Effects of cigarette smoke on Haemophilus influenzae-induced otitis media in a rat model.

Exposure to cigarette smoke (CS) is a factor that could delay or worsen the recovery of otitis media (OM) by causing inflammatory swelling of the Eustachian tube (ET). However, despite the suggested relationship, little is known about the association between OM and CS. Therefore, we aimed to evaluate the effects of CS on the development, progression, and recovery of OM, as well as the histological and molecular changes caused by CS exposure, by using a rat model of OM infected with non-typeable Haemophilus influenzae (NTHi). Eighty Sprague-Dawley rats with normal middle ears (MEs) were divided into four groups (n = 20 rats/group): control, CS, OM, and CS + OM. The CS and CS + OM groups were exposed to CS for 2 weeks. The inflammatory reaction to NTHi was more intense and lasted longer in the CS + OM group than in the other groups. Goblet cell proliferation and mucus secretion in the ET were more significant in the CS and CS + OM groups than in the other groups. These findings suggested that because CS directly affects the ET and ME mucosa, bacterial OM can become more severe and may resolve more slowly in the presence of CS exposure rather than in its absence.

[Observation of mucosa of eustachian tube with scanning electron microscope on spontaneous otitis media in mice].

OBJECTIVE: To investigate the ultrastructural changes of the mucosa of eustachian tube in mice and to reveals the influence of eustachian tube on middle ear function and its relavence with otitis media. METHOD: 12 wild type and 12 mutant mice were divided into two groups by age to observe the the ultrastructural changes of the mucosa of eustachian tube. RESULT: Wild type mice exhibited a thick lawn of morphologically normal, distributed cilia in the mucosa of the middle ear at both time points. The cilia of mucosa of middle ear in mutant mice were short, impaired and disrupted. The impairment of the cilia progressed to a much great severity at 6 months compared to 3 months. CONCLUSION: Otitis media occurs not only the ciliated cells decreased and the goblet cells increased. More importantly, the ciliary structure was damaged, leading to the dysfunction of the mucociliary transport system and causing otitis media.

Otitis media in sperm-associated antigen 6 (Spag6)-deficient mice.

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.

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.

Histologic changes in the auditory tube mucosa of rats after long-term exposure to cigarette smoke.

PURPOSE: The aim of the study was to investigate the effect of cigarette smoke on the auditory tube and middle ear mucosa after long-term exposure (4 and 6 months). MATERIALS AND METHODS: Fifteen rats were divided into 3 groups. The experimental groups were exposed to cigarette in a smoking chamber for 4 and 6 months (n = 5 each). A control group (n = 5) was placed in the same chamber without exposure to cigarette smoke. Histologic changes of the auditory tube mucosa were observed through light and electron microscopes. Histologic changes of the middle ear mucosa were also observed through light microscopes. RESULTS: The histologic changes consisted of a proliferation of goblet cells and an increase of mucus secretion in auditory tube. Squamous metaplasia was paradoxically decreased according to the duration of exposure in auditory tube. The number of goblet cell was gradually increased according to the duration of exposure in the auditory tube and middle ear. CONCLUSIONS: Long-term passive smoke directly affects the auditory tube and middle ear mucosa. Histologic changes of auditory tube mucosa consisted of goblet cell proliferation and excessive mucus secretion.

Protective effect of pulmonary surfactant on cilia of Eustachian tube in otitis media with effusion.

