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Objective: The efficiency of laryngovideostroboscopy (LVS) in detecting premalignancies of the vocal fold and early glottic cancer was determined in a prospective monocentric study. In addition, the recovery rate of the mucosal membrane on the vocal fold after surgical intervention was determined by LVS. Methods: We included 159 patients with a leukoplakia of the vocal folds and 50 healthy controls. Clinicopathological data and LVS characteristics (amplitude, mucosal wave, nonvibratory segment, glottic closure, phase symmetry, periodicity) at the lesion site were obtained and compared with the histopathological results. LVS parameters were recorded before cordectomy and in a 12-month follow-up interval. Patients who had prior laryngosurgery, radiotherapy, or laryngeal scarring were excluded. Results: Absent or greatly reduced mucosal waves were found in all patients with an invasive carcinoma, in 94% with a severe intraepithelial neoplasia (SIN III), in 38% with a moderate squamous intraepithelial neoplasia (SIN II), in 32% with a mild squamous intraepithelial neoplasia (SIN I), and in 23% with a hyperkeratosis without dysplasia. The sensitivity and specificity of LVS in predicting an invasive carcinoma based on the absence or reduction of mucosal waves was 0.96 and 0.90, respectively. Following surgical intervention, the recovery rate of the mucosal wave and amplitude was 12% in the invasive carcinoma group, 36% in the SIN III group and up to 80% for both these parameters in the SIN I, SIN II, and hyperkeratosis groups. Conclusion: LVS is a valid tool to identify early glottic carcinoma and its high risk premalignancy carcinoma in situ (CIS). Even when there is no definitive differentiation between SIN I and II, the invasive character of a CIS and an invasive glottic carcinoma can be identified. Especially strobosopic signs of abnormal amplitude and/or mucosal waves, particularly phoniatric halt, are an early indication for a CIS or an invasive carcinoma.

Introduction: Phonomicrolaryngeal surgery involves the surgical treatment of benign disorders of the vocal folds. Postoperative scar tissue in vocal fold mucosa is undesired because mucosal hypodynamia may lead to prolonged impairment in voice quality. This study aims to present and share the outcomes of a new endolaryngeal suture technique. Methods: This study consists of patients who underwent vocal fold surgery followed by endolaryngeal microscopic suturation with a technique that we call the “mini-microsuture technique” between January 2018-December 2019. Videolaryngoscopic images were examined to observe the tissue healing process (suture elimination time, mucosal scar status, and wave pattern) at the first and the fourth week postoperatively. Results: A retrospective analysis was made in 144 (57 males (39.5%) and 87 females (60.5%)) patients who underwent phonomicrosurgery with “Mini-microsuture technique.” Mean age was 40.61 ± 13.54 (10-78) years. There were multiple pathological lesions in 41.66% (n: 60) of our patients, and 58.33% (n: 84) of them had single-lesion. 63.88% (n: 92) of the patients had bilateral vocal fold lesions where 36.22% (n: 52) of the patients had pathology in one vocal fold. Conclusions: The “mini-microsuture technique” is an easy and functional procedure that can be performed by a single surgeon under microscopy, which minimizes tissue trauma, prevents mucosal hypodynamia, and provides a better anatomical structure postoperatively for a symmetrical vibration. The technique does not significantly prolong operation time when mastered and is a cost-effective method in which surgery which can be concluded using a single suture material.

Objectives: Bicarbonate is critical for acid-base tissue homeostasis. In this study we investigated the role of bicarbonate ion transport in vocal fold epithelial defense to acid challenges. Acidic insults to the larynx are common in gastric reflux, carcinogenesis and metastasis, and acute inflammation. Methods: Ion transport was measured in viable porcine vocal fold epithelium. First, 18 vocal folds were exposed to either the carbonic anhydrase antagonist acetazolamide or to vehicle. Second, 32 vocal folds were exposed to either a control buffer or a bicarbonate-free buffer on their luminal or basolateral surface or both. Third, 32 vocal folds were challenged with acid in the presence of bicarbonate-free or control buffer. Results: The vocal fold transepithelial resistance was greater than 300 Ω*cm2, suggesting robust barrier integrity. Ion transport did not change after exposure to acetazolamide (p > 0.05). Exposure to bicarbonate-free buffer did not compromise vocal fold ion transport (p > 0.05). Ion transport increased after acid challenge. This increase approached statistical significance and was the greatest for the control buffer and for the bicarbonate-free buffer applied to the basolateral surface. Conclusions: Bicarbonate secretion may contribute to vocal fold defense against acid challenge. Our data offer a potential novel role for bicarbonate as a therapeutic agent to reduce pH abnormalities in the larynx and prevent associated pathological changes.

Objectives: Previous studies have indicated that although normal wound healing requires low levels of reactive oxygen species (ROS), excessive amounts of ROS impair wound healing. In injured vocal folds, this excess may result in dysphonia due to scarring that is difficult to treat. However, the expression of ROS during vocal fold wound healing has yet to be investigated. In this study, we assessed the expression and localization of ROS in injured vocal folds by immunohistochemical analysis. Methods: Vocal folds of Sprague-Dawley rats were unilaterally injured by stripping the mucosa under transoral endoscopy. The larynges were harvested at specific time points after injury and were immunohistochemically examined for 4-hydroxy-2-nonenal (4-HNE), an ROS marker, and for the presence of inflammatory cells. Results: We found that 4-HNE–immunopositive cells were significantly increased in the lamina propria of the injured vocal folds as compared to the normal vocal folds on postinjury days 1 and 3. More than half of the 4-HNE–immunopositive cells were also immunopositive for a macrophage- and granulocyte-specific antibody. Conclusions: This study suggests that a large amount of ROS is produced during early-phase wound healing, until postinjury day 3, and that this period may be crucial for regulating ROS levels. The results also suggest that inflammatory cells may contribute to ROS generation.

