabbits. The same laser energy with the same intensity and frequency were used in all cases, and we evaluated outcomes via surface scanning electron microscopy (SEM) and histological examination 1 day after injury. Histological and high-speed vocal fold vibration examinations were evaluated 1 month after injury. Surface injury roughness grading was done via SEM, and the Acute injury ratio and lamina propria (LP) ratio were also calculated. The dynamic glottal gap was measured through functional analyses based on recordings from a high-speed digital camera. The Holmium laser caused significantly more vocal fold damage than did the KTP and CO2 lasers as revealed by SEM and evaluation of acute and chronic injuries. Functional analysis with high-speed digital camera indicated that the holmium laser reduced dynamic glottal gap compared with the normal vocal fold, where the other lasers did not. The histological and functional analyses of rabbit vocal fold experiments indicated that fiber-based laryngeal laser surgery for vocal fold lesions could be performed relatively safely using a KTP or CO2 laser.

Long-term outcomes of lateral vocal fold autologous fat injection for unilateral vocal fold paralysis

Objective: To investigate the long-term outcomes of patients with unilateral vocal fold paralysis resulting in dysphonia treated with lateral vocal fold autologous fat injection. To analyze the factors that may affect the long-term efficacy of the procedure. Methods: From July 2003 to June 2020, 163 patients (86 males and 77 females), aged 9-73 years (mean (34.50±12.94) years) with unilateral vocal fold paralysis resulting in dysphonia underwent transoral laryngoscopic injection of autologous fat into the lateral vocal folds. Subjective auditory perception assessment (GRBAS scale), objective acoustic assessment, voice handicap index (VHI) evaluation and stroboscopic laryngoscopy were compared before and after the surgery. Patients were followed up for 1 to 18 years, with median follow-up time of 6 years. SPSS 22.0 software was used for statistical analysis. Results: Of 163 patients, 17 patients (10.4%) had mild hoarseness (G1) and 146 patients (89.6%) had moderate to severe hoarseness (G2-3). Stroboscopic laryngoscopy revealed an arch-shaped vocal fold on the affected side, fixed in the paramedian position or abduction position, with obvious glottic closure fissure. Postoperatively, voice recovered to normal (G0) in 139 patients (85.3%), mild hoarseness (G1) in 18 patients (11.0%) and moderate hoarseness (G2) in 6 patients (3.7%). Of these, 131 patients (80.4%) showed significant improvement in hoarseness, 29 patients (17.8%) showed mild improvement and 3 patients (1.8%) showed no significant improvement in hoarseness. Objective acoustic parameters of Jitter, Shimmer, NHR and MPT improved significantly, as did VHI scores. Stroboscopic laryngoscopy showed medialization of the affected vocal folds, improved vocal fold closure and normal or nearly normal vocal fold mucosal waves. With a fat injection volume of 3.0-4.5 ml, the patient's subjective auditory perception scores of G, R, B and A improved more significantly within 3 months after surgery, and both VHI and MPT were significantly better since 1 year after surgery. With bilateral vocal fold injection, the B and A scores improved significantly from 1 month postoperatively compared to unilateral injections(unilateral vs. bilateral injection 1 month post-operation, tB scores=1.42,tA scores=1.51,P<0.05). Conclusions: The long-term efficacy of autologous fat injection in the paraglottic space for the treatment of unilateral vocal fold paralysis was stable. The efficacy of the surgery was related to the amount of fat injected, unilateral or bilateral of the injection.

Using Diffusion Tensor Imaging to Explore the Changes in the Microstructure of Canine Vocal Fold Scar Tissue

