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OBJECTIVES/HYPOTHESIS We previously developed an instrument called the Aerodynamic Vocal Fold Driver (AVFD) for intraoperative magnified assessment of vocal fold (VF) vibration during microlaryngoscopy under general anesthesia. Excised larynx testing showed that the AVFD could provide useful information about the vibratory characteristics of each VF independently. The present investigation expands those findings by testing new iterations of the AVFD during microlaryngoscopy in the canine model. STUDY DESIGN Animal model. METHODS The AVFD is a handheld instrument that is positioned to contact the phonatory mucosa of either VF during microlaryngoscopy. Airflow delivered through the AVFD shaft to the subglottis drives the VF into phonation-like vibration, which enables magnified observation of mucosal-wave function with stroboscopy or high-speed video. AVFD-driven phonation was tested intraoperatively (n = 26 VFs) using either the original instrument design or smaller and larger versions three-dimensionally printed from a medical grade polymer. A high-fidelity pressure sensor embedded within the AVFD measured VF contact pressure. Characteristics of individual VF phonation were compared with typical two-fold phonation and compared for VFs scarred by electrocautery (n = 4) versus controls (n = 22). RESULTS Phonation was successful in all 26 VFs, even when scar prevented conventional bilateral phonation. The 15-mm-wide AVFD fits best within the anteroposterior dimension of the musculo-membranous VF, and VF contact pressure correlated with acoustic output, driving pressures, and visible modes of vibration. CONCLUSIONS The AVFD can reveal magnified vibratory characteristics of individual VFs during microlaryngoscopy (e.g., without needing patient participation), potentially providing information that is not apparent or available during conventional awake phonation, which might facilitate phonosurgical decision making. LEVEL OF EVIDENCE NA Laryngoscope, 130: 1980-1988, 2020.

OBJECTIVES/HYPOTHESIS Photoangiolytic precision afforded by the 532-nm potassium-titanyl-phosphate (KTP) laser relies on predictable energy delivery. Inadequate energy output can cause vessel rupture, and excessive energy can cause thermal damage. The quality of the cleaved surface and carbon deposits from ablated tissue are two factors that could negatively impact fiber performance. The effects of these on energy output and blood vessel coagulation were assessed using a chorioallantoic membrane (CAM) model. STUDY DESIGN Comparative analysis. METHODS Laser fibers with carbon debris, optimal fiber cleaving, and suboptimal cleaving were inspected at three times magnification, and the light dispersion pattern of each fiber was rated. The average energy output from consecutive pulses through each fiber configuration was recorded. The effect of these fiber conditions on clinical efficacy was estimated by measuring vessel coagulation versus rupture in the CAM model. Repeated measures analysis of variance compared results. RESULTS Carbon debris and suboptimal cleaving resulted in decreased energy output in comparison to optimal cleaving ([-Δ244 mJ, d = 4.31, P

OBJECTIVES/HYPOTHESIS Lubricin/proteoglycan-4 (PRG4) lubricates connective tissues such as joints and tendon sheaths, enabling them to better withstand shearing and frictional forces during motion. We wondered whether PRG4 might play a role in phonation, as normal vocal folds withstand repetitive, high-velocity deformations remarkably well. As a first step, we tested whether PRG4 is expressed in vocal folds. STUDY DESIGN Laboratory study. METHODS Anatomical and molecular methods were applied to 47 larynges from humans, macaque (Macaca fascicularis), canines, pigs, calves, and rats. Immunohistochemistry (IHC), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) methods were used to test for the presence of PRG4. RESULTS In all species, the true vocal fold lamina propria (TVF-LP) was positive for PRG4 by IHC, whereas immunoreactivity of the false vocal fold was weak or absent, depending on the species. Human TVF-LP was strongly stained across all layers. Immunoreactivity was seen variably on the vocal fold surface and within the vocal fold epithelium, in the conus elasticus and thyroglottic ligament, and at the tip of vocal process. Western blots of four humans and six pigs demonstrated immunoreactivity at appropriate molecular weight. qRT-PCR of pig tissues confirmed PRG4 mRNA expression, which was highest in the TVF-LP. CONCLUSIONS PRG4 was found in phonatory tissues of six mammals. We suggest it might act as a lubricant within the lamina propria and possibly on the vocal fold surface, limiting phonation-related damage to vocal fold extracellular matrix and epithelium, and enhancing vocal efficiency by reducing internal friction (viscosity) within the vocal fold. LEVEL OF EVIDENCE NA Laryngoscope, 129:E229-E237, 2019.