Objectives: The primary objective of this study was to quantitatively analyze ex vivo porcine, fetal human, and adult human vocal folds by use of optical coherence tomography (OCT). A secondary objective was to quantitatively discriminate among 1-, 2-, and 3-layer lamina propria structures. Methods: We performed an analysis of the vocal folds of 10 adult pig, 3 adult human, and 2 fetal human vocal fold specimens using OCT and histologic techniques. We present a quantitative comparison of the OCT results and histologic findings. Results: We found that OCT allowed for the visualization of the subepithelial vocal fold architecture of all imaged tissue, and that it revealed distinct characteristic signal intensities for each type of specimen. Conclusions: Optical coherence tomography was developed for in vivo imaging of biological microstructures. This study demonstrates the ability of OCT to differentiate between the vocal fold architectures of 3 histologically distinct types of vocal folds. Future studies aim to develop a quantitative optical imaging algorithm that can be used to facilitate an in vivo longitudinal clinical investigation of the changes that occur in this layered structure over time and maturation.

Objectives: Little is known about the function of the true vocal folds (TVFs) during cough. The objective of this study was to determine the reliability of measuring TVF movements during cough and to obtain preliminary normative data for these measures. Methods: Sequential glottal angles associated with TVF adduction and abduction across the phases of cough were analyzed from laryngeal videoendoscopy records of 38 young healthy individuals. Results: The intraobserver and interobserver reliability of 3 experienced measurers was high (intraclass correlation of at least 0.97) for measuring sequential and maximum glottal angles. The TVF abduction velocity during expulsion was significantly higher than the precompression adduction velocity (p = 0.002), but there were no significant differences in maximum angle. No statistically significant differences were seen in maximum TVF angle and velocity when they were compared between the sexes and between the levels of cough strength. True vocal fold closure following expulsion occurred in 42% of soft coughs and in 57% of moderate to hard coughs. Conclusions: The TVF abduction angles during cough can be reliably measured from laryngeal videoendoscopy in young healthy individuals. The TVF movements are faster for expulsion abduction than for precompression adduction, but the extents of abduction are similar. To validly determine the cough phase duration, simultaneous measures of airflow are needed.

Objectives: We sought to better characterize pathologic changes that occur in the human vocal fold after radiotherapy for head and neck cancer. Methods: In a blinded, controlled study of archived tissue, we evaluated postirradiation salvage laryngectomy vocal fold tissue without evidence of malignant disease. Clinical and demographic patient data were collected. In a blinded fashion, irradiated tissue was compared to nonirradiated, benign control tissue. Histomorphometric analysis was used to assess muscle and collagen organization, superficial lamina propria (SLP) and vocal ligament thickness, vocalis muscle fiber area, collagen content, and hyaluronic acid content. Immunohistochemical analysis was used to assess the content of type I collagen, type IV collagen, vimentin, fibronectin, α–smooth muscle actin, matrix metalloproteinase 9, and laminin. Results: Twenty irradiated vocal folds were evaluated and compared to control specimens. Collagen and muscle disorganization was noted in the irradiated specimens. The SLP and vocal ligament thicknesses and the mean muscle fiber diameters did not differ significantly. The SLP fibronectin and the vocalis muscle and SLP collagen content were significantly increased in the irradiated vocal folds, and the SLP collagen content increased significantly with time between irradiation and resection. The laminin content of irradiated vocalis muscles was significantly decreased. Conclusions: Radiotherapy results in significant vocal fold tissue changes. Having more precisely defined these changes, we plan continued investigation seeking targeted preventive and therapeutic interventions for improved vocal quality following radiotherapy.

Objectives: A basic function of the larynx is to provide sphincteric protection of the lower airway, initiated by a brain stem–mediated glottal closure reflex. Glottal closing force is defined as the measured pressure generated between the vocal folds during glottal closure. One of the factors thought to affect the glottal closure reflex is a variation in core body temperature. Methods: Four adult male Yorkshire pigs were used in this study. The subjects were studied under control conditions (37°C), hyperthermic conditions (38°C to 41°C), and hypothermic conditions (36°C to 34°C). Results: We demonstrated that the glottal closing force increased significantly with an increase in core body temperature and also decreased significantly with decreased core body temperature. These results are supported by neurophysiological changes demonstrated by other studies in pups and adult dogs in response to altered core body temperatures. The mechanism for these responses is thought to reside centrally, rather than in the peripheral nervous system. Conclusions: We hope that a better understanding of these aspects of glottal closure will alter the care of many patients with postanesthesia hypothermia and many sedated inmates and will also further enhance preventive measures needed to decrease the incidence of sudden infant death syndrome in overheated or febrile infants.

Objectives: The purpose of this study was to determine whether the videostroboscopic finding of vibratory asymmetry in mobile vocal folds is a reliable predictor of vocal fold paresis. In addition, the ability of experienced reviewers to predict the distribution (left/right/bilateral) of the paresis was investigated. Methods: This is a retrospective chart review of all patients who presented to our clinic during a 3-year period with symptoms suggestive of glottal insufficiency (vocal fatigue or reduced vocal projection) accompanied by the videostroboscopic findings of bilateral normal vocal fold mobility and vibratory asymmetry. Twenty-three of these patients underwent diagnostic laryngeal electromyography of the thyroarytenoid and cricothyroid muscles to determine the presence of vocal fold paresis. Results: Nineteen of the 23 patients (82.6%) were found to have electrophysiological evidence of vocal fold paresis, either unilaterally or bilaterally, when videostroboscopic asymmetry was present in mobile vocal folds. However, the three expert reviewers' ability to predict the distribution (left/right/bilateral) of the paresis was poor (26.3%, 36.8%, and 36.8%, respectively). Conclusions: The videostroboscopic finding of vibratory asymmetry in mobile vocal folds is a reliable predictor of vocal fold paresis in most cases. However, the ability of expert reviewers to determine the distribution (left/right/bilateral) of the paresis using videostroboscopic findings is poor. This study highlights the value of laryngeal electromyography in arriving at a correct diagnosis in this clinical situation.

