Utilization potential of intraluminal optical coherence tomography for the Eustachian tube.

Imaging the Eustachian tube is challenging because of its complex anatomy and limited accessibility. This study fabricated a fiber-based optical coherence tomography (OCT) catheter and investigated its potential for assessing the Eustachian tube anatomy. A customized OCT system and an imaging catheter, termed the Eustachian OCT, were developed for visualizing the Eustachian tube. Three male swine cadaver heads were used to study OCT image acquisition and for subsequent histologic correlation. The imaging catheter was introduced through the nasopharyngeal opening and reached toward the middle ear. The OCT images were acquired from the superior to the nasopharyngeal opening before and after Eustachian tube balloon dilatation. The histological anatomy of the Eustachian tube was compared with corresponding OCT images, The new, Eustachian OCT catheter was successfully inserted in the tubal lumen without damage. Cross-sectional images of the tube were successfully obtained, and the margins of the anatomical structures including cartilage, mucosa lining, and fat could be successfully delineated. After balloon dilatation, the expansion of the cross-sectional area could be identified from the OCT images. Using the OCT technique to assess the Eustachian tube anatomy was shown to be feasible, and the fabricated OCT image catheter was determined to be suitable for Eustachian tube assessment.

Cilia distribution and polarity in the epithelial lining of the mouse middle ear cavity.

The middle ear conducts sound to the cochlea for hearing. Otitis media (OM) is the most common illness in childhood. Moreover, chronic OM with effusion (COME) is the leading cause of conductive hearing loss. Clinically, COME is highly associated with Primary Ciliary Dyskinesia, implicating significant contributions of cilia dysfunction to COME. The understanding of middle ear cilia properties that are critical to OM susceptibility, however, is limited. Here, we confirmed the presence of a ciliated region near the Eustachian tube orifice at the ventral region of the middle ear cavity, consisting mostly of a lumen layer of multi-ciliated and a layer of Keratin-5-positive basal cells. We also found that the motile cilia are polarized coordinately and display a planar cell polarity. Surprisingly, we also found a region of multi-ciliated cells that line the posterior dorsal pole of the middle ear cavity which was previously thought to contain only non-ciliated cells. Our study provided a more complete understanding of cilia distribution and revealed for the first time coordinated polarity of cilia in the epithelium of the mammalian middle ear, thus illustrating novel structural features that are likely critical for middle ear functions and related to OM susceptibility.

Histological identification of nasopharyngeal mechanoreceptors.

The auditory tube plays a fundamental role in regulating middle ear pressure. A "system" sensitive to a pressure gradient between the middle ear and the ambient environment is necessary. The presence of mechanoreceptors in the middle ear and the tympanic membrane has been studied, but the presence of these receptors in the nasopharyngeal region remains unclear. The aim of this study is to confirm the presence of pressure sensitive corpuscles in the nasopharynx. An experimental study was conducted on five fresh and unembalded human cadavers. The pharyngeal ostium of the auditory tube and its periphery was removed in one piece by video-assisted endonasal endoscopy. Samples were fixed in formaldehyde solution, embedded in paraffin, and cut. Slides were analyzed by HES (Hematoxyline Eosine Safran) coloration, by S100 protein and neurofilament protein immunostaining. Encapsulated nerve endings were researched and identified by slides analysis. Eight samples were included in our study. On seven samples, Ruffini corpuscles were identified in the mucosa of the posterior area of the pharyngeal ostium, with a higher concentration in the pharyngeal recess and in the posterior nasopharyngeal wall. Our study identified nasopharyngeal mechanoreceptors that could detect the nasopharyngeal pressure and, by extension, the atmospheric pressure. These findings support the theory of the neuronal reflex arc of isobaric system of the middle ear, based on the existence of a "system" sensitive to a pressure gradient between the middle ear and the ambient environment. Understanding of this system has been helpful in the diagnosis and management of middle ear diseases.

Morphology and ciliary motion of mucosa in the Eustachian tube of neonatal and adult gerbils.

