Chemical receptors of the arytenoid: A comparison of human and mouse.

OBJECTIVES/HYPOTHESIS: The larynx is a highly responsive organ exposed to mechanical, thermal, and chemical stimuli. Chemicals elicit responses both in intraepithelial nerve fibers and in specialized chemosensory cells, including scattered solitary cells as well as taste cells organized into taste buds. Activation of both chemosensory cells and taste buds in the larynx elicit cough, swallow, or apnea with exposure to sour or bitter substances, and even by water or sweet-tasting chemicals. In an effort to begin understanding their function, we sought to compare the distribution, density, and types of chemosensory cells and chemoresponsive nerve fibers in laryngeal epithelium of humans and mice. STUDY DESIGN: Animal and human laboratory analysis. METHODS: Using immunohistochemistry, we identified taste cells and polymodal nociceptive nerve fibers in the arytenoid area of the laryngeal epithelium of the following: 1) infants undergoing supraglottoplasty for laryngomalacia, and 2) a cadaveric specimen procured from a 34-year-old donor. We then compared these findings to both preweanling and mature mouse tissue. RESULTS: Arytenoid tissue from both human and mouse contained many taste buds containing type II taste cells-bitter, sweet, or umami sensing-which were innervated by nerve fibers expressing P2X3 type adenosine triphosphate receptors. Type III cells (acid responsive) were also present, but they were fewer in human tissue than in equivalent tissue from mice. In both species, the epithelium was densely innervated by free nerve endings. CONCLUSIONS: Our findings suggest that from a standpoint of chemosensation, human and mouse larynges are biologically similar. This suggests that a murine model can be used effectively in laryngeal chemosensory research. LEVEL OF EVIDENCE: NA Laryngoscope, 130:423-430, 2020.

Quantifying the Subharmonic Mucosal Wave in Excised Larynges via Digital Kymography.

OBJECTIVES: In this paper, a nonlinear least squares fitting method was proposed to quantify subharmonic mucosal waves. STUDY DESIGN AND METHODS: Subharmonic mucosal waves from 10 excised canine larynges were recorded using digital kymography and analyzed using nonlinear least squares and linear least squares methods. Amplitudes of fundamental and subharmonic mucosal wave components of right-upper, right-lower, left-upper, and left-lower vocal fold lips were calculated. Lastly, phase differences of fundamental and subharmonic components of the left and right vocal folds were compared. RESULTS: The results showed that the nonlinear least squares analysis method provides a more effective complement to the linear fitting method for subharmonic mucosal wave extraction. There was a significant difference in amplitudes between the subharmonic and the fundamental components of mucosal waves (P < 0.05). The phase differences of the fundamental and the subharmonic components of the right and left vocal folds were not significantly different. CONCLUSIONS: The application of the nonlinear least squares analysis method in digital kymography is useful for the characterization of subharmonic mucosal waves.

Effect of saline inhalation on vocal fold epithelial morphology evaluated by optical coherence tomography.

OBJECTIVES/HYPOTHESIS: Examination of tissue structures by optical coherence tomography (OCT) has been shown to be useful on mucous membranes of the vocal folds, but so far its application to the human larynx has been limited because it is technically cumbersome and usually needs to be performed with sedation. Here a newly developed, noninvasive combined laryngoscopy and OCT procedure is described and its suitability for ambulatory OCT studies evaluated. Because inhalation therapies utilizing saline solutions are commonly used as a treatment option for disorders of the airways, and vocal fold epithelium is most likely to be affected due to its superficial positioning, epithelial thickness was chosen as a relevant test parameter and evaluated before and after saline inhalation. METHODS: Seven vocally healthy participants performed a 10-minute inhalation of saline solution and underwent a combined laryngoscopy and OCT before and after the inhalation therapy. Endoscopy was performed using a newly developed combined laryngoscopy and OCT device. The OCT images were used to estimate the epithelial thickness of the vocal folds. RESULTS: Epithelial thickness measured in all participants before treatment was comparable in size reported in previous studies. Statistical differences before and after inhalation were not detected. CONCLUSION: The newly developed combined laryngoscopy and OCT procedure enables rapid investigation of the vocal fold epithelium. Inhalation of saline solution did not appear to affect the thickness of the epithelium of the vocal folds in vocally healthy subjects, as evaluated by OCT. LEVEL OF EVIDENCE: N/A. Laryngoscope, 126:E332-E336, 2016.

