Immunohistochemical distribution of proteins involved in glutamate release in subepithelial sensory nerve endings of rat epiglottis.

To elucidate the efferent functions of sensory nerve endings, the distribution of calretinin and vesicular glutamate transporter 1 (VGLUT1) in laryngeal laminar nerve endings and the immunohistochemical distribution of proteins associated with synaptic vesicle release, i.e., t-SNARE (SNAP25 and syntaxin 1), v-SNARE (VAMP1 and VAMP2), synaptotagmin 1 (Syt1), bassoon, and piccolo, were examined. Subepithelial laminar nerve endings immunoreactive for Na+-K+-ATPase α3-subunit (NKAα3) were largely distributed in the whole-mount preparation of the epiglottic mucosa, and several endings were also immunoreactive for calretinin. VGLUT1 immunoreactivity was observed within terminal part near the outline of the small processes of NKAα3-immunoreactive nerve ending. SNAP25, syntaxin 1, and VAMP1 immunoreactivities were detected in terminal parts of calretinin-immunoreactive endings, whereas VAMP2 immunoreactivity was only observed in a few terminals. Terminal parts immunoreactive for calretinin and/or VGLUT1 also exhibited immunoreactivities for Syt1, Ca2+ sensor for membrane trafficking, and for bassoon and piccolo, presynaptic scaffold proteins. The presence of vesicular release-related proteins, including SNARE proteins, in the terminals of laryngeal laminar endings indicate that intrinsic glutamate modulates their afferent activity in an autocrine-like manner.

Localization and expression of TRPV1 and TRPA1 in the human oropharynx and larynx.

BACKGROUND: Previous studies have found that TRPV1 and TRPA1 receptor agonists improve swallow response in patients with oropharyngeal dysphagia (OD), but little is known about the expression of these receptors in the human oropharynx. The aim of this study was to assess the expression and localization of TRPV1 and TRPA1 in human samples from the oropharynx of healthy patients, to provide the basis for new pharmacological treatments for OD. METHODS: Samples from oropharyngeal regions innervated by cranial nerves V, IX, and X (tongue, pharynx, and epiglottis) were obtained during ENT surgery and processed either for mRNA (21 patients) or for immunohistochemical assays (seven patients). The expression analysis was performed with RT-qPCR using ACTBh as reference gene. Hemotoxylin and eosin staining was used to study the histology; the immunohistochemical assay used (i) neuron-specific enolase to detect nerve fibers or (ii) fluorescent probes to locate TRPV1 and TRPA1. RESULTS: TRPV1 was expressed in the three studied regions, with higher levels in CN V region (tongue) than in CN X region (epiglottis; p < 0.05), and was localized at epithelial cells and nociceptive fibers in all studied regions. TRPA1 was also expressed in all studied regions, but was always localized below the basal lamina. No immunoreactivity for TRPA1 was found on epithelial cells. CONCLUSIONS & INFERENCES: TRPV1 and TRPA1 are widely expressed in the human oropharynx with two distinct patterns. Our study further confirms that TRPV1/A1 receptors are promising therapeutic targets to develop active treatments for OD patients.

Proton pump (H+/K+-ATPase) expression in human larynx.

OBJECTIVE: The goal of our study was to investigate and to identify the existence of proton pump in different parts of larynx. The presence of acidic content in this area is known to be connected to several laryngeal diseases. It is mostly developed by upward recurrence of acidic gastric content, but there are some signs that the acid can be produced in the larynx as well, because of the proton pump activity in laryngeal mucosa. METHODS: The study was performed on two types of specimens: (1) 50 cadaver larynges and (2) 11 surgical larynges obtained after laryngectomy. Samples were taken from supraglottis, glottis and subglottic areas and immunohistochemistry for the beta subunit of the proton pump was done. RESULTS: The presence of proton pump was proved in seromucous glands in laryngeal supraglottic area, but it was also, for the first time, found in human chondrocytes in the thyroid and epiglottic cartilage. CONCLUSION: These new findings could encourage further research that would illuminate better the etiopathogenesis not only of laryngopharyngeal reflux, but also the pathophysiology of cartilaginous disorders.

Supraglottic laryngeal tumor microenvironmental factors facilitate STAT3 dependent pro-tumorigenic switch in tumor associated macrophages to render utmost immune evasion.

Content of tumor microenvironment (TME) is varied greatly among different types of laryngeal tumors, namely, supraglottic, glottic and subglottic tumors. These three different TMEs shape infiltrating monocytes/macrophages toward M2 genotypes in variable degrees. Results obtained from in vitro studies demonstrated extent of expression of M2 phenotypic features on macrophages was maximum after their exposure to supraglottic laryngeal tumor cell lysates (SLTCL) than glottic or subglottic lysates. Moreover, M2 macrophages generated under influence of SLTCL show less nitric oxide production, greater IL-10: IL-12 ratio and poor antigen presentation. Co-culture of such M2 macrophages with T cells from healthy donors resulted decreased activation of T cells and T cell mediated tumor cell cytotoxicity, than, glottic or subglottic. SLTCL mediated macrophage polarization is STAT3 dependent and might be one of the major factors for severe immune paralysis leading to poor prognosis of supraglottic laryngeal tumor bearer following standard treatment.