Objectives: Most cases of irresolvable hoarseness are due to deficiencies in the pliability and volume of the superficial lamina propria of the phonatory mucosa. By using a US Food and Drug Administration–approved polymer, polyethylene glycol (PEG), we created a novel hydrogel (PEG30) and investigated its effects on multiple vocal fold structural and functional parameters. Methods: We injected PEG30 unilaterally into 16 normal canine vocal folds with survival times of 1 to 4 months. High-speed videos of vocal fold vibration, induced by intratracheal airflow, and phonation threshold pressures were recorded at 4 time points per subject. Three-dimensional reconstruction analysis of 11.7 T magnetic resonance images and histologic analysis identified 3 cases wherein PEG30 injections were the most superficial, so as to maximally impact vibratory function. These cases were subjected to in-depth analyses. Results: High-speed video analysis of the 3 selected cases showed minimal to no reduction in the maximum vibratory amplitudes of vocal folds injected with PEG30 compared to the non-injected, contralateral vocal fold. All PEG30-injected vocal folds displayed mucosal wave activity with low average phonation threshold pressures. No significant inflammation was observed on microlaryngoscopic examination. Magnetic resonance imaging and histologic analyses revealed time-dependent resorption of the PEG30 hydrogel by phagocytosis with minimal tissue reaction or fibrosis. Conclusions: The PEG30 hydrogel is a promising biocompatible candidate biomaterial to restore form and function to deficient phonatory mucosa, while not mechanically impeding residual endogenous superficial lamina propria.

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

Objectives: The management of vocal fold scarring and sulcus vocalis is challenging. These disorders are thought to be fibroplastic anomalies in the cover portion of the vocal fold that cause deterioration of the vibratory properties of the vocal fold mucosa. Histologic studies have revealed disorganization of extracellular matrix that needs to be addressed in the treatment of scarred vocal folds. Replacement of scar tissues with an appropriate implant may lead to regeneration of the vocal fold mucosa and its tissue properties. This retrospective case study examined the feasibility of using an atelocollagen sheet as a regenerative implant. Methods: Six patients with a post-cordectomy scar or sulcus vocalis underwent implantation of an atelocollagen sheet into the lamina propria of the vocal folds. The procedure consisted of elevation of a microflap, dissection and removal of scar tissue, implantation of the material, and wound closure. Vocal function was evaluated before and after surgery by stroboscopic examination and by aerodynamic and acoustic analyses. Results: The postoperative changes of aerodynamic and acoustic parameters varied among patients; however, gradual improvement was seen in most cases over a year. Stroboscopic findings also revealed gradual improvement of vibratory properties in most cases. Conclusions: Implantation of an atelocollagen sheet may have restorative effects on vocal fold scarring and sulcus vocalis in terms of tissue properties and function of the mucosa.

Objectives We characterized various recurrent laryngeal nerve (RLN) injuries in dogs. Methods Sixteen dogs were classified as having complete injuries (transection) or incomplete injuries (ligation, half-section, and crush). The characteristics of nerve injuries were evaluated by endoscopic examination, laryngeal electromyography (LEMG), and histopathologic examination at 0 to 12 months after the injury. Results After the RLN injury, the average muscle fiber diameter and the average muscle bundle diameter of the affected muscles were decreased, and the average number of muscular cell nuclei per square inch increased. Fibrillation potentials were found 1 to 3 months after injury, and reinnervation potentials appeared 3 to 6 months after incomplete injury. For nerve transection and ligation, there was no reaction with LEMG instantly after injury involving vocal fold fixation. Vocal fold motion did not improve in members of the complete injury group, whereas all of the vocal folds in the members of the nerve ligation subgroup had limited activity in the later period. Various forms of vocal fold mobility were observed after injury in the half-section subgroup. Animals in the crush subgroup had normal EMG signals combined with abnormal LEMG signals with lower amplitudes after injury. Vocal fold fixation was not observed in this subgroup. Conclusions We found the causes of nerve injury, in order of decreasing severity, to be transection, ligation, half-section, and crush.

Glottal dysplasia is likely the most common laryngeal disease with a discernible lesion; however, investigations describing its initial anatomic geographic presentation are rare. To examine this, we identified 52 patients who did not have significant prior treatment or glottal cancer. Thirty-one patients had bilateral disease, so there were 83 vocal folds with precancerous dysplasia. The phonatory mucosa was the dominant disease site in all; the epicenter was on the superior surface in 65 of the 83 folds and on the medial surface in 18 of the 83 folds. The arytenoid mucosa was involved in 8 of the 83 folds. Nineteen of the 52 patients had direct anterior-commissure involvement, and none had interarytenoid mucosal disease. The investigation established the commonly held principle that glottal dysplasia occurs primarily on phonatory mucosa. Given the frequent occurrence and recurrence of glottal dysplasia, treatment goals should focus on disease control to prevent malignant degeneration while preserving the subepithelial superficial lamina propria, necessary for phonatory mucosal pliability, vocal fold vibration, and optimal vocal function.