To compare the physical characteristics of 3 laser-protected endotracheal tubes (LPETs) commonly used in endoscopic laser surgery. To report potential intraoperative problems related to LPET use and suggest practical solutions.Comparative analysis.Academic laboratory.Physical characteristics of the Mallinckrodt Laser-Flex (MTL), Medtronic Laser-Shield II (ML-II), and Rusch LaserTubus (RL) were compared. The effect of bending LPETs on airflow resistance was estimated with a pressure transducer. The force required to pull each tube through the glottis and the pressure exerted during this maneuver were measured in a fresh cadaveric human larynx.The design features and physical characteristics of LPETs differ, including varying balloon-tip lengths. Bending LPETs to acute angles caused significant pressure increase within the RL tube (Δ 3.42 cm HThe ML-II tube had the most favorable characteristics, with minimal pressure during extubation and resistance to kinking. The RL tube kinks readily with a resultant increase in resistance to airflow. The MTL tube extends farther into the trachea due to a relatively elongated balloon-tip configuration. Future LPET designs should incorporate features that avoid intraoperative difficulties related to airway protection and ventilation.

The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p

Objectives/Hypothesis: Vocal fold (VF) injections of viscous materials are typically performed using hand-operated syringes or injection guns; however, these methods can be imprecise due to accumulation of pressure, effort-related tremor, and poor feedback regarding injection volume and rate. Study Design: Apparatus development with laboratory bench-top and animal model testing. Methods: A foot pedal-triggered device for dispensing viscous materials was modified by adding a linear transducer and display for monitoring dispensed volume. In bench tests, bovine VFs were injected with fluids/materials of different viscosities (saline, glycerol, hydrogel, and liposuctioned fat) through narrow-bore needles using a range of driving pressures and air pulse durations. The device was further evaluated in >50 in vivo VF injection experiments. Results: Device function was repeatable, with high correlations (typically R2 > 0.98) between the readout and direct measures of volume, even for small volumes (

Objectives/Hypothesis: Early glottic cancer has been involuted by treatment with the 532 nm pulsed potassium-titanyl-phosphate (KTP) laser in initial clinical studies. Selective photoangiolysis of the sublesional circulation that allows for relative sparing of surrounding tissue is the presumed mechanism. No prior controlled animal-model study has analyzed the ability of selective coagulation of lesional microvasculature coagulation with the KTP laser to involute malignant lesions. This study tests the efficacy of photoangiolysis with the KTP laser in treating squamous cell carcinoma in an established animal model. Study Design: In vivo. Methods: Malignant lesions were induced unilaterally in the cheek pouches of 21 hamsters by applying 9,10-dimethyl-1,2-benzanthrancene. The contralateral cheek pouch served as a control. Weekly lesion photodocumentation and pulsed KTP laser (30 W, 15 msec pulse width, 2 pulses/sec) treatments were done. The endpoint of each treatment was a uniform white-blanching of the lesion. Hamsters were sacrificed 1 week after the last treatment and cheek pouches were analyzed histologically. Results: Carcinoma was confirmed in 19 hamsters, and lesions that were initially 2 mm (P = .0004). Every lesion (10/10) that initially measured 5 mm resolved after completion of the treatment period. Conclusions: Pulsed KTP laser photoangiolysis can effectively involute small malignant lesions, but may be less effective at involuting larger (>2 mm) lesions.