The distribution of transient receptor potential melastatin-8 in the rat soft palate, epiglottis, and pharynx.

Immunohistochemistry for transient receptor potential melastatin-8 (TRPM8), the cold and menthol receptor, was performed on the rat soft palate, epiglottis and pharynx. TRPM8-immunoreactive (IR) nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant in the posterior portion of the soft palate and at the border region of naso-oral and laryngeal parts of the pharynx. The epiglottis was free from such nerve fibers. The double immunofluorescence method demonstrated that TRPM8-IR nerve fibers in the pharynx and soft palate were mostly devoid of calcitonin gene-related peptide-immunoreactivity (CGRP-IR). The retrograde tracing method also demonstrated that 30.1 and 8.7 % of sensory neurons in the jugular and petrosal ganglia innervating the pharynx contained TRPM8-IR, respectively. Among these neurons, the co-expression of TRPM8 and CGRP-IR was very rare. In the nodose ganglion, however, pharyngeal neurons were devoid of TRPM8-IR. Taste bud-like structures in the soft palate and pharynx contained 4-9 TRPM8-IR cells. In the epiglottis, the mucous epithelium on the laryngeal side had numerous TRPM8-IR cells. The present study suggests that TRPM8 can respond to cold stimulation when food and drinks pass through oral and pharyngeal cavities.

Pituitary adenylatecyclase-activating polypeptide-immunoreactive nerve fibers in the rat epiglottis and pharynx.

The distribution of pituitary adenylatecyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers was studied in the rat epiglottis and pharynx. PACAP-IR nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant on the laryngeal side of the epiglottis and in the dorsal and lateral border region between naso-oral and laryngeal parts of the pharynx. PACAP-IR nerve fibers were also detected in taste buds within the epiglottis and pharynx. In addition, many PACAP-IR nerve fibers were found around acinar cells and blood vessels. The double immunofluorescence method demonstrated that distribution of PACAP-IR nerve fibers was similar to that in CGRP-IR nerve fibers in the epithelium and taste bud. However, distributions of PACAP-IR and CGRP-IR nerve fibers innervating mucous glands and blood vessels were different. The retrograde tracing method also demonstrated that PACAP and CGRP were co-expressed by vagal and glossopharyngeal sensory neurons innervating the pharynx. These findings suggest that PACAP-IR nerve fibers in the epithelium and taste bud of the epiglottis and pharynx which originate from the vagal and glossopharyngeal sensory ganglia include nociceptors and chemoreceptors. The origin of PACAP-IR nerve fibers which innervate mucous glands and blood vessels may be the autonomic ganglion.

Connexins within the rat larynx.

OBJECTIVES/HYPOTHESIS: To determine the types and localization of connexins within the rat larynx. STUDY DESIGN: Quantitative real time polymerase chain reaction (qRT-PCR) of the epiglottis and laryngeal mucosa was used to identify connexins (Cx). Immunohistochemical labeling was then used to localize the Cxs within the larynx. METHODS: Twelve larynges from 3 to 4 month old Fisher-344 rats were used. RNA was extracted (N = 8) and cDNA produced. Primers for Cx26, Cx30, Cx32, Cx37, Cx40, and Cx43 were added and qRT-PCR performed. Others larynges were serially sectioned for immunohistochemistry. RESULTS: qRT-PCR revealed Cx43, Cx32, and Cx30 within the epiglottis and Cx43 in the vocal folds and Cx43 and Cx32 within the subglottic mucosa. Immunohistochemical staining confirmed these results. CONCLUSION: The rat epiglottis is rich in Cx43, Cx32, and Cx30 whereas the vocal folds contain Cx43 and the subglottic mucosa Cx43 and Cx32. Their localizations suggest involvement in secretion for protective purposes and they may play a key role in laryngeal pathoses.

Age-dependent increases of calcium and phosphorus in human epiglottal cartilage.

To elucidate compositional changes of the elastic cartilage with aging, the authors investigated age-related changes of elements in the epiglottal cartilages by inductively coupled plasma-atomic emission spectrometry. After the ordinary dissection by medical students at Chiang Mai University was finished, the epiglottises were resected from the subjects. The epiglottal cartilages were isolated and the element contents were determined. The subjects consisted of 11 men and 14 women, ranging in age from 39 to 92 yr old. It was found that although the extent of accumulation of calcium and phosphorus was slight, calcium and phosphorus increased progressively in the epiglottal cartilages with aging. In contrast, sulfur, magnesium, zinc, iron, and sodium did not change significantly in them. Regarding the relationships among elements, it was found that there were significant correlations among calcium, phosphorus, magnesium, and sodium in the epiglottal cartilages, with one exception between calcium and sodium contents. In comparison between men and women, no significant differences were found in the predominant elements such as calcium, sulfur, and phosphorus in the epiglottal cartilages.