To apply diffusion tensor imaging (DTI) in measurement of the diffusion characteristics of water molecules in vocal fold scar tissue, combined with the analysis of textural characteristics of collagen fibers in the cover layer of the vocal folds to explore the feasibility of DTI in the qualitative and quantitative diagnosis of vocal fold scars and the evaluation of microstructural changes of vocal fold scar tissue. A unilateral injury was created using micro-cup forceps in the left vocal fold of six beagles. The contralateral normal vocal fold was used as a self-control. Five months postinjury, the larynges were excised and placed into a magnetic resonance imaging (MRI) system (9.4T BioSpec MRI, Bruker, German) for scanning and extraction of the diffusion parameters, fractional anisotropy (FA) and tensor trace in the anterior, middle, and posterior portions of the vocal fold cover layer. These parameters were then analyzed for statistical significance between the scarred vocal fold and the normal vocal fold. After MRI scanning, the tissue of the vocal folds was divided into anterior, middle, and posterior parts for sectioning and staining with hematoxylin and eosin, and samples were subsequently digitally scanned for texture analysis. The irregularity parameters, energy, contrast, correlation, and homogeneity, of collagen fibers of the vocal folds and the mean gray value of collagen fibers were calculated by the gray-level co-occurrence matrix (GLCM) texture analysis method. The differences in the mean value of the two sides of the vocal fold were compared. In addition, Pearson correlation analysis was performed between DTI parameters and irregularity parameters. The FA of the left vocal fold cover layer was significantly lower compared to the self-control group (P=0.0366), and the tensor trace value on the left vocal fold cover layer was significantly higher compared to the self-control group (P=0.0353). The FA was significantly higher in the anterior part of the right vocal fold cover layer compared to the middle and posterior parts of the same side (P=0.0352), and the tensor trace was significantly lower in the anterior part of the right vocal fold cover layer compared to the middle and posterior parts of the same side (P=0.0298). There were no significant differences in FA and tensor trace between the middle and posterior parts of the vocal fold cover layer. The mean gray value of the left vocal folds cover layer was significantly smaller than the right vocal fold cover layer (P=0.0219), the energy of the left vocal fold cover layer was significantly smaller than that of the right vocal fold cover layer (P < 0.0001), the contrast of the left vocal folds cover layer was significantly larger than that of the right vocal fold cover layer (P=0.0002), the correlation of the left vocal folds cover layer was significantly smaller than the right vocal fold cover layer (P=0.0002), and the homogeneity of the left vocal folds cover layer was significantly smaller than the right vocal fold cover layer (P=0.0003). Pearson correlation analysis yielded values of r=0.926, P=0.000 between the FA and mean gray value; r=-0.918, P=0.000 between FA and energy; r=-0.924, P=0.000 between the FA and homogeneity, r=-0.949, P=0.000 between tensor trace and mean gray value; r=0.893, P=0.000 between the tensor trace and energy; and r=0.929, P=0.000 between the tensor trace and homogeneity. FA and tensor trace can be used as effective parameters to reflect microstructural changes in vocal fold scars. DTI is an objective and quantitative method of analyzing vocal fold scarring, and it noninvasively evaluates the microstructure of vocal fold collagen fibers.

Metabolic Roles of Colony-Forming Tissue Stem Cells in the Maculae Flavae of Newborn Vocal Fold In Vivo

Tissue stem cells in the maculae flavae (a stem cell niche) of the human vocal fold form colonies in vivo like stem cells in vitro. However, the roles of colony-forming aggregated cells in the maculae flavae in vivo have not yet been determined. This study investigated the glycolysis, of the colony-forming aggregated cells in the maculae flavae of the human newborn vocal fold in vivo. Three normal newborn vocal folds were investigated under light microscopy with immunohistochemistry and transmission electron microscopy. Colony-forming aggregated cells in the newborn maculae flavae strongly expressed glucose transporter-1 and glycolytic enzymes (hexokinase II, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase A). The colony-forming aggregated cells did not express phosphofructokinase-1 (rate-limiting enzyme of regular glucose metabolism pathway) but expressed glucose-6-phosphate dehydrogenase (rate-limiting enzyme) indicating the cells relied more on the pentose phosphate pathway. The colony-forming aggregated cells' strong expression of lactate dehydrogenase A indicated that they rely more on anaerobic glycolysis in an anaerobic microenvironment. Mitochondrial cristae of the colony-forming aggregated cells in the newborn maculae flavae were sparse. Consequently, the microstructural features of the mitochondria suggested that their metabolic activity and oxidative phosphorylation were low. The colony-forming aggregated cells in the maculae flavae of the newborn vocal fold seemed to rely more on anaerobic glycolysis using the pentose phosphate pathway for energy supply in vivo. Microstructural features of the mitochondria and the glycolytic enzyme expression of the colony-forming aggregated cells suggested that the oxidative phosphorylation activity was low. Already at birth, in the anaerobic microenvironment of the macular flavae in vivo, there is likely a complex cross-talk regarding metabolism between the colony-forming aggregated cells along the adhesion machinery and chemical signaling pathways which reduces toxic oxygen species and is favorable to maintaining the stemness and undifferentiated states of the tissue stem cells.

Polypositional transcutaneous ultrasound assessment of vocal folds mobility. Ultrasonographic sign of laryngeal dysfunction

To increase the effectiveness of the diagnosis of laryngeal dysfunction using transcutaneous laryngeal ultrasonography. Patients underwent laryngeal ultrasonography and videolaryngoscopy before and after thyroid or parathyroid surgery. Ultrasound was performed polypositionally in the transverse and oblique planes. Functional tests with breathing and breath holding were used. Qualitative (the smile or flying bird signs, the vertical closing line of the vocal folds, synchronicity and symmetry movement of the arytenoid cartilages) and quantitative (the length contraction of the vocal cord, the rotation angle of the arytenoid cartilage) ultrasonic parameters determin the normal vocal folds mobility. 996 patients were included in the study. Vocal folds paresis was detected in 106 (10.6%) patients. In 72 (7.2%) cases partial impaired mobility of the vocal folds (laryngeal dyskinesia) were detected. The echographic patterns of these patients were analyzed. Qualitative ultrasound signs of laryngeal dysfunction were identified: a crooked smile or falling bird signs, a closing line deformation of the vocal folds, an arytenoid immobility. Quantitative ultrasound signs included: a decrease in the length contraction of the vocal cord and a reduction of rotation angle of the arytenoid cartilage. Unilateral laryngeal paresis was diagnosed in 101 (10.1%) patients. In unilateral disorders the rotation angle of the arytenoid on the affected side was 0-14° and the length contraction of the vocal cord was 0-1.8 mm. A crooked smile or falling bird signs, a closing line deformation of the vocal folds and immobility of the arytenoid cartilages were also determined. In 5 (0.5%) cases bilateral laryngeal paresis was revealed, in which on both sides the rotation angles of the arytenoid were 0-14°, and the length contraction of the vocal cords was 0-1.8 mm. At the same time there was no a smile or flying bird signs and a closing line of the vocal folds. Laryngeal dyskinesia was characterized by a crooked smile or falling bird signs and a closing line deformation of the vocal folds. At the same time, partial mobility of the arytenoid cartilage was noted in comparison with the contralateral side (there was a difference in the rotation angle of the arytenoid between the right and left sides of 15 ° or more degrees). The sensitivity and specificity polypositional ultrasound of the vocal folds in women were 100% and 99.8%, in men - 85.7% and 99.2%, respectively.