The Eustachian tube is a small canal that connects the tympanic cavity with the nasal part of the pharynx. The epithelial lining of the Eustachian tube contains a ciliated columnar epithelium at the tympanic cavity and a pseudostratified, ciliated columnar epithelium with goblet cells near the pharynx. The tube serves to equalize air pressure across the eardrum and drains mucus away from the middle ear into the nasopharynx. Blockage of the Eustachian tube is the most common cause of all forms of otitis media, which is common in children. In the present study, we examined the epithelial lining of the Eustachian tube in neonatal and adult gerbils, with a focus on the morphological and functional development of ciliated cells in the mucosa. The length of the tube is ∼8.8 mm in adult gerbils. Scanning electron microscopy showed that the mucosal member near the pharyngeal side contains a higher density of ciliated cells and goblet cells than that near the tympanic side. The cilia beat frequency is 11 Hz. During development, the length of the Eustachian tube increased significantly between postnatal day 1 (P1) and P18. Scanning electron microscopy showed that the mucosa contained a high density of ciliated cells with a few goblet cells at P1. The density of ciliated cells decreased while the density of goblet cells increased during development. At P18, the mucosa appeared to be adult-like. Interestingly, the ciliary beat frequency measured from ciliated cells at P1 was not statistically different from that measured from adult animals. Our study suggests that the Eustachian tube undergoes significant anatomical and histological changes between P1 and P18. The tube is morphologically and functionally mature at P18, when the auditory function (sensitivity and frequency selectivity) is mature in this species.

Cholinergic chemosensory cells in the auditory tube.

The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(ß2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCß2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.

Localization of aquaporins, water channel proteins, in the mouse eustachian tube.

CONCLUSION: Immunolocalization of the subtypes of water channel proteins, aquaporins (AQPs), was detected in the mouse eustachian tube (ET). AQPs are located continuously from the serous glands and the capillary vessels to the luminal side in the ET epithelium and may play an important role in the transportation of water to the surface of the ET lumen through the epithelium. OBJECTIVES: Although the water supply to the surface of the ET lumen is considered to be essential for closing of the ET, the pathway of the water in the ET is not fully understood. Since AQPs, a group of water transport proteins, have been reported to regulate water homeostasis, we examined the location of AQPs in the mouse ET. MATERIALS AND METHODS: Nine subtypes of AQPs were examined in paraffin embedded ETs of adult mice using the avidin-biotin peroxidase complex method of immunohistochemistry. RESULTS: Four subtypes of AQPs were detected in the mouse ET. AQP-1 was detected in fibroblasts, endothelial cells of capillary vessels and cartilage cells. AQP-3 was distinctly detected in the basal membrane of epithelial cells. AQP-4 was detected in the basal membrane of epithelial cells. AQP-5 was expressed in the luminal side of the ET epithelial cells and also in the apical surface of the cells of the serous glands.

Glycoprofile of the different cell types present in the mucosa of the horse guttural pouches.

Histochemical characterization of the equine guttural pouches was performed using lectins combined with sialidase digestion and deglycosylation pre-treatments. The goblet cells contained O- and N-linked oligosaccharides with alpha-Fuc, GlcNAc moieties whereas beta-GalNAc, beta-Gal-(1-3)-GalNAc, beta-Gal-(1-4)-GlcNAc and alpha-Gal residues belonged only to O-linked glycoproteins. The acinar and ductal cells expressed alpha-Man/alpha-Glc in N-linked oligosaccharides, GlcNAc in both O- and N-glycoproteins and beta-GalNAc, beta-Gal-(1-3)-GalNAc, beta-Gal-(1-4)-GlcNAc and alpha-Gal residues included in O-linked glycoproteins. The Golgi area of the epithelial lining expressed alpha-Fuc in O-linked glycoproteins, internal GlcNAc in N-linked glycoproteins and large amounts of sialic acid residues linked to subterminal beta-GalNAc, Galbeta1,4GlcNAc and Galbeta1,3GalNAc. High amounts of sulpho-carbohydrates and of sialic acids (alpha2,3-6), linked to-alpha/beta-Gal and sialic acids (alpha2-6) linked to beta-GalNAc, were also demonstrated. Such diversity of the mucin saccharide residues may be implicated in the binding of macromolecules such as those of bacterial or viral etiology, thus playing a role in the organism's host-defense mechanism in the guttural pouches.

Differentiation of glycans in equine guttural pouches.

The aim of this study was to investigate the carbohydrate composition of mucosubstances in the equine guttural pouches using conventional histochemical tests in conjunction with glycolytic digestion to degrade different classes of glycosoaminoglycans. In the goblet cells, the mucopolysaccharides contained chondroitin sulfate B, heparin, heparan sulfate and sialic acid residues. The acinar cells also expressed these substances (except for heparin), whereas the ductal cells produced chondroitin sulfate B and sialic acid. Neutral sugars were also found in each cell type. The diversity of the glycocomponents found in the auditory tube suggests that they may have important functional roles. Indeed, the glycosoaminoglycans provide a hydrophilic environment that prevents dehydration and desiccation of the guttural membranes during air passage. Additionally, these glycomolecules may be involved in the pathogenesis of some bacterial disease in horses, such as equine strangles.

A member of the cathelicidin family of antimicrobial peptides is produced in the upper airway of the chinchilla and its mRNA expression is altered by common viral and bacterial co-pathogens of otitis media.