Objectives: Although total excision remains the standard treatment for vocal fold retention cysts, postoperative deficits and damage to the vocal folds still occur. Marsupialization is a more conservative technique and can prevent these complications. Methods: In this prospective clinical series, 25 patients underwent the marsupialization procedure. Under a direct laryngomicroscope, the cystic wall margin was retracted medially with microforceps. An incision was made with microscissors encircling the equator of the cyst. The cyst contents drained from the cystic cavity when the capsule was sectioned. For 7 patients with concomitant marked vocal fold atrophy, strap muscle transposition laryngoplasty was simultaneously performed. Results: All patients had complete preoperative and postoperative voice parameter analyses. A subjective improvement in voice quality was reported by 23 of the 25 patients (92%). A small recurrent vocal fold cyst was detected in 1 patient. Small vocal fold deficits and sulcus vocalis were detected in 2 and 4 patients, respectively. Only 1 patient described a worse voice after operation. No other complications were noted. Conclusions: Marsupialization of vocal fold retention cysts is a simple, relatively safe, and effective surgical treatment. Voice improvement, a low incidence of recurrence, and minimal vocal fold deficits demonstrate the validity of this technique. Marked preoperative vocal fold atrophy or postoperative glottal gap can be managed with medialization laryngoplasty.

Objectives: The present study was conducted to determine the effects of irradiation on the human laryngeal glands. Methods: Light and transmission electron microscopic observations were made. Results: Granular endoplasmic reticula and Golgi apparatuses were sparse in the cytoplasm of serous and mucous cells in cases with a short duration after radiotherapy. The secretory granules in serous cells had decreased in number. The secretory granules were less electron-dense compared to those in non-irradiated specimens, but were electron-lucent. The mucigen droplets in mucous cells were not as numerous as those in non-irradiated specimens. The discharge of secretory granules and mucigen droplets had decreased. In cases with a long duration after radiotherapy, there were some granular endoplasmic reticula and Golgi apparatuses in the cytoplasm. However, the transmission electron microscopy findings of secretory granules and mucigen droplets were the same as those in the irradiated glands with a short duration after radiotherapy. Conclusions: Morphological changes in the irradiated laryngeal glands influenced not only the amount but also the quality of secretions. The above changes lessened the lubrication of the vocal folds, thus causing a voice disorder to some extent. Local immunity and mucociliary transport were also affected. The effects of irradiation on the laryngeal glands partially altered the laryngeal functions.

Objectives: We determined the expression of matrix metalloproteinase (MMP)–1, MMP-9, collagen types I and III, and fibronectin from rabbit vocal folds after injury. Methods: Thirty rabbits were involved in this study. Five animals were assigned to each time period. Noninjured vocal fold specimens were collected as a control. Gene expression was analyzed at 2, 4, 8, 24, 48, and 72 hours after injury by use of real-time polymerase chain reaction. Results: Compared to 2 hours after injury, MMP-1 expression was increased at 4, 8, 24, 48, and 72 hours. Compared to 4 hours, MMP-1 expression was increased at 8, 24, 48, and 72 hours. Compared to the control specimens, MMP-9 expression was increased at 4, 8, 24, 48, and 72 hours. Compared to 2 hours, MMP-9 expression was increased at 4, 8, 24, 48, and 72 hours. Compared to 2 and 4 hours, collagen type I expression was increased at 72 hours. Collagen type III expression was increased at 72 hours compared to 2, 4, and 8 hours. Compared to 2 hours, fibronectin expression was increased at 24, 48, and 72 hours. Conclusions: Results revealed time-dependent changes in expression of MMP-1, MMP-9, collagen types I and III, and fibronectin from rabbit vocal folds after injury. Future experiments are planned to investigate the effects of phonation on expression of these genes after injury.

Objectives: This study proposes the use of a new parameter of vocal aerodynamics, phonation threshold flow (PTF). The sensitivities of the PTF and the phonation threshold pressure (PTP) were quantitatively compared to the percent of vocal fold elongation from physiologic length. Methods: Ten excised canine larynges were mounted on a bench apparatus capable of controlling vocal fold elongation. Subglottal airflow was gradually increased until the onset of phonation. Elongation of the vocal folds was varied from +0% (physiologic length) to +15%, and the PTF and PTP were measured. Results: The mean PTFs at physiologic vocal fold length ranged from 101 to 217 mL/s. No statistically significant relationship was found to exist between the size of the larynx and the measured PTF values (p = .404). The average percent change of PTF compared to the magnitude of elongation was found to be statistically significant (p < .001). The data indicated that the PTF was proportional to the percent of vocal fold elongation. Conclusions: The PTF was positively correlated with vocal fold elongation and the PTP for small magnitudes of elongation. The results suggest that the PTF may be indicative of the biomechanical properties of the vocal folds, thus providing a possibly valuable tool in the clinical evaluation of laryngeal function.

Objectives: The development of the scanning system AcuBlade has considerably enhanced carbon dioxide laser energy delivery, improving cutting and ablation modes. The scanning system can be applied with the 2 available high-powered pulsed waves, SuperPulse and UltraPulse. This study was conducted to determine whether there are any differences in phonosurgery between the SuperPulse and UltraPulse lasing applications with regard to thermal diffusion into the surrounding tissues, healing time, and clinical results. Methods: Thirteen patients with bilateral and similar vocal fold lesions underwent operation — One side in SuperPulse mode and the other side in UltraPulse mode. The parameters for phonosurgery were depth of 0.2 mm, 10 W, single pulse, and 0.10 second for SuperPulse, and 2 passes, 10 W, single pulse, and 0.10 second for UltraPulse. Results: Incisions were sharper with UltraPulse, making the surgery easier, but at the first postoperative follow-up visit, after 8 to 10 days, no differences were observed in the presentation, the healing, or the vibration of the 2 vocal folds. Coagulation along the incision line was 25 μm for SuperPulse and 15 μm for UltraPulse (median values). Conclusions: In comparison with SuperPulse, the UltraPulse carbon dioxide laser made the procedure easier, but did not improve the clinical outcome.