Objectives/Hypothesis: Optical coherence tomography (OCT) can provide high-resolution (! 10‐15 lm/pixel) images of vocal fold microanatomy, as demonstrated previously. We explored physiologically triggered Fourier-domain OCT for imaging vocal folds during phonation. The goal is to visualize dynamic histological cross sections and four-dimensional data sets where multiple planes are displayed in synchronized motion. If feasible, this approach could be a useful research tool and spur development of new clinical instrumentation. Study Design: A Fourier-domain, triggered OCT system was created and tested in experiments on excised calf larynges to obtain preliminary observations and characterize important factors affecting image quality. Methods: Larynges were imaged during phonation driven by warm, humidified air. A subglottal pressure signal was used to synchronize the OCT system with the phonatory cycle. Image sequences were recorded as functions of anatomical location or subglottal pressure. Implant materials were also imaged during vibration, both in isolation and after injection into a vocal fold. Results: Oscillations of epithelium and lamina propria were observed, and parameters such as shape, amplitude, and velocity of the vocal fold mucosal waves were found to be measurable. Ripples of mucosal wave as small as 100 lm in vertical height were clearly visible. Internal strain was also observed in normal and implanted vocal folds. Conclusions: Four-dimensional OCT of the vocal fold may help to more directly relate biomechanics to anatomy and disease. It may also be useful for assaying the functional rheology of implants in the context of real tissue. With further development, this technology has potential for clinical endoscopic application.

Objectives/Hypothesis: To explore whether adipose-derived stem/stromal cells (ASCs) have therapeutic potential for treating scarred superficial lamina propria through the effects of secreted hepatocyte growth factor (HGF) on scar fibroblasts. Study Design: In vitro study using coculture system. Methods: Scar fibroblasts (SFs) were isolated from ferret vocal folds electrocauterized 2 weeks previously. ASCs were isolated from ferret lipoaspirated subcutaneous abdominal fat. For coculture experiments, the two cell types were combined in Transwell plates for 6 days, followed by 1 or 3 days of monoculture after removing the upper chamber. Assays were then performed on cells and media from the bottom chamber. We measured: 1) the production of healuronic acid (HA), collagen and HGF via enzymelinked immunosorbent assays, 2) the expression of α-smooth muscle actin (α-SMA), 3) cell proliferation, and 4) apoptosis of SFs (2, 3, and 4 via flow cytometry). Other experiments examined the effects of HGF on SFs and the effects of HGF neutralization in the coculture system. Results: Coculture led to significant decreases in SF collagen production (P < .05), cell proliferation (P < .05), and α-SMA expression (P < .05), whereas HA production increased (P < .05). Coculture also increased HGF secretion from ASCs (P < .05). Neutralization of HGF abolished the inhibitory effects of ASCs on SF collagen synthesis (P < .05). Conclusions: ASCs influence SFs to adopt a less fibrotic profile. It appears that HGF is at least one of the soluble factors responsible for this effect. Implanted ASCs could potentially ameliorate vocal fold scar by acting as long-term, intrinsic sources of HGF.

