Investigation of Anti-Hyaluronidase Treatment on Vocal Fold Wound Healing

SUMMARY: Phytochemical constituents of medicinal plants demonstrate inhibition of tissue and bacterial hyaluronidase. Echinacoside is a caffeoyl conjugate of Echinacea with known anti-hyaluronidase properties. The purpose of this study was to investigate the wound healing effects of Echinacea on vocal fold wound healing and functional voice outcomes. Pig animal model. Methods: Vocal fold injury was induced in 18 pigs by unilateral vocal fold stripping. The uninjured vocal fold served as control. Three groups of six pigs randomly received a topical application of 300, 600, or 1,200 mg of standardized Echinacea on the injured side. Animals were euthanized after 3, 10, and 15 days of wound healing. Phonation threshold pressure and vocal economy measurements were obtained from excised larynges. Treatment outcomes were examined by comparing the animals receiving treatment with a set of 19 untreated and 5 historical controls. Treatment effects on wound healing were evaluated by histologic staining for hyaluronan and collagen. Treated larynges revealed improved vocal economy and phonation threshold pressure compared with untreated larynges. Histologically, treated vocal folds revealed stable hyaluronan content and no significant accumulation of collagen compared with control. Findings provide a favorable outcome of anti-hyaluronidase treatment on acute vocal fold wound healing and functional measures of voice.     

Elastic models of vocal fold tissues

Elastic properties of canine vocal fold tissue (muscle and mucosa) were obtained through a series of experiments conducted in vitro and were modeled mathematically. The elastic properties play a significant role in quantitative analysis of vocal fold vibrations and theory of pitch control. Samples of vocalis muscle and mucosa were dissected and prepared from dog larynges a few minutes premortem and kept in a Krebs solution at a temperature of 37 +/- 1 degrees C and a pH of 7.4 +/- 0.05. Samples of muscle tissue and mucosa were stretched and released in a slow, sinusoidal fashion. Force and displacement of the samples were measured with a dual-servo system (ergometer). After digitization, stress-strain data for samples of muscle tissue and cover tissue were averaged. The stress-strain data were then fitted numerically by polynomial and exponential models.     

Contact Telescopy Reveals Blood Vessel Alterations of Vocal Fold Mucosa in Reinke''s Edema

AIM: To evaluate contact telescopy findings for estimation of blood vessel changes in vocal fold mucosa in patients with Reinke's edema. Histological features significant for diagnosis of microvascular vocal fold alteration were correlated with clinical findings. METHODS: In 80 patients with Reinke's edema, laryngoscopy and video-telescopy image analysis of vocal folds were performed. Vocal fold mucosa biopsies were histologically analyzed and compared with contact telescopy findings. An interesting aspect of vocal fold microcirculation found both by contact telescopy imiging and by histological specimens was described. RESULTS: Contact telescopy in vivo revealed different forms of pathological blood vessel networks with unusual appearance of loops or branching. Some dilated varicose vascular channels had very thin walls, and within atypical capillaries, partial erythrocyte accumulation was found. Details of blood flow are also visible, showing multidirectional and discontinuous blood flow in neighboring vessels. CONCLUSION: The noninvasive contact telescope technique is very useful as an additional diagnostic tool for defining a condition of a subepithelial Reinke's space in a very short period of time. The great advantage of contact telescopy is systematic in vivo and in situ observation of microvascular details in the vocal folds. The contact technique allows dynamic follow-up of the microcirculation in Reinke's edema as well as simultaneous consultation of a pathologist in the operating theater.     

Laryngeal injuries after short-versus long-term intubation

Forty-five patients were seen over a 5-year period with laryngeal injuries following endotracheal intubation (ETI). The mean duration of ETI was 5.6 days (2 hours to 37 days). Patients intubated for less than 24 hours were most likely to present with a vocal fold immobility or an anterior glottic web. Long-term intubation was associated with the development of subglottic stenoses and granulomas. Patients with vocal fold immobility were seen more often after ETI for surgical reasons and had a significantly higher incidence of previous intubation and tobacco usage. Subglottic stenoses were seen in younger patients intubated for medical reasons and associated with nasogastric tubes and longer periods of intubation.     