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

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

Objectives: Evolving photoangiolytic laser techniques for treating vocal fold lesions motivated the development of a model for research and surgical training. The chick chorioallantoic membrane (CAM), which is composed of a microvasculature suspended within the egg albumen, simulates the vocal fold microcirculation within the superficial lamina propria (SLP). To characterize this model, we compared measurements of vessel diameters to superficial vessels in human vocal folds. Methods: The diameters of first-, second-, and third-order CAM vessels were measured in fertilized chicken eggs. The superficial blood vessels of the human vocal fold were measured from intraoperative images. Results: According to the branching pattern, vessel segments were identified as first-, second-, or third-order, with average diameters of 0.035 mm (0.02 to 0.1 mm), 0.18 mm (0.12 to 0.41 mm), and 0.8 mm (0.6 to 0.98 mm), respectively. The total vessels measured included 362 first-order, 119 second-order, and 82 third-order vessels. In 10 adult human vocal folds, an average vessel diameter of 0.04 mm (0.015 to 0.1 mm) was observed in 50 vessels. Conclusions: The CAM microvasculature suspended in albumen provides a useful surgical model simulating the microcirculation within the SLP of the human vocal fold. Although first-order CAM vessels best approximate the size of normal vocal fold subepithelial vessels seen at surgery, second- and third-order vessels resemble the vascular abnormalities frequently encountered during microsurgery for phonotraumatic lesions.

Objectives: The 2-μm-wavelength thulium laser has recently been shown to be an effective cutting instrument in endolaryngeal surgery, although there is increased thermal trauma as compared with the carbon dioxide laser. This study investigated temperature changes and thermal trauma during thulium laser dissection of laryngeal tissue, with and without air cooling, in an ex vivo model. Methods: A continuous-wave thulium laser (400-μm fiber, 4-W continuous power, 4-second duration) was used to incise 10 calf vocal folds. Paired cooled and uncooled cuts were made in each fold with a dermatologic cooling device. A thermistor inserted into the glottic subepithelium was used to measure tissue temperatures. Thermal damage was analyzed histologically by measuring the depth of the zone of lactate dehydrogenase inactivation surrounding the mucosal incision. Results: The initial vocal fold temperature averaged 24.3°C without cooling and 4.4°C with cooling. The peak temperature during cutting averaged 59.1°C without cooling and 28.0°C with cooling. The thermal damage zone surrounding the cooled incisions was approximately 27% less than that surrounding the uncooled incisions. Conclusions: Air cooling can reduce the extent of thermal trauma associated with thulium laser surgery of the vocal folds, and the high-temperature plume generated during laser cutting is effectively cleared.

Objectives: Laryngostroboscopy is used to check the oscillatory patterns of the vocal folds. However, the use of one single flash timing cannot give a clear view of abnormal vocal fold oscillations that have multiple fundamental frequencies. Visualization of such complex vocal fold movements will be helpful in the diagnosis of diplophonia, a pathological condition in which the vocal folds produce multiple tones at the same time. Methods: We developed a new stroboscopy-based technique using multiple light-emitting diodes (LEDs) and image analysis. Specific flash timings for each LED, suitable for accurate visualization, were determined on a computer according to an algorithm based on frequency analysis. The image analysis extracted the necessary parts of the captured images to yield a clear slow-motion view of the oscillations. The series of visualization procedures took advantage of the narrow-spectrum light property of LEDs, thereby yielding a degradation-free picture. Results: Feasibility tests using a mechanical vocal fold model demonstrated that this computer-assisted system allows observation of the pathological oscillatory patterns as one single video. They would not be clearly visualized by conventional stroboscopy. Conclusions: Because of its relatively simple use and inexpensive construction, the proposed technique can become one potential option for clinical assessment of pathological vocal fold oscillations.

Objectives: Changes in voice are commonly associated with aging (presbyphonia). Age-related voice change significantly impairs elderly individuals' ability to communicate meaningfully with others and affects their quality of life. With changing age demographics in our society and increasing emphasis on quality of life, treatment of presbylaryngis is becoming more paramount. Methods: We used 9 aged and 9 young mice to validate a mouse model for the aging larynx. We stained the larynges with Alcian blue to determine the hyaluronic acid content, trichrome stain to determine the collagen content, and immunohis-tochemical stain for α smooth muscle actin to determine the myofibroblast content. Morphometric measurements were performed for muscle area, muscle thickness, and muscle fiber diameter. Results: Statistically significant differences in the density measurements of hyaluronic acid and collagen reflected decreased hyaluronic acid and increased collagen content in the aging larynx. We found α smooth muscle actin–labeled myofibroblasts only in the aged larynges. No statistically significant differences were found in the morphometric measurements. Conclusions: Aged mice may make a practical model for the age-related changes in the vocal folds that can be used further in studies aiming to correct these changes.

Objectives: The aim of this study was to analyze glottal velocity and glottal opening and closure. For this purpose, we developed a miniature, flexible, hot-wire probe that can make truly instantaneous measurements of the human larynx in vivo. Methods: A miniature hot-wire tip was inserted into a flexible transnasal endoscope. Fiberscopic examination was performed transnasally so that we could observe glottal vibration using high-speed imaging. The tip of the hot-wire probe was placed just above the glottis. The position of the probe was carefully monitored and checked with another flexible endoscope. Results: Changes in velocity were recorded periodically. The velocity was higher in close proximity to the vocal folds. High-speed motion pictures were taken at a rate of 2,000 frames per second with an auxiliary light source. Conclusions: Quantitative analysis of glottal velocity is required to improve our understanding of the relationship between laryngeal physiology and acoustics in humans. To solve the problem of synchronization inaccuracy, glottal velocity was captured instantaneously in the high-speed imaging system's processor memory.