Objectives/Hypothesis: High-resolution imaging of vocal folds that distinguishes vocal fold (VF) layered microstructure and VF implants would provide a key experimental tool for translational research investigating biomaterial-based interventions to treat vocal fold scar. To establish proof of concept, we studied whether 11.7 Tesla (T) magnetic resonance (MR) microimaging provides the needed resolution to resolve vocal fold tissue architecture. Study Design: We performed ex vivo MR microimaging of fixed ferret and canine larynges to determine whether changes in the layered architecture can be detected in the presence of scar and subsequent to biomaterial injections into the vocal folds. Serial section histological analyses were done to corroborate MR microimaging findings. Methods: Multiple axial and transverse/coronal 300-μm slices were obtained using an 11.7 T MR spectrometer/500 MHz for proton with gradient-recalled echo and rapid acquisition with relaxation enhancement imaging sequences. Results: High-resolution (39 μm/pixel) MR microimages distinguished VF epithelium, lamina propria, muscle, and cartilage in ferret and canine larynges. In ferret scarred VFs (n = 25), collagen-rich dense scar tissue was distinguishable from contralateral nonscarred VFs and from normal ferret VFs (n = 25), as confirmed on histology. MR microimaging accurately detected injected autologous fat, hyaluronic acid-based and polyethylene glycol (PEG)-based implants injected into both ferret and canine VFs. Importantly, MRI accurately showed resorption of PEG implants in ferrets and canines, as confirmed on histology. Additionally, ex vivo MR spectroscopy distinguished fat from PEG-based implants. Conclusions: Ex vivo 11.7 T MR microimaging provided high-resolution images of ferret and canine laryngeal tissue microstructure, although the superficial lamina propria could not be distinguished. Histology confirmed MR microimaging findings, indicating utility of MR microimaging of modeled scar, implant residence time, and tissue responses, thus providing integrative insight relevant to translational research. Laryngoscope, 2009

Objectives/Hypothesis: Optical coherence tomography (OCT) is a promising new imaging modality that can help discern the layered microstructure of vocal folds. In the future, subepithelial injections of implants will improve vocal fold pliability where there is stiffness of phonatory mucosa. Using OCT both to delineate the depth of subepithelial injections real-time and to serially image the implant over time would be valuable, and has not been demonstrated previously. Study Design: Ex vivo study using excised calf larynges and survival study using canines in vivo. Methods: An investigation was done employing real-time OCT imaging of subepithelial injection pulses into phonatory mucosa of four calf larynges ex vivo to track the presence of subepithelial implants in phonatory mucosa in a survival study using an in vivo canine model. Results: OCT readily identified polyethylene glycol (PEG)-based hydrogel in the subepithelial plane in both the ex vivo calf study and the in vivo canine study. Ex vivo calf images correlated with histological specimens obtained immediately postinjection. Images obtained in this study provide confirmation of the hydrogel injection depth in real time, and allow for the implant to be tracked during a canine survival study. Conclusions: OCT can confirm subepithelial placement of hydrogel implant in the vocal fold with sufficient resolution to provide instantaneous feedback of an injection pulse. Survival studies in an in vivo canine model indicate that OCT can potentially be helpful in monitoring rheologically appropriate implants within the superficial lamina propria. © 2009 The American Laryngological, Rhinological and Otological Society, Inc.

Objectives: Selective vascular ablation (photoangiolysis) using pulsed lasers that target hemoglobin is an effective treatment strategy for many vocal fold lesions. However, vessel rupture with extravasation of blood reduces selectivity for vessels, which is frequently observed with the 0.45-ms, 585-nm pulsed dye laser. Previous studies have shown that vessel rupture is the result of vaporization of blood, an event that varies with laser pulse width and pulse fluence (energy per unit area). Clinical observations using a 532-nm wavelength pulsed potassium-titanyl-phosphate (KTP) laser revealed less laser-induced hemorrhage than the pulsed dye laser. This study investigated settings for the pulsed KTP laser to achieve selective vessel destruction without rupture using the avian chorioallantoic membrane under conditions similar to flexible laryngoscopic delivery of the laser in clinical practice. Study Design: The chick chorioallantoic membrane offers convenient access to many small blood vessels similar in size to those targeted in human vocal fold. Using a 532-nm pulsed KTP laser, pulse width, pulse energy, and working distance from the optical delivery fiber were varied to assess influence on the ability to achieve vessel coagulation without vessel wall rupture. Methods: Thirdorder vessels (n = 135) were irradiated: Energy (471-550 mJ), pulse width (10, 15, 30 ms), and fiber-to-tissue distance (1 mm, 3 mm) were varied systematically. Results: Selective vessel destruction without vessel wall rupture was more often achieved by increasing pulse width, increasing the fiber-to-tissue distance, and decreasing energy. Vessel destruction without rupture was consistently achieved using 15- or 30-ms pulses with a fiber-to-tissue distance of 3 mm (pulse fluence of 13-16 J/cm 2 ). Conclusions: This study substantiates our clinical observation that a 532-nm pulsed KTP laser was effective for ablating microcirculation while minimizing vessel wall rupture and hemorrhage.