Characterization of chronic vocal fold scarring in a rabbit model

The purpose of the current study was to assess the histologic and rheologic properties of the scarred vocal fold lamina propria during a chronic phase of wound repair in a rabbit model. Eighteen rabbit larynges were scarred using a procedure that involved stripping the vocal fold lamina propria down to the thyroarytenoid muscle, using 3-mm microforceps. The approximate dimension of injury to the vocal fold was 3 x 1.5 x 0.5 mm [length x width x depth]. At 6 months postoperatively, histologic analysis of the scarred and control lamina propria in eight of these rabbits was completed for collagen, procollagen, elastin, and hyaluronic acid. Compared with control samples, scarred tissue samples revealed fragmented and disorganized elastin fibers. Additionally, collagen was significantly increased, organized, and formed thick bundles in the scarred vocal fold lamina propria. Measurements of the viscoelastic shear properties of the scarred and control lamina propria in the remaining 10 rabbits revealed increased elastic shear modulus (G') in 8 of 10 scarred samples and increased dynamic viscosity (eta') in 9 of 10 scarred samples. Although rheologic differences were not statistically significant, they revealed that on average, scarred samples were stiffer and more viscous than the normal controls. Histologic data are interpreted as indicating that by 6 months postinjury, the scarred rabbit vocal fold has reached a mature phase of wound repair, characterized by an increased, organized, and thick bundle collagen matrix. Rheologic data are interpreted as providing support for the potential role of increased, thick bundle collagen, and a disorganized elastin network on shear stiffness and dynamic viscosity in the chronic vocal fold scar. Based on these results, a 6-month postoperative time frame is proposed for future studies of chronic vocal fold scarring using the rabbit animal model.     

Measurements of vocal fold tissue viscoelasticity: approaching the male phonatory frequency range

Viscoelastic shear properties of human vocal fold tissues have been reported previously. However, data have only been obtained at very low frequencies (< or = 15 Hz). This necessitates data extrapolation to the frequency range of phonation based on constitutive modeling and time-temperature superposition. This study attempted to obtain empirical measurements at higher frequencies with the use of a controlled strain torsional rheometer, with a design of directly controlling input strain that introduced significantly smaller system inertial errors compared to controlled stress rheometry. Linear viscoelastic shear properties of the vocal fold mucosa (cover) from 17 canine larynges were quantified at frequencies of up to 50 Hz. Consistent with previous data, results showed that the elastic shear modulus (G'), viscous shear modulus (G"), and damping ratio (zeta) of the vocal fold mucosa were relatively constant across 0.016-50 Hz, whereas the dynamic viscosity (eta') decreased monotonically with frequency. Constitutive characterization of the empirical data by a quasilinear viscoelastic model and a statistical network model demonstrated trends of viscoelastic behavior at higher frequencies generally following those observed at lower frequencies. These findings supported the use of controlled strain rheometry for future investigations of the viscoelasticity of vocal fold tissues and phonosurgical biomaterials at phonatory frequencies.     

Collagen injection in the scarred vocal fold

Scar tissue in the vocal fold can impair vibration and voice quality. The association of scarring and glottic insufficiency prompted our use of injectable bovine collagen in a variety of pathologic conditions exhibiting vocal fold scarring. Incremental augmentation and improved glottic function in several treated patients seemed to be facilitated by softening of scar tissue. Although the use of bioimplants in the larynx is associated with immunologic risks, the proclivity to soften scar tissue is one potential benefit of such materials. The biological activity of injectable collagen seems to alter local collagen metabolism by promoting ingrowth of host fibroblasts that are active in collagen degradation and synthesis. Further research to study the mechanisms whereby injectable collagen promotes remodeling of connective tissue might have significant therapeutic implications in the management of laryngeal scarring.     

Laser Doppler Measurements of the Vocal Fold Blood Micro-Circulation

The authors have developed a laser Doppler method, referred to as LDF+LS (laser Doppler flowmetry by laryngostereometry), for measuring the vocal fold micro-circulation. The vocal folds of 103 patients were examined during general anesthesia. A laser Doppler probe was placed on defined positions at the vocal fold edge: midmembranous position (MP), 2 mm and 4 mm behind, and 1 mm anterior to MP. Three parameters were derived, ie, the concentration (CMBC) and velocity (V) of moving blood cells, and the product of both resulting in perfusion: P = CMBCxV. The results of 53 subjects with normal vocal fold status showed that the V at MP was significantly lower than 2 mm behind MP (P < 0.05). Men had significantly higher velocity than women (P < 0.05). Subjects older than 65 years had higher perfusion and CMBC as compared with younger subjects (P < 0.01). Smokers with normal vocal folds had higher velocity than non-smokers (P < 0.05). Measurements of healthy vocal folds were compared with benign vocal fold pathology. Vocal fold polyps had significantly higher perfusion than normal vocal folds (P < 0.01). In conclusion, the laser Doppler measurement is a sensitive tool estimating superficial blood flow in both normal and pathological vocal folds. The blood flow in normals differs with respect to gender and age probably due to variations in micro-circulation of the mucosa and lamina propria. Vocal fold pathology and external factors such as smoking result in blood flow changes.     