Objectives: Acquired anterior glottic webs occur most commonly after endoscopic resection of laryngeal papilloma involving the anterior vocal folds. Treatment of anterior glottic webs has included a tracheotomy with laryngofissure and placement of a laryngeal stent or keel. We have used an endoscopic technique of web lysis and placement of a laryngeal keel without tracheotomy over the past 7 years. Methods: A retrospective chart review was conducted of all cases of endoscopic web lysis and keel placement performed by the senior author (S.A.B.). Results: Over the past 7 years, 10 patients underwent the procedure, with a mean follow-up of 18 months. The length of the anterior web was up to two thirds of the membranous vocal fold. Outcomes analysis revealed a recurrence in 1 patient and 2 minor complications necessitating treatment. Conclusions: Endoscopic web lysis and keel placement offers superior results with less morbidity compared to open techniques.

Objectives: In humans, a decrease in transglottal pressure ( Pt) causes an increase in the fundamental frequency of phonation ( F0) only at a specific voice pitch within the modal register, the mechanism of which remains unclear. Methods: In the present study, numerical analyses were performed to investigate the mechanism of the voice pitch-dependent positive change of F0 due to Pt decrease. The airflow and the airway, including the vocal folds, were modeled in terms of mechanics of fluid and structure. Results: Simulations of phonation using the numerical model indicated that Pt affects both the average position and the average amplitude magnitude of vocal fold self-excited oscillation in a non-monotonous manner. This effect results in voice pitch-dependent responses of F0 to Ft decreases, including the positive response of F0 as actually observed in humans. Conclusions: The findings of the present study highlight the importance of considering self-excited oscillation of the vocal folds in elucidation of the phonation mechanism.

Objectives: The purpose of this study was to examine the aerodynamic and acoustic effects of the false vocal folds and the epiglottis on excised larynx phonation. Methods: Several canine larynges were prepared and mounted over a tapered tube that supplied pressurized, heated, and humidified air. Glottal adduction was accomplished either by using two-pronged probes to press the arytenoids together or by passing a suture to simulate lateral cricoarytenoid muscle activation. First, the excised larynx with false vocal folds and epiglottis intact was subjected to a series of pressure-flow experiments with longitudinal tension and adduction as major control parameters. Then, the epiglottis and finally the false vocal folds were removed and the experiment was repeated. The subglottal pressure and the electroglottographic, flow rate, audio, and sound pressure signals were recorded during each experiment. Glottal flow resistance was calculated from the pressure and flow signals. The electroglottographic signal was used to extract the fundamental frequency. Results: It was found that the false vocal folds and the epiglottis offer a positive contribution to the glottal resistance and sound intensity of the larynx. Also, vocal fold elongation and glottal medial compression caused an increase in glottal resistance. The pressure-flow relationships were approximately linear regardless of the structure. Conclusions: The addition of the supraglottic laryngeal structures has a significant impact on both aerodynamic and acoustic characteristics of excised larynges.

Objectives: Vocal fold scarring is the major cause of voice disorders after voice surgery or laryngeal trauma. The role of inflammatory factors in vocal fold wound healing and fibrosis has not been adequately investigated. Scarless wound healing has been associated with decreased inflammatory responses. To understand scar formation and develop reliable treatments, it is necessary to control extracellular matrix production and inflammation. Thus, we examined the inflammation profile and extracellular matrix production in wounded vocal folds in the acute phase of wound healing. Methods: Vocal fold stripping was performed on 30 Sprague-Dawley rats. Vocal fold tissue was collected at 5 time points (4, 8, 16, 24, and 72 hours). We examined the in vivo messenger RNA expression profile of inflammatory factors interleukin 1β, interferon γ, tumor necrosis factor a, nuclear factor Kβ, transforming growth factor β, and cyclooxygenase 2, as well as hyaluronic acid synthases 1 and 2, procollagen subtypes I and III, and elastin synthase in scarred vocal folds after injury, compared to normal vocal folds, using real-time reverse transcription-polymerase chain reaction. Results: The inflammatory factors showed a time-dependent sequence of expression peaks, starting with interleukin 1β, nuclear factor Kβ, tumor necrosis factor a (4 and 8 hours), and transforming growth factor β (72 hours). Interferon y decreased at 24 hours. Correspondingly, hyaluronic acid synthase 1 expression peaked first (4 and 8 hours), whereas hyaluronic acid synthase 2 expression peaked at 16 hours and again at 72 hours. Procollagen I expression peaked at 72 hours, whereas procollagen III decreased from 8 to 16 hours but peaked at 72 hours. Cyclooxygenase 2 expression was elevated, whereas elastin expression remained constant. Conclusions: The results show a clear profile of vocal fold inflammation with corresponding changes in extracellular matrix production.

Objectives: We sought to further describe the development of the 3-layered human vocal fold in children and to quantify macrophage and myofibroblast concentrations in each layer. Methods: We used an optical analysis software package to examine 8 longitudinally sectioned human vocal folds that had been fixed in formalin (ages 2 days to 14 years). Results: The 2-day-old vocal fold contained only a monolayer of cells. This became a bilayer by 5 months, and a trilayer began to become evident by 7 years. The percent of total depth represented by the superficial layer of the lamina propria (SLP) gradually decreased with age. The SLP made up 22% of the total lamina propria by age 7 years; this percentage approximates that in the adult vocal fold. Macrophages and myofibroblasts were predominately found in the SLP, and began to be apparent by 11 months of age. Conclusions: These results help describe the development of human voice and may have implications as to when phonosurgical therapy can be considered for children.

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

This study evaluates how extraesophageal reflux affects membranous vocal fold healing in a canine model. We created membranous vocal fold injuries in the animals and randomly assigned them to topical application of acid and pepsin at pH 2 or pH 6 or of normal saline solution every other day for 12 days. The experimental vocal folds were compared to uninjured, control vocal folds from animals painlessly sacrificed for other reasons. Hematoxylin and eosin, fibronectin, and procollagen I staining were performed for histologic analysis. The injured specimens had three times greater cellular infiltrate (p ≤ .001, analysis of variance) and twice as much fibronectin and procollagen I (p ≤ .001, analysis of variance) as did the specimens from the control animals. No significant differences or trends were identified for cellular infiltrate, fibronectin, or procollagen I within the injured groups (p > .05, Bonferroni t-test). Acute wound healing did not appear to be influenced by the presence of acid and pepsin at pH 2 or 6 as compared to saline solution.