OBJECTIVE: The aim of this study was to evaluate the effect of pulmonary surfactant (PS) on cilia of Eustachian tube (ET) in guinea pigs suffering from otitis media with effusion (OME). METHODS: A nonviable heat-killed pneumococci (HKP) solution was injected into the tympanum of guinea pig via a transtympanic approach therefore establishing models of OME (effusion in tympanum and hearing loss). After being injected with physiologic saline solution or pulmonary surfactant transtympanically, the response threshold of each guinea pig was taken by ABR. Changes of effusion and ciliary ultrastructure were observed. RESULTS: Normal group (A), the response threshold is 14.00+/-3.08 dBHL. Abundant and intact cilia were observed, ciliary morphology was normal, cilia regularly arranged in concord, no goblet cells was observed. Model-control group (B0), 5 days following the inoculation of HKP, serous effusion was present in tympanum. The response threshold is 45.00+/-5.67 dBHL. Cilia lost and arranged irregularly, hyperplasia of the goblet cells was observed. Saline group, the previously observed serous effusion of tympanum was increased or unchanged. The response threshold is 65.50+/-6.85 dBHL. Cilia were coalescing and swelling. A lot of cilia lost and arranged disorderly. A number of compound cilia of the bulging type were formed by cytoplasmic protuberances of ciliated cells. Massive mucoid secretions stuck on the surface of the ET. PS group, the previously observed serous effusion of tympanum was reduced in amount or disappeared completely. The response threshold is 23.53+/-6.32 dBHL. Ciliary morphology was generally normal. Cilia comparatively arrange in concord. A mucus deposits was adhering to the tips of cilia. CONCLUSION: The findings of the study suggest that pulmonary surfactant plays an important role in protecting of cilia of ET of guinea pigs suffering from OME.

Histologic changes in the eustachian tube mucosa of rats after short-term exposure to cigarette smoke.

OBJECTIVE AND BACKGROUND: It is generally accepted that cigarette smoke is a major risk factor for middle ear disease. However, the literature is void of articles addressing the direct relationship between cigarette smoke exposure and middle ear disease. Furthermore, there are many conflicting opinions concerning the role of cigarette smoke in the pathogenesis of middle ear disease. The purpose of this study was to evaluate the effects of cigarette smoke on the Eustachian tube mucosa. MATERIALS AND METHODS: Thirty healthy 150 to 230 g Sprague Dawley rats with normal middle ears were used. The animals were divided into six groups of five. Five experimental groups (N=5 each) were exposed to a domestic cigarette (This, tar 7.0 mg, nicotine 0.75 mg) every 30 minutes (total 2.5 hours, 5 cigarettes total) on a daily basis in a smoking chamber for 1, 2, 4, 6, or 8 weeks. A control group (N=5) was placed in the same chamber without exposure to cigarette smoke. After exposure, the animals were sacrificed and cross sections of the Eustachian tubes were prepared. Histologic changes of the Eustachian tube mucosa were observed through light and electron microscopes. RESULTS: Loss of cilia, goblet cell depletion, and squamous metaplasia of the Eustachian tube mucosa were observed following exposure to smoke. The one- and two-week exposure groups demonstrated the greatest decrease in goblet cell counts. The eight-week exposure group showed recovery from this decrease. Squamous metaplasia was observed in all experimental groups and was most prominent in the eight-week exposure group. CONCLUSION: These findings suggest that cigarette smoke directly affects Eustachian tube mucosa in the early stages of exposure. Some of the mucosal changes, however, were reversed during the latter stages of exposure. A mechanism different from that which occurs in the nasal cavity and trachea may be activated in the Eustachian tube after exposure to passive smoke. The protective function of the Eustachian tube may play some role in this mechanism.

[Histopathological and ultracytochemical observation of mucosa on the eustachian tube and middle ear with experimental secretory otitis media].

OBJECTIVE: To further explore the pathogenesis of the secretory otitis media and provide basis for its clinical treatment. METHOD: A model of otitis media with effusion was established by coagulating the pharyngeal orifice of the eustachian tube under an endoscope with microwave. Orifice and its cartilage-mucosa of the eustachian tube and the mucosa in the middle ear were observed and compared, dark-granulated cells near the middle ear and surfactant-like lamellar bodies were observed. RESULT: Under light microscope, the eustachian tube in all the specimens were unblocked. The thick-measurements and pathology observations of the cartilage-mucosa on eustachian tube with otitis media revealed that there were no obviously inflammatory change and no significantly difference in comparison with normal control group. In all the pathologic groups, no surfactant-like lamellar bodies were found in the secretory cells, which showed impaired characters with little secretory granules. CONCLUSION: The cartilage mucosa on eustachian tube is possessed of very strong function of natural defence for inflammatory disease. Surfactant-like substance are important in maintaining the physiological function of the eustachian tube. These studies provide a basis for further experimental research and clinical treatment for secretory otitis media.