The role of contact endoscopy in screening for premalignant laryngeal lesions: a study of 141 patients.

At their earliest stage, pathologic lesions of the laryngeal epithelium are macroscopically invisible. Ideally, these lesions should be detected before their clinical manifestations appear so that prompt management can be initiated. However, most diagnostic modalities are unable to detect early premalignant lesions. We conducted a retrospective study of the use of contact endoscopy in analyzing the vocal fold mucosal epithelium in adults who had been operated on at our hospital under general anesthesia for various nonlaryngeal diseases. After we identified 71 such patients who were smokers, we chose an almost equal number of nonsmokers (n = 70) for comparison purposes. In all, our study population was made up of 141 patients--51 men and 90 women, aged 21 to 78 years (mean: 52). All patients had normal findings on preoperative laryngeal endoscopy. Our goal was to determine if the routine use of this diagnostic modality is justified in selected cases. Contact endoscopy identified dysplastic vocal fold lesions in 4 patients and chronic laryngitis in 3; all 7 of these patients were smokers. Since early laryngeal lesions are not macroscopically evident, early detection of these changes by other means is associated with a better prognosis and easier management. Our study demonstrates that the use of contact endoscopy during general anesthesia as a standard diagnostic method in long-time cigarette smokers is fully justified.

[Lymphatic tissue of the nose (NALT) and larynx (LALT) in species comparison: human, rat, mouse]. / Das lymphatische Gewebe der Nase (NALT) und des Kehlkopfes (LALT) im Speziesvergleich: Mensch, Ratte, Maus.

Nose- and larynx associated lymphatic tissues (NALT and LALT) vary markedly between humans, rats and mice. NALT of rats and mice is formed by paired lymphoid aggregates in the nasal cavity, while it consists of individual mucosa associated lymphoid follicles throughout the nose in humans. In addition to NALT, tonsils are present in humans, but not in rats and mice. In the larynx, LALT can be found in humans, but not in rats. Size and functionality of NALT, tonsils and LALT vary with age. The extrapolation of data obtained from rodents to humans should be carefully evaluated due to these differences. The term common mucosal immune system should replaced by the term "integrated" MALT and the immunological differences between respiratory and digestive tract should always be considered.

[Principle differences of the vocal folds in humans and swine: a few essential morphologic criteria]. / Prinzipielle Unterschiede am Stimmapparat von Mensch und Schwein: Einige wesentliche morphologische Kriterien.

The histological composition of the Lamina propria in pigs - especially in the cranial fold of the glottis - gives reason to expect other phoniatric properties than in man. Age-related changes must be considered.

Gradation of stiffness of the mucosa inferior to the vocal fold.

During phonation, energy is transferred from the subglottal airflow through the air/mucosa interface that results in the propagation of the mucosal wave in the vocal fold. The vocal fold is soft, and the subglottal mucosa is stiff. We hypothesize that it is highly improbable that there is a rigid boundary between the tissue structures, with a sudden drop in stiffness; and that a gradual change would be more likely to support the efficient transfer of energy from the airflow to the mucosal wave. Our objective was to test this hypothesis by quantifying the change in mucosa stiffness with respect to anatomical position. In this initial study, using five pig larynges, a series of point-specific measurements of mucosa stiffness were taken in a line from the midpoint of the vocal fold toward the trachea. A modified linear skin rheometer adapted for laryngeal elasticity measurement applied shear stress to a series of seven positions at 2-mm intervals starting from the midmembranous vocal fold medial surface. A sinusoidal shear force of 1g was applied at each point, and resultant displacement curve logged. Using a regression algorithm, the stiffness of the tissue was derived in units of grams force per millimeter displacement. Five readings were taken at each position. The results indicate that there is a linear increase in stiffness with respect to position, increasing as the measurements are taken further from the vocal fold. There is a gradual change in stiffness of the subglottal mucosa of a pig larynx.