Cationic antimicrobial peptides (AMPs), a component of the innate immune system, play a major role in defense of mucosal surfaces against a wide spectrum of microorganisms such as viral and bacterial co-pathogens of the polymicrobial disease otitis media (OM). To further understand the role of AMPs in OM, we cloned a cDNA encoding a cathelicidin homolog (cCRAMP) from upper respiratory tract (URT) mucosae of the chinchilla, the predominant host used to model experimental OM. Recombinant cCRAMP exhibited alpha-helical secondary structure and killed the three main bacterial pathogens of OM. In situ hybridization showed cCRAMP mRNA production in epithelium of the chinchilla Eustachian tube and RT-PCR was used to amplify cCRAMP mRNA from several other tissues of the chinchilla URT. Quantitative RT-PCR analysis of chinchilla middle ear epithelial cells (CMEEs) incubated with either viral (influenza A virus, adenovirus, or RSV) or bacterial (nontypeable H. influenzae, M. catarrhalis, or S. pneumoniae) pathogens associated with OM demonstrated distinct microbe-specific patterns of altered expression. Collectively, these data showed that viruses and bacteria modulate AMP messages in the URT, which likely contributes to the disease course of OM.

[Endoscopic examinations of the eustachian tube]. / Endoskopische Untersuchungen der Eustachi-Röhre.

BACKGROUND AND OBJECTIVE: Endoscopy of the eustachian tube allows direct examination of endoluminal alterations in the course of the organ. The aim of this study was to describe anatomic and functional findings in healthy awake subjects. PATIENTS/METHODS: Ten eustachian tubes in six healthy individuals with no history of tube dysfunction were examined under local anesthesia using different types of rigid and flexible endoscopes. RESULTS: Nine of ten tubes presented with no pathologic finding. The motility of the tube cartilage could be visualized in all cases and showed a wide variety. Prior anesthesia of the mucosa with the subject in the supine position, if necessary supplemented by a tube catheter, rendered the procedure more comfortable. CONCLUSION: The eustachian tube shows a wide spectrum of anatomic and functional varieties in normal subjects. The method allows comprehensive evaluation of anatomic and functional stenoses of the eustachian tube. The transnasal-transpharyngeal approach allows only limited evaluation of the middle ear structures.

Immortalization of rat eustachian tube epithelial cells by adenovirus 12-simian virus 40 hybrid virus.

The eustachian tube epithelial cells play an important role in the initial pathogenesis of otitis media. In order to study the role of the eustachian tube epithelial cells in the pathogenesis of otitis media, we have established a rat eustachian tube epithelial cell line. The cell line was derived by infecting primary cultures of eustachian tube epithelial cells with the adenovirus 12-simian virus 40 (Adl2-SV40) hybrid virus. The immortalized cells have retained the morphological characteristics of the parental cells and show positive staining with anti-cytokeratin antibodies (a marker for epithelial cells), but not with anti-vimentin antibodies (a fibroblast marker). The cells have been in continuous culture for more than 10 months and have undergone 38 passages. Western blotting and cell staining have confirmed the expression of the SV40 T antigen and p53. Chromosomal analysis indicates that the cell line is aneuploid and derived from male rat epithelial cells. Together, our results suggest that the cell line originated from eustachian tube epithelial cells from a male rat and was successfully immortalized by the Ad12-SV40 virus.

A model of active Eustachian tube function in the rat: effect of modulating parasympathetic innervation.

The effect of altering secretion into the Eustachian tube by modulating cholinergic innervation was studied in the anaesthetized rat. Active properties of the Eustachian tube were determined by measuring the ability of reflex-induced swallowing to equalize against an increased pressure level in the bulla. Reflex-induced swallowing was initiated by electrically stimulating the superior laryngeal nerve. Passive properties of the Eustachian tube were determined by increasing middle ear pressure until the Eustachian tube spontaneously opened. Blocking cholinergic neurotransmission with atropine had no effect on active or passive properties of the Eustachian tube. Potentiating cholinergic neurotransmission with neostigmine significantly impaired the ability of active swallowing to equilibrate middle ear pressure, but had no effect on passive properties of the Eustachian tube. The findings show that cholinergic nerve transmission, most likely from the parasympathetic division of the autonomic nervous system, can influence Eustachian tube function. We hypothesize that this effect is due to changes in surface tension in the Eustachian tube as a result of changes in secretion.

Cytological and microbiological findings in guttural pouch lavages of clinically normal horses with head restraint.