To examine the functional effects of hyaluronan and collagen alterations in acute vocal fold scar, we injured 15 pig larynges by vocal fold mucosa stripping. At 3, 10, and 15 days after operation, we performed excised larynx experiments to measure phonation threshold pressure (PTP) and vocal economy (an acoustic output-cost ratio; OCR), and then performed hyaluronan and collagen assays. Five uninjured larynges were used as excised controls. Hyaluronan was reduced in the scarred vocal folds through 15 days of wound healing. Collagen was increased at day 15. The PTP was increased and OCR was decreased in scarred larynges, indicating decreased vocal efficiency and ease of phonation. Thus, PTP and OCR were sensitive to the biomolecular changes in acute vocal fold scar. Hyaluronan was more susceptible than collagen to acute tissue ultrastructural alterations. These findings may provide a rationale for increasing hyaluronan in acute vocal fold scar to improve postoperative vocal outcomes.

Vocal fold scarring remains a therapeutic challenge. Hepatocyte growth factor (HGF) has strong antifibrotic activity and has proved to have therapeutic potential in restoration of scar tissues such as liver cirrhosis and lung fibrosis. The present study aimed to clarify the effects of HGF injection into scarred vocal folds in a canine model. Canine vocal folds were stripped unilaterally and treated with intracordal injection of saline solution (sham group), HGF (HGF group), or HGF with cultured autologous normal vocal fold fibroblasts (Fb/HGF group) 1 month after injury. The larynges were harvested 6 months after the initial injury and then subjected to vibratory and histologic examination. The results of vibratory examinations in the excised larynx setup revealed that phonation threshold pressure significantly increased and vocal efficiency was significantly reduced in all treated groups as compared to normal data obtained from normal canine larynges. However, the HGF group presented much better results than both the sham and Fb/HGF groups in terms of mucosal wave amplitude and incidence of vocal fold bowing, glottal incompetence, and phase asymmetry. The histologic data indicated a significant increase of collagen in both the sham and Fb/HGF groups, while normal levels of collagen were found in the HGF group. Tissue contraction of the lamina propria was also observed in both the sham and Fb/HGF groups, but was barely detectable in the HGF group. Although the HGF-treated vocal folds appeared to require more driving forces for vibration, HGF might prevent excessive collagen deposition and tissue contraction and thus reduce the effects of scarring on the vibratory properties of the vocal folds. From these data it is concluded that HGF has considerable potential in the treatment of vocal fold scarring.

This study seeks to determine the ability of enzyme-linked immunosorbent assays of vocal fold secretions to detect and describe the inflammatory response in the vocal folds. Vocal fold and palatal secretions were collected during operation from patients with a range of vocal fold disorders and from control patients. The secretions were subjected to assays for interleukin-1β, prostaglandin E2, and transforming growth factor β. The results indicate a differential expression of mediators associated with the wound healing cascade in the vocal folds. The prostaglandin E2 levels clearly differentiated vocal fold secretions associated with laryngeal disease versus control sites. Furthermore, the interleukin-1β concentrations were significantly elevated in subjects with epithelial lesions of the vocal folds as opposed to lesions of the lamina propria. Although still in its infancy, such analysis may ultimately hold scientific and clinical utility in the study and management of patients with vocal fold disease.

The aim of this study was to regenerate the injured vocal fold by means of selective cultured autologous mesenchymal stem cells (MSCs). Eight adult beagle dogs were used for this experiment. Selective incubation of MSCs from bone marrow was done. These MSCs were submitted to 3-dimensional incubation in 1% hydrochloric acid atelocollagen. Three-dimensional incubated MSCs were injected into the left vocal fold, and atelocollagen only was injected into the right vocal fold of the same dog as a control. Four days after injection, the posterior parts of the vocal folds were incised. The regeneration of the vocal fold was estimated by morphological and histologic evaluations. Our results showed that 3-dimensional incubated MSCs were useful in the regeneration of the injured vocal fold. This study shows that damaged tissues such as an injured vocal fold would be able to be regenerated by tissue engineering.

The voice of aged persons is usually different from that of younger adults — a fact indicating that aging induces changes in the human larynx. The aim of this study was to perform a histomorphometric analysis of the vocal fold and of a fragment of skin in order to determine the modifications related to aging and possible differences between genders. Twenty cadavers, 10 men and 10 women, were studied. Stereological methods were used for histologic analysis. In the vocal folds, we observed a reduction of the lamina propria thickness (p < .001) and of epithelial cell density (p < .001) as a function of age. In the skin, there was a reduction of chorion thickness (p = .02) and of epithelial cell density (p = .01). The decrease in the thickness of the lamina propria of the vocal folds with aging may contribute to the vocal pattern present in the elderly. These changes were more evident in men.

The current treatment options for dysphonia secondary to vocal fold scarring are limited. Few studies address changes in the lamina propria, which is critical to vocal fold biomechanical properties and voice production. Using rheological and histological measures of homologous collagen matrix (HCM)–injected vocal folds, we assessed HCM's potential for providing bulk and restoring biomechanical performance. Twenty rabbits underwent bilateral vocal fold scarring. After 10 weeks of scar maturation, the rabbits had unilateral injections of HCM or saline solution. Ten weeks after the injections, histological studies revealed well-defined collagen globules distributed throughout the lamina propria and underlying muscular tissue. Significantly more procollagen was observed in the HCM-treated group. No significant differences in elastic shear modulus or dynamic viscosity were found between the treatment groups. This study demonstrates that HCM is an inert, relatively stable injectate that may serve well for medialization but does not appear to improve the dynamic properties of the lamina propria.