Development of the tubotympanum in the rat.

OBJECTIVE: To investigate the relationship between the anatomical maturation of the middle ear and that of the eustachian tube and paratubal muscles in the rat. DESIGN: Wistar rats ranging from gestational day 12 to postnatal day 40 were used. METHODS: Tissue specimens were examined with routine light microscopy and electron microscopy. Epithelial differentiation was studied immunohistochemically with antibodies to different cytokeratins. RESULTS: The epithelial lining of the tubotympanum showed differentiation-related cytokeratin expression throughout the whole developmental period. The mucociliary epithelium reached mature features around birth. A dorsal extension and its framing cartilage started forming around 5 days after birth. This extension became lined by stratified nonciliated epithelium and attained maturity around 10 days after birth concurrently with the attachment of the dilatory muscles. This process was immediately followed by aeration of the middle ear cavity. CONCLUSIONS: The continuous expression of cytokeratins demonstrates that the epithelial lining of the tubotympanum is only derived from the embryonal endoderm. Furthermore, this study demonstrates that the eustachian tube shows a two-stage postnatal development. First, the mucociliary system matures, providing protection/clearance when the animal starts respiration and swallowing. Subsequently, the dorsal part attains maturity. The features of the epithelial lining of the dorsal part of the eustachian tube and the coincidence of the maturation of this part with the attachment of the dilating muscle fibers and the aeration of the middle ear indicates that this part provides ventilation. These findings support the authors' hypothesis that different parts of the eustachian tube serve different purposes: clearance, protection and ventilation.

Induction of temporary otitis media in specific-pathogen-free pigs by intratympanic inoculation of Mycoplasma hyorhinis.

OBJECTIVE: To examine whether Mycoplasma hyorhinis inoculated into the tympanic cavity can cause otitis media in pigs. ANIMALS: 17- or 22-day-old specific-pathogen-free pigs. PROCEDURE: Histologic and bacteriologic examinations were performed on specimens from the tympanic cavity and auditory tube at 0, 7, 14, and 25 days after intratympanic inoculation of M hyorhinis (auditory tube cloning strain 14). RESULTS: In M hyorhinis-inoculated pigs, mild to moderate inflammation of the auditory tube and tympanic cavity first appeared at postinoculation day (PID) 7. In pigs euthanatized at PID 14, the degree of inflammation was aggravated. Immunohistochemical analysis revealed M hyorhinis antigens on the luminal surface of the auditory tube and tympanic cavity. By PID 25, lesions had lessened. By use of transmission and scanning electron microscopic examinations, mycoplasmal organisms were identified among the cilia in the auditory tubes at PID 14 but not at PID 25. Results of bacteriologic examination indicated that 10(4) to 10(6) color-changing units of M hyorhinis were isolated from the tympanic cavity at PID 0. Variable numbers of M hyorhinis were isolated at PID 7 and 14, and numbers were decreased at PID 25. CONCLUSIONS: M hyorhinis inoculated into the tympanic cavity can cause a self-limiting otitis media in SPF pigs.

Scanning electron microscopic view of auditory tonsil in turkey.

The auditory tonsil of the turkey was studied by scanning electron microscopy. It is a sizable mass of lymphatic tissue dorsal to the infundibular opening in the auditory tube. The infundibular opening is in the roof of the turkey's mouth, caudal to the nasal cleft, and is the external orifice of the auditory tube that connects with the air spaces of the pneumonic bones in the head. Folds of the auditory tonsil that project toward or into the infundibular opening contained numerous lymphatic nodules in their lamina propria. The epithelial surface of the folds was covered with ciliated and nonciliated columnar cells, goblet cells with microvilli, and ductal openings of mucous glands located in the area. The lymphoid nodules were either oval or round