Liquid accumulation in vibrating vocal fold tissue: a simplified model based on a fluid-saturated porous solid theory.

The human vocal fold is treated as a continuous, transversally isotropic, porous solid saturated with liquid. A set of mathematical equations, based on the theory of fluid-saturated porous solids, is developed to formulate the vibration of the vocal fold tissue. As the fluid-saturated porous tissue model degenerates to the continuous elastic tissue model when the relative movement of liquid in the porous tissue is ignored, it can be considered a more general description of vocal fold tissue than the continuous, elastic model. Using the fluid-saturated porous tissue model, the vibration of a bunch of one-dimensional fibers in the vocal fold is analytically solved based on the small-amplitude assumption. It is found that the vibration of the tissue will lead to the accumulation of excess liquid in the midmembranous vocal fold. The degree of liquid accumulation is positively proportional to the vibratory amplitude and frequency. The correspondence between the liquid distribution predicted by the porous tissue theory and the location of vocal nodules observed in clinical practice, provides theoretical evidence for the liquid accumulation hypothesis of vocal nodule formation (Jiang, Ph.D., dissertation, 1991, University of Iowa).

Microendoscopy of Reinke's space.

OBJECTIVES: Contemporary surgical treatment of the superficial layer of the lamina propria or Reinke's space is most commonly performed through an incision in the overlying vocal fold epithelium. This approach may disrupt normal tissue, induce scarring, and allow extrusion of implanted materials. Previously reported external approaches to Reinke's space required either a laryngofissure or a "minithyrotomy" for access. These surgical approaches were performed without direct imaging of Reinke's space. Instruments placed below the vocal fold epithelium via this external approach were visualized through the translucent vocal fold epithelium. We designed this study to identify the feasibility of limited-access surgery of the lamina propria using microendoscopes placed into Reinke's space through an external approach. METHODS: A cadaveric human larynx was dissected, and microendoscopes were directly advanced into Reinke's space through a subepithelial puncture of the cricothyroid membrane, as well as lateral fenestration through the thyroid cartilage. RESULTS: Photodocumentation of the undersurface of vocal fold epithelium, the opposing surface of the vocal ligament, and the intervening Reinke's space was successfully accomplished. CONCLUSIONS: Advances in both microendoscopes and accompanying instrumentation permit access to the superficial layer of the lamina propria without disrupting the overlying epithelium. This approach to microendoscopy of Reinke's space may allow for more effective surgical treatment of cysts, chronic edema, vascular abnormalities, atrophy, scarring, and sulcus vocalis.

Expression and distribution of CD44 and hyaluronic acid in human vocal fold mucosa.

OBJECTIVES: Expression of CD44 (a cell surface receptor for hyaluronic acid) and the distribution of hyaluronic acid were examined in the human vocal fold mucosa. METHODS: Light microscopic investigation was carried out on 10 normal larynges of newborn, infant, younger adult, and older adults with Alcian blue staining, a hyaluronidase digestion study, and immunohistochemistry for CD44. RESULTS: Before the appearance of hyaluronic acid in the newborn vocal fold mucosa, CD44 was expressed on the stellate cells in the macula flava and on the fibroblasts in Reinke's space. During infancy, hyaluronic acid appeared and was distributed in the vocal fold mucosa. Many more stellate cells in the macula flava showed CD44 expression, and a large amount of hyaluronic acid was present around the infant stellate cells; however, the fibroblasts in Reinke's space expressed little CD44. During adulthood, hyaluronic acid was distributed in the vocal fold mucosa. Almost all of the stellate cells in the macula flava showed CD44 expression, and much hyaluronic acid existed around the stellate cells in the adult macula flava. However, fibroblasts in the adult Reinke's space expressed little CD44, and hyaluronic acid density in that space was lower than that in the macula flava. CONCLUSIONS: Stellate cells in the macula flava and CD44 cooperatively play important roles in maintaining hyaluronic acid in the human vocal fold mucosa as a vibrating tissue.