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

ObjectivesThere 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. MethodsThree 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. ResultsCapillaries 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. ConclusionsThere 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.

Comparison of convolutional neural networks for classification of vocal fold nodules from high-speed video images.

Deep learning is in this study used through convolutional neural networks (CNN) to the determination of vocal fold nodules. Through high-speed video (HSV) images and computer-assisted tools, a comparison of convolutional neural network models and their accuracy will be presented. The data have been collected by an Ear Nose Throat (ENT) specialist with a 90° rigid scope in the years from 2007 to 2019, where 15.732 high-speed videos have been collected from 7909 patients. A total of 4000 images have been carefully selected, 2000 images were of normal vocal folds and 2000 images were of vocal folds with varying degrees of vocal fold nodules. These images were then split into training-, validation-, and testing-data set, for use with a CNN model with 5 layers (CNN5) and compared to other models: VGG19, MobileNetV2, and Inception-ResNetV2. To compare the neural network models, the following evaluation metrics have been calculated: accuracy, sensitivity, specificity, precision, and negative predictive values. All the trained CNN models have shown high accuracy when applied to the test set. The accuracy is 97.75%, 83.5%, 91.5%, and 89.75%, for CNN5, VGG19, MobileNetV2, and InceptionResNetV2, respectively. Precision was identified as the most relevant performance metric for a study that focuses on the classification of vocal fold nodules. The highest performing model was MobilNetV2 with a precision of 97.7%. The average accuracy across all 4 neural networks was 90.63% showing that neural networks can be used for classifying vocal fold nodules in a clinical setting.

PSAT383 Tension-free Thyroidectomy in the Treatment of Thyroid Cancer and Follicular Neoplasia: Experience of the First 100 Cases

Abstract Background and aims. The safety of thyroid surgery in terms of recurrent laryngeal nerve palsy and hypoparathyroidism was increasing in the last decade. In this study, we present a new method of tension-free thyroidectomy (TFT), which could be used to further decrease the complication rate after thyroidectomy. Patients and Methods The procedure is based on the medial approach to the recurrent laryngeal nerve and the parathyroid glands after the division of isthmus and successive total dissection of Barry ligament. One hundred consecutive patients (128 nerves at risk) underwent "tension-free thyroidectomy" (TFT) from August to November 2021 performed by one surgeon. There were 80 females and 20 male patients (ratio 4: 1) with a mean age of ninety-two 46,4 (range from 17 to 75). Lobectomy was carried out in 74 (74%) patients, total thyroidectomy in 26 (26%). In 42 cases patients additionally underwent central or/and lateral neck dissection. Indications for surgery were papillary carcinoma (N=52), medullary cancer (N=2), follicular neoplasia - Bethesda IV group after fine-needle biopsy (N=46). Mean thyroid nodule size was 25,3 mm (ranged 7 - 120 mm). Intraoperative transient neuromonitoring was used in all the cases (5 mA), in 20 cases permanent neuromonitoring was also used. Translaryngeal untrasound or direct laryngoscopy were routinely used prior and after surgery to evaluate vocal cords mobility. Calcium and parathormone level were measured in patients after thyroidectomy on the first, 14th and 30th postoperative days. Results The mean operating time of lobectomy was 80±39 (range: 25-120), for total thyroidectomy 80 ±36 (range: 55-130). There was no conversion to the conventional lateral-to-medial approach. No fluctuation of electric activity of laryngeal nerves during surgery was revealed. Intraoperative loss of signal (LOS) due to thermal effect of electrocautery and subsequent transient unilateral laryngeal nerve palsy occurred in 3 cases (2,3% from the total number of nerves at risk). In all these patients normal vocal fold function was confirmed on the 30th day after surgery. No permanent nerve palsy cases revealed. Three patients (out of 26 in the total thyroidectomy group – 11,5%) exhibited a decrease of parathyroid hormone level on the postoperative day 1 which was resolved in two weeks under the substitution therapy with calcium and alfacalcidol. No cases of permanent hypoparathyroidism occurred. Conclusion TFT (tension-free thyroidectomy) can be considered a safe and feasible operation in patients with thyroid cancer and follicular neoplasia. Larger and comparative (randomized) studies with conventional dissection technique should be performed to investigate the hypothesis that this approach can provide a lower complication rate. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.