Immunohistochemical study on distribution of mast cell phenotypes in human laryngeal mucosa: evidence for laryngeal type I allergy.

It is controversial whether or not type I allergic reactions can occur in the human laryngeal mucosa. To clarify this issue, we studied the distribution of mast cell phenotypes in the human laryngeal mucosa using the immunohistochemical staining method with antihuman tryptase and chymase antibodies. A large number of mast cells are present in the superficial layer of subepithelial connective tissue (SECT) of the epiglottis, arytenoid, and subglottis. Although mast cells containing both tryptase and chymase are predominant in the deep layer of the SECT, the majority of mast cells containing tryptase alone are located in both the epithelial layer and the superficial layer of the SECT. We conclude that the human laryngeal mucosa has the potential to induce type I allergic reaction.

Effects of various ion transport inhibitors on the water response in the superior laryngeal nerve in rats.

The effects of inhibitors [acetazolamide, an inhibitor of carbonic anhydrase; amiloride, an inhibitor of the Na channel; furosemide, an inhibitor of the Na/K/2Cl transporter; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the Cl channel] on the water response in the superior laryngeal nerve (SLN) were investigated using whole nerve recordings from the SLN of anesthetized and paralyzed rats. Changes in spontaneous activity in the SLN after i.v. injection of a hypo- or hypertonic solution were also investigated. The water response to higher concentration amiloride solutions (0.1, 1, 5 and 10 mM) were significantly smaller in comparison with the control, i.e. the water response to deionized water (88-59% of the control, Fisher's PLSD, P < 0.05). DIDS suppressed the water response significantly at concentrations of 0.5 and 2 mM by 18 and 33%, respectively (P < 0.05). Likewise, acetazolamide (2 mM) and furosemide (5 mM) significantly suppressed the water response by 9 and 40%, respectively (P < 0.05). An i.v. bolus injection of a hypertonic solution (1 ml of 1.5 M NaCl or 1.0 M mannitol) depressed spontaneous activity of the SLN. In contrast, an i.v. injection of a hypotonic solution (0.015 M NaCl) increased spontaneous activity. These results suggest that several ion transporters and ion channels, as well as carbonic anhydrase, that may exist in the dorsal surface in the epiglottis may regulate the water response in the SLN and that osmotic changes in the dorsal surface of the epiglottis and in the interstitial space can affect nerve activity in the SLN.

Glycoconjugate in rat taste buds.

The taste buds of the fungiform papillae, circumvallate papilla, foliate papillae, soft palate and epiglottis of the rat oral cavity were examined by lectin histochemistry to elucidate the relationships between expression of glycoconjugates and innervation. Seven out of 21 lectins showed moderate to intense staining in at least more than one taste bud. They were succinylated wheat germ agglutinin (s-WGA). Dolichos biflorus agglutinin (DBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I) and Phaseolus vulgaris agglutinin-L (PHA-L). UEA-I and BSL-I showed moderate to intense staining in all of the taste buds examined. They strongly stained the taste buds of the epiglottis, which are innervated by the cranial nerve X. UEA-I intensely stained the taste buds of the fungiform papillae and soft palate, both of which are innervated by the cranial nerve VII. The taste buds of circumvallate papilla and foliate papillae were innervated by the cranial nerve IX and strongly stained by BSL-I. Thus, UEA-I and BSL-I binding glycoconjugates, probably alpha-linked fucose and alpha-D-galactose, respectively, might be specific for taste buds. Although the expression of these glycoconjugates would be related to the innervation of the cranial nerve X, the differential expression of alpha-linked fucose and alpha-D-galactose might be related to the innervation of the cranial nerve VII and IX, respectively.

[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.

[Scanning electron microscopy studies concerning the epiglottis epithelia of rats (author's transl)]. / Rasterelektronenmikroskopische Studien zur Feinstruktur des Epiglottisepithels von Ratten.

The epithelial surface of the epiglottis in adult male and female albino rats is studied by scanning electron microscopy. Prominent differences exist between the surface morphology as observed in the males and that observed in the females sacrificed in the estrus phase of the ovarial cycle. The differences are most pronounced in respect to the amount of secretory vesicles, microvilli- and microridge-cells and in respect to the observable number of desquamating cells. The existence and the importance of the microridge-cells are discussed. Special attention is also paid to the results indicating that at least some parts of the extrapulmonary airway epithelia act as possible target organs for sex hormones. Finally the importance of the observed differences is discussed for the correct interpretation of pathomorphological alterations in the extrapulmonary airway epithelia that have been observed under experimental conditions and during infections.