OBJECTIVE: To evaluate the cytological and microbiological contents of guttural pouch washes of ten randomly selected horses restrained so as to prevent them lowering their heads, and to assess the possible effects on the guttural pouch environment in these horses. PROCEDURE: Cytological and microbiological studies were performed on guttural pouch washes of ten clinically normal horses restrained in a standing position so as to prevent them from lowering their heads below normal, as would happen during transportation on long journeys. They were restrained for 12 or 24 h and cytological, bacteriological and mycological findings in guttural pouch washes were recorded. RESULTS: The cytological gradings and neutrophil concentrations of guttural pouch washings were higher in horses that had their heads restrained for a longer period. Washings from these horses were more likely to contain cultivable bacteria and were the only washes yielding potentially pathogenic bacterial species. CONCLUSION: Variation in the cytological differential counts and bacterial cultures of guttural pouch lavages may be found in clinically normal horses which have had their heads restrained in an elevated position for periods from 12 to 24 h. This should be considered when examining this site and care must be taken when interpreting cytology of guttural pouch lavages in samples taken after transportation for more than 12 h. Restriction of head movement could also affect the normal pouch enviroment and predispose it to disease.

Surfactant proteins A and D in Eustachian tube epithelium.

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.

Characterization of mucins in human middle ear and Eustachian tube.

Mucins are important glycoproteins in the mucociliary transport system of the middle ear and Eustachian tube. Little is known about mucin expression within this system under physiological and pathological conditions. This study demonstrated the expression of MUC5B, MUC5AC, MUC4, and MUC1 in the human Eustachian tube, whereas only MUC5B mucin expression was demonstrated in noninflamed middle ears. MUC5B and MUC4 mucin genes were upregulated 4.2- and 6-fold, respectively, in middle ears with chronic otitis media (COM) or mucoid otitis media (MOM). This upregulation of mucin genes was accompanied by an increase of MUC5B- and MUC4-producing cells in the middle ear mucosa. Electron microscopy of the secretions from COM and MOM showed the presence of chainlike polymeric mucin. These data indicate that the epithelium of the middle ear and Eustachian tube expresses distinct mucin profiles and that MUC5B and MUC4 mucins are highly produced and secreted in the diseased middle ear. These mucins may form thick mucous effusion in the middle ear cavity and compromise the function of the middle ear.

Expression and localization of lung surfactant protein B in Eustachian tube epithelium.

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.

Histological and morphometrical studies on the mucosa of the equine guttural pouch (auditory tube diverticulum).

The present study attempted to clarify the characteristics of the guttural pouch mucosa in equines and to evaluate its foreign substance clearance ability. The specimens were collected from nine regions (eight in the guttural pouch mucosa, and one in the nasopharynx mucosa). We first examined the pouch mucosa by light and electron microscopy. We then measured the frequency of goblet cells per 200 epithelial cells, the length of the cilia, the thickness of the epithelial cell layer and lamina propria and statistically analyzed the data. The guttural pouch mucosa consisted of stratified columnar epithelia with brush-like cilia, and there were almost no histological differences between adults and foals. The morphometrical study revealed significant differences in goblet cell frequency (p < 0.001) and the thickness of lamina propria (p < 0.05). By contrast, no statistically significant difference was detected in the length of the cilia or the thickness of the epithelial cell layer. These findings suggest that the guttural pouch mucosa provides foreign substance clearance ability, but that its ability varies among different regions of the epithelium.

Cell biology of tubotympanum in relation to pathogenesis of otitis media - a review.

The sterility of the eustachian tube and tympanic cavity of normal individuals is maintained not only by the adaptive immune system, but also by the mucociliary system and the antimicrobial molecules of innate immunity. Mucin production and periciliary fluid homeostasis are essential for normal mucociliary function and dysfunction of this system is an important risk factor for otitis media. The secreted antimicrobial molecules of the tubotympanum include lysozyme, lactoferrin, beta defensins, and the surfactant proteins A and D (SP-A, SP-D). Defects in the expression or regulation of these molecules may also be the major risk factor for otitis media.

Surfactant protein A and D expression in the porcine Eustachian tube.

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT-PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody-independent protection of the middle ear.

Cytological and bacteriological findings in guttural pouch lavages of clinically normal horses.

Percutaneous washes of the guttural pouches were obtained from two groups of 15 clinically normal horses, one lightly exercised and the other heavily exercised. Microbiological and cytological studies showed a wide variation in the differential cell counts. The cytological pattern of the normal lavages (< 5 per cent neutrophils) was characterised by a large proportion of ciliated columnar epithelial cells, a few non-ciliated cuboidal epithelial cells, and less than 1 per cent monocytes, lymphocytes, and eosinophils. Abnormal lavages (with more than 5 per cent neutrophils) had higher levels of bacterial growth than normal lavages. There were significant differences between the bacterial growth and total cell count, and also between the neutrophil contents of the lavages from the two groups of horses.