Vocal fold fibrotic scar is characterized by fibrosis of the lamina propria and epithelium, and is difficult to treat. Hepatocyte growth factor (HGF) has antifibrotic activity and has received attention as a possible therapeutic alternative to treat fibrosis. In this study, in order to clarify whether HGF can be involved in vocal fold scarring, we examined the existence of HGF and its receptor, c-Met, in rat vocal folds, and then the activity of HGF in rabbit injured vocal folds, using immunohistochemistry and enzyme-linked immunosorbent assay. We found HGF and c-Met on epithelial cells and gland cells of the rat vocal folds. On the injured vocal folds of rabbits, little HGF was observed immediately after injury, but prominent activity occurred simultaneously with reepithelialization of the vocal fold mucosa on days 10 to 15. The activity of HGF was observed on fibroblasts in the lamina propria, as well as the epithelium. It is suggested that HGF in the vocal folds is produced by the fibroblasts and delivered to the epithelium. The implication of these findings is that HGF is involved in wound healing of the vocal fold, and may provide an alternative approach in preventing and treating vocal fold scarring.

We investigated wound healing of vocal fold mucosal excisions using a canine model, comparing cold steel, the continuous wave (CW) carbon dioxide (CO2) laser, and a new, microsecond-pulsed CO2 laser. The thermal injury to the surrounding normal tissues produced by the laser increases the risk for scar formation and poor functional voice outcome. This injury may be reduced with a pulsed CO2 laser. Acute, 2-week, and 6-week studies of 15 dogs were made with blinded observers and nonparametric statistical analyses. Histologic comparison showed less thermal injury and scar formation in the vocal folds treated with the pulsed CO2 laser than with the CW CO2 laser. Functional outcomes as studied with laryngeal videostroboscopy revealed better vibratory characteristics with the pulsed CO2 laser as compared with the CW CO2 laser. No differences were seen on these measures between the pulsed laser and cold techniques. Pulsed CO2 lasers may offer the ease of laser laryngology and the superior wound healing of cold steel.

The mucus layer on the vocal folds was examined by videostrobolaryngoscopy in patients with laryngeal tension-fatigue syndrome, a chronic functional dysphonia due to vocal abuse and misuse. Besides the findings in previous reports (such as abnormal glottal closure, phase or amplitude asymmetry, and the irregular mucosal wave), the vocal folds during vibration had an uneven mucus surface. The occurrence of an uneven mucus layer on vocal folds was significantly greater in subjects with this voice disorder (83% or 250 of 301 patients in this series) than in those without voice disorders (18.5% or 5 of 27). The increase of mucus viscosity, mucus aggregation, and the formation of rough surfaces on the vocal folds alter the mechanical properties that contribute to vibration of the cover of the vocal folds, and thereby worsen the symptoms of dysphonia in patients with laryngeal tension-fatigue syndrome.

Classically, the formation of a mucosal wave is dependent on the pliable mucosa present in the vocal fold. The supracricoid partial laryngectomy with cricohyoidoepiglottopexy is an organ preservation surgical technique in which both true vocal folds, both false vocal folds, both paraglottic spaces, and the entire thyroid cartilage are resected. The functional goal is speech and swallowing without a permanent tracheostomy. In an effort to further study voice production in these patients, we performed laryngeal stroboscopy in 5 patients. Analysis was performed with a modified Bless grading system. In addition, speech and voice parameters were also measured and correlated with stroboscopic findings. The key finding in this study was the presence of a periodic mucosal wave on the anterior aspect of the arytenoid cartilage, where it abuts the epiglottic cartilage. Patients with lower periodicity and symmetry scores tended to have lower jitter and shimmer percentages.

Some new anatomic data on the laryngeal cartilage framework have been obtained for the biomechanical modeling of the larynx. This study attempted to define and measure some biomechanically important morphometric features of the laryngeal framework, including both the human and the canine laryngeal frameworks, because the canine larynx has been frequently used as an animal model in gross morphology and in physiological experiments. The larynges of 9 men, 7 women, and 9 dogs were harvested and dissected after death. Linear and angular geometric measurements on the thyroid cartilage, the cricoid cartilage, and the arytenoid cartilage were made with a digital caliper and a protractor, respectively. The results are useful for constructing quantitative biomechanical models of vocal fold vibration and posturing (abduction and adduction), eg, continuum mechanical models and finite-element models of the vocal folds.

A prospective, blinded study was designed to determine whether placement of a microsuture in epithelial defects created on canine vocal folds results in histologically demonstrable improved healing. Epithelial defects similar to those created during microflap removal were made by means of traditional microsurgical techniques on both vocal folds of 4 adult dogs. One vocal fold defect on each dog was then closed with a single microsuture placed through the laryngoscope. The larynges were harvested approximately 6 weeks later, and blinded histologic quantification of scar formation was performed. Microsutures resulted in less scarring in all but 1 of the larynges studied. Unsutured vocal folds exhibited a 75% larger average scar cross-sectional area. Although the sample size was insufficient to establish statistical significance, the observed difference in scar formation between microsutured and unsutured vocal folds suggests that primary closure with a microsuture in the canine model results in less scarring than when healing occurs by secondary intention.

The relationship between fundamental frequency of phonation (Fo) and the change in Fo per unit change of transglottal pressure (dF/dP) was investigated in 3 excised canine larynges. The effects of the length and depth of vocal fold vibration on the relationship between dF/dP and Fo were evaluated. A positive correlation between dF/dP and Fo was observed when an increase in Fo was accompanied by a decrease in the depth of vibration while the length of vibration reached a plateau. Although a negative correlation between dF/dP and Fo was observed in only 1 case, this relationship seemed relevant to the length of vibration. These results help to clarify the adjustments of length and depth of vibration involved in regulating Fo.