Traumatic vocal fold avulsion injury in a newborn

A full-term newborn developed respiratory compromise in the immediate postparturition period requiring urgent intubation. Evaluation of post-extubation stridor later the same day revealed an avulsion injury extending from the left vocal fold into the lateral glottic musculature. Primary repair was accomplished with anatomic realignment of the torn vocal fold and muscle. Endotracheal intubation was utilized for stenting and the patient was extubated following 3 days of paralysis with sedation. Follow-up examination revealed a reparative granuloma, which was lasered. Eight-week follow-up examination revealed normal vocal fold architecture. At 18 months the patient continues to have a normal voice and normal laryngeal development.     

Simulated effects of cricothyroid and thyroarytenoid muscle activation on adult-male vocal fold vibration

Adjustments to cricothyroid and thyroarytenoid muscle activation are critical to the control of fundamental frequency and aerodynamic aspects of vocal fold vibration in humans. The aerodynamic and physical effects of these muscles are not well understood and are difficult to study in vivo. Knowledge of the contributions of these two muscles is essential to understanding both normal and disordered voice physiology. In this study, a three-mass model for voice simulation in adult males was used to produce systematic changes to cricothyroid and thyroarytenoid muscle activation levels. Predicted effects on fundamental frequency, aerodynamic quantities, and physical quantities of vocal fold vibration were assessed. Certain combinations of these muscle activations resulted in aerodynamic and physical characteristics of vibration that might increase the mechanical stress placed on the vocal fold tissue.     

Vocal Warm-up Increases Phonation Threshold Pressure in Soprano Singers at High Pitch

Vocal warm-up is thought to optimize singing performance. We compared effects of short-term, submaximal, vocal warm-up exercise with those of vocal rest on the soprano voice (n = 10, ages 19-21 years). Dependent variables were the minimum subglottic air pressure required for vocal fold oscillation to occur (phonation threshold pressure, Pth), and the maximum and minimum phonation fundamental frequency. Warm-up increased Pth for high pitch phonation (p = 0.033), but not for comfortable (p = 0.297) or low (p = 0.087) pitch phonation. No significant difference in the maximum phonation frequency (p = 0.193) or minimum frequency (p = 0.222) was observed. An elevated Pth at controlled high pitch, but an unchanging maximum and minimum frequency production suggests that short-term vocal exercise may increase the viscosity of the vocal fold and thus serve to stabilize the high voice.     

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

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

The vocal athlete and endotracheal intubation: A management protocol

Endotracheal intubation is associated with significant laryngeal sequelaethat range in severity from mild hoarseness to life-threatening tracheal stenosis. Although the most severe trauma appears to be related to prolonged intubation, even short-term intubation (< 1 day) can adversely affect laryngeal and vocal function. Concern is warranted for all intubated patients, but particularly for the vocal athlete whose livelihood and identity depend on optimal vocal function. It is proposed that the vocal athlete faced with endotracheal intubation risk warrants careful multidisciplinary management. A number of intubation risk factors have been identified in the literature; however, clinical management of vocal athletes who undergo intubation has not been addressed. In medical settings where adverse intubation outcomes can lead to litigation, this clinical management protocol is expected to improve the probability of favorable voice outcome following endotracheal intubation.     

A constitutive model of the human vocal fold cover for fundamental frequency regulation

The elastic as well as time-dependent mechanical response of the vocal fold cover (epithelium and superficial layer of the lamina propria) under tension is one key variable in regulating the fundamental frequency. This study examines the hyperelastic and time-dependent tensile deformation behavior of a group of human vocal fold cover specimens (six male and five female). The primary goal is to formulate a constitutive model that could describe empirical trends in speaking fundamental frequency with reasonable confidence. The constitutive model for the tissue mechanical behavior consists of a hyperelastic equilibrium network in parallel with an inelastic, time-dependent network and is combined with the ideal string model for phonation. Results showed that hyperelastic and time-dependent parameters of the constitutive model can be related to observed age-related and gender-related differences in speaking fundamental frequency. The implications of these findings on fundamental frequency regulation are described. Limitations of the current constitutive model are discussed.     