Objectives: Intraoperative assessment of vocal fold vibration during phonomicrosurgery performed under general anesthesia may enhance surgical decision-making. We therefore developed and bench-tested a new device we refer to as the aerodynamic vocal fold driver (AVFD). Materials and Methods: The AVFD comprises a hand-held probe that uses airflow to drive individual vocal folds into phonatory-like vibration. This permits stroboscopic visualization of mucosal waves with simultaneous control of subglottal air pressure. In initial experiments to validate the technique, AVFD driven phonation and conventional whole-larynx phonation were compared using excised canine larynges (n = 14). Results: Single vocal fold phonation using the AVFD and whole larynx phonation yielded similar, positive correlations between subglottal pressure and both amplitude and frequency of vibration. Experiments simulating vocal fold scar-related mucosal stiffening by subepithelial injection of fixative showed the expected elevation of phonation threshold pressures as measured with the AVFD. Likewise, unilateral tissue compression injury disrupted vocal fold vibration, and the AVFD was useful for quantifying improvement in the damaged vocal fold after repair with injection of cross-linked hyaluronic acid gel. Conclusions: These results show that this new instrument has the potential to provide novel and useful information for laryngeal experimentation and to improve phonosurgery.

We identified a 'semicircular' compartment of the rat thyropharyngeus muscle at the pharyngoesophageal junction and used the glycogen depletion method to determine how the fibers of this muscle (as well as all others of the pharynx and larynx) are innervated by different cranial nerve branches. The semicircular compartment appears anatomically homologous to the human cricopharyngeus muscle, an important component of the upper esophageal sphincter. While we found very little overlap in the muscle targets of the pharyngeal, superior laryngeal and recurrent laryngeal nerves within the pharynx and larynx, the semicircular muscle receives a dual, interdigitating innervation from two vagal branches: the pharyngeal nerve and a branch of the superior laryngeal nerve we call the dorsal accessory branch. After applying horseradish peroxidase to either of these two nerves, we compared the distribution and number of cells labeled in the brainstem. The dorsal accessory branch conveys a more heterogeneous set of efferent fibers than does the pharyngeal nerve, including the axons of pharyngeal and esophageal motor neurons and parasympathetic preganglionic neurons. The observed distribution of labeled motor neurons in nucleus ambiguus also leads us to suggest that the semicircular compartment is innervated by two subsets of motor neurons, one of which is displaced ventrolateral to the main pharyngeal motor column. This arrangement raises the possibility of functional differences among semicircular compartment motor neurons correlated with the observed differences in brainstem location of cell bodies.

Injectable hydrogels may potentially be used for augmentation/regeneration of the lamina propria of vocal fold tissue. In this study, hyaluronic acid (HA) and dextran were chemically modified and subsequently crosslinked via formation of hydrazone bonds in phosphate buffer. Swelling ratios, degradation, and compressive moduli of the resulting hydrogels were investigated. It was found that the properties of HA-dextran hydrogels were variable and the trend of variation could be correlated with the hydrogel composition. The biocompatibility of three injectable HA-dextran hydrogels with different crosslinking density was assessed in the vocal fold region using a ferret model. It was found that HA-dextran hydrogels implanted for three weeks stimulated mild foreignbody reactions. Distinct tissue-material interactions were also observed for hydrogels made from different formulations: the hydrogel with the lowest crosslinking density was completely degraded in vivo; while material residues were visible for other types of hydrogel injections, with or without cell penetration into the implantation depending on the hydrogel composition. The in vivo results suggest that the HA-dextran hydrogel matrices can be further developed for applications of vocal fold tissue restoration. © 2010 Wiley Periodicals, Inc.