Types and patterns of response in the larynx following inhalation.

The laryngeal mucosa responds to insult similarly to other epithelial tissues but the response depends on location within the larynx since important anatomic differences exist, even within rodent species. Although dogs and nonhuman primates are also utilized for inhalation toxicology studies, little published information is available regarding sites of injury from inhaled toxicants in these species. Accurate and consistent fixation, trimming, and microtomy of laryngeal sections allow the pathologist to compare the morphology of laryngeal mucosa from exposed and control animals and detect and interpret subtle differences resulting from inhalation exposure. There are anatomic landmarks that are keys to providing consistent sections through important areas of the laryngeal mucosa. Repeated inhalation of toxic concentrations of chemicals, drugs, or environmental contaminants induces a wide range of responses, depending on the physical properties and concentration of the toxic substance and duration of exposure. Responses include edema, acute to chronic inflammation, fibrosis, mucosal ulceration, degeneration, and necrosis. Attempts at repair include regeneration, hyperplasia, squamous metaplasia, hyperkeratosis, and neoplasia. Awareness of normal histology and zones of transition from squamous to respiratory epithelium in different areas of the larynx in different species is critical to avoid confusing normal epithelium with metaplasia or hyperplasia. Microscopic examination of laryngeal mucosa from animals exposed via inhalation and necropsied following a recovery period provides the opportunity to determine the degree of regression or progression of exposure-induced laryngeal lesions.

Age related changes in the distribution of laryngeal glands in the human adult larynx.

OBJECTIVE: To understand the defense status of glandular tissue in the human adult larynx, we examined the glandular distribution, concentration and the ratio of the mucous glands in the subglottic and supraglottic larynx. METHODS: One-hundred and seven adult larynges were examined. The area of the mucosa and glands were measured using an image analyzing system. The correlations of the concentration as well as the ratio of the mucous glands with age were analyzed. RESULTS: The ratio of the areas occupied by the glandular acini of the subglottis was almost equal to that of infants as we reported earlier. The concentration of the laryngeal glands decreased in higher-aged adults at the level of the supraglottis, but not at the level of the subglottis. CONCLUSIONS: In comparison to infants, the ratio of laryngeal glands of the human subglottic larynx is rather constant in human adults. However, the supraglottic larynx seems to be influenced by increasing age. The changes deserved in the laryngeal glands predominantly occur in the supraglottic larynx.

Lateral phase mucosal wave asymmetries in the clinical voice laboratory.

Anecdotally, in some persons it has been observed by the Senior Author (K.K.) that asymmetries of the mucosal wave exist when examined videostroboscopically. In the vast majority of these people, no pathology is ever discovered. Mucosal wave asymmetries could cause concern for the otolaryngologist, who may consider them to be a forewarning of subclinical pathology and subject the patient to unnecessary, expensive, and anxiety-provoking investigations or interventions. The purpose of this study was to establish the prevalence of mucosal wave asymmetries in an asymptomatic population lacking laryngeal pathology. Acoustic spectral analysis is also utilized to determine if the presence of subharmonics might be associated. A hospital-based, cross-sectional study design was used. The subjects had no known vocal or medical pathologies, and were nonsmoking. The study group was composed of 30 males aged 35-50 years and 30 women between 22-55 years. Each of the males underwent acoustic spectral analysis; and all subjects completed a medical questionnaire, subjective talkativeness rating, and videostroboscopic laryngeal examination. 10.5% of the subjects (exact 95% CI = 4.0-21.5%) exhibited mucosal wave variations at stroboscopy, characterized as periodic lateral phase asymmetries found consistently in both the modal and upper registers. There was no association with the chosen acoustic spectral parameters, talkativeness scales, or questionnaire-based variables. Mucosal wave asymmetries may be a variance of normal, and are likely to be far more common in the general population than previously believed. The prevalence detected here is expected to be important in the clinical laryngology practice, where these asymmetries may be frequently encountered and influencing management decisions. There has been little normative data published for variations of the mucosal wave specifically for epidemiological purposes. Clinically, in the absence of such data, otolaryngologists may over interpret videostroboscopic findings, leading to unnecessary investigations or interventions.

Growth of laryngeal glands in infants: a comparative study of the sub- and supraglottic larynx.

It is well established that laryngeal secretions, produced in the glandular tissue, contribute to the local defence system of the larynx. In this study, the distribution of glandular tissues in the subglottic and supraglottic larynx was examined in 55 infants, using an image analysing system. The average area of the mucosa or glandular acini at the level of the false vocal fold was almost three times larger than that of the subglottis. At both levels, the area occupied by the glands increased according to the age at death and became more evident according to the total development score (TDS) which was estimated by adding the age at death and the gestational period. The results indicate that the glandular structure of the level of the sub- or supraglottic larynx develops constantly and equally after birth.

Fine structure of the human newborn and infant vocal fold mucosae.

This study was carried out to determine the fine structure of the membranous portion of the human newborn and infant vocal fold mucosae and to clarify how the human vocal fold mucosa begins to grow after birth. Light and electron microscopic observations were made on the vocal fold mucosae excised from human newborn and infant larynges. Our findings are summarized as follows. 1) Reinke's space of the newborn vocal fold is a loose structure composed of ground substance and sparse fibers. 2) Ground substance is abundant and glycoproteins (fibronectin) are present in the lamina propria. 3) In the maculae flavae of newborn vocal folds, fibroblasts begin to form collagenous, reticular, and elastic fibers. 4) In the infant maculae flavae, fibroblasts are activated to become stellate in shape and synthesize extracellular matrices such as collagenous and reticular fibers. 5) After birth, the collagenous and reticular fibers synthesized in the maculae flavae extend toward the middle of the membranous portion of the vocal fold mucosa. 6) Glycoprotein (fibronectin) determines the orientation of collagenous and reticular fiber deposition. 7) Glycoprotein (fibronectin) decreases with the increase of the fibrous components in Reinke's space. 8) Elastic fibers increase after collagenous and reticular fibers appear in Reinke's space. 9) Much hyaluronic acid (glycosaminoglycan) is actively produced in the infant maculae flavae. 10) The collagenous and reticular fibers serve as stabilizing scaffolds for the other extracellular matrices, such as elastic fibers and glycosaminoglycan. During infancy, the extracellular matrices synthesized in the maculae flavae appear in the membranous portion of the vocal fold, each at a different time, so as to initiate the 3-dimensional formation of the vocal fold mucosa.

[A histochemical study of the glandular system of the human epiglottis. The clinical implications]. / Estudio histoquímico del sistema glandular de la epiglotis humana. Implicaciones clínicas.

The paper deals about classical histologic and histochemical study for mucinoid substances through 7 laryngeal pieces coming from clinic autopsies. The gland units occurring in the two lower thirds of the epiglottis were formed by a great number of serous acini and mucinous tubuli, which secretion is acid and neutral mucous substances, as sialomucins, serving as lubricant and protective means. This glandular system convey through the epiglottic perforans holes, without break, the pre-epiglottic and epiglottic spaces, with the clinical repercussion that suggest the chance of encroachment for incipient epiglottic cancer to pre-epiglottic room.

Histology of laryngeal mucosa.

The larynx is a complex tubular segment of the respiratory system formed by irregularly shaped plates of hyaline and elastic cartilage. The mucosa form two pairs of folds, false and true vocal cords, which extend into the lumen of the larynx. The laryngeal epithelium corresponding to the mechanically exposed areas consists of stratified squamous nonkeratinized epithelium. Suprabasally in this epithelium, dendritic antigen-presenting Langerhans cells (LCs) can be found. In the rest of the larynx, the epithelium is ciliated columnar pseudostratified with a rich population of goblet cells. Except in the true vocal cords, lamina propria consists of rather loose connective tissue and contains groups of small, branched tubuloalveolar glands.