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

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

Spatial arrangement of the structural elements ofvocal fold layers: An adjustment to the vibration process

It is well established that the multilayered structure of the vocal fold is highly adjusted to the requirements of the vibration process during phonation. There is also some partial data indicating that the spatial arrangement of each vocal fold layer corresponds to the functional requirements, and thus facilitate the phonation process. Nevertheless, all reports on the spatial arrangement of the vocal fold structures deal only with an individual element of the vocal fold histologic structure. The present study encompasses the spatial histologic analysis of all major elements of the vocal fold layers. It was demonstrated that the vocal fold epithelial cells, the connective and muscle fibers, and even the blood vessels run parallel to the vocal fold free edge, which indicates a high adjustment to the phonation requirements and the vibration process.     

Determination of superior surface strains and stresses, and vocal fold contact pressure in a synthetic larynx model using digital image correlation

Stresses and strains within the vocal fold tissue may play a critical role in voice fatigue, in tissue damage and resulting voice disorders, and in tissue healing. In this study, experiments were performed to determine mechanical fields on the superior surface of a self-oscillating physical model of the human vocal folds using a three-dimensional digital image correlation method. Digital images obtained using a high-speed camera together with a mirror system were used to measure displacement fields, from which strains, strain rates, and stresses on the superior surface of the model vocal folds were computed. The dependence of these variables on flow rate was established. A Hertzian impact model was used to estimate the contact pressure on the medial surface from superior surface strains. A tensile stress dominated state was observed on the superior surface, including during collision between the model folds. Collision between the model vocal folds limits the medial-lateral stress levels on the superior surface, in conjunction with compressive stress or contact pressure on the medial surface.     

Dual Intracordal Unilateral Vocal Fold Cysts: A Perplexing Diagnostic and Therapeutic Challenge

SUMMARY: A 52-year-old professional voice user, a director of international sales and marketing and sports coach, described a 40-year history of severe hoarseness, with recent progressive dysphonia. He could not be heard on a cell phone and was unable to raise the volume or intensity of his voice. Strobovideolaryngoscopy revealed a perplexing set of dual intracordal lesions in the left vocal fold and a posterior glottal gap. The amplitude and magnitude of the left vocal fold mucosal waves were barely perceptible and were severely decreased in the right. There was marked phase asymmetry and aperiodicity bilaterally. A computed tomography scan of the neck with contrast and multiplanar reformatted reconstructions and a magnetic resonance imaging (MRI) of the neck with and without contrast revealed the nodular appearing masses of the left vocal fold without any definite evidence of paraglottic or supraglottic extension and no cervical lymphadenopathy. He had a hyperfunctional larynx with laryngopharyngeal reflux and chronic reflux laryngitis. Voice restoration required antireflux therapy, voice therapy, and phonomicrosurgical excision of the lesions. One benign epidermoid malformation cyst was lined by squamous epithelium. The second was a retention cyst lined with cuboidal epithelium. Finally, after 40 years, he has a normal voice.     

The shear modulus of the human vocal fold in a transverse direction

The aim of this study was to measure the shear modulus of the vocal fold in a human hemilarynx, such that the data can be related to direction of applied stress and anatomical context. Dynamic spring rate data were collected using a modified linear skin rheometer using human hemilarynges, and converted to estimated shear modulus via application of a simple shear model. The measurement probe was attached to the epithelial layer of the vocal fold cover using suction. A sinusoidal force of 3g was applied to the epithelium, and the resultant displacement logged at a rate of 1kHz. Force measurement accuracy was 20microg and position measurement accuracy was 4microm. The force was applied in a transverse direction at the midmembranous point between the vocal process and the anterior commissure. The shear modulus of the three female vocal folds ranged from 814 to 1232Pa. The shear modulus of the three male vocal folds ranged from 1021 to 1796Pa. These data demonstrate that it is possible to obtain estimates for the shear modulus of the vocal fold while preserving anatomical context. The modulus values reported here are higher than those reported using parallel plate rheometry. This is to be expected as the tissue is attached to surrounding structures, and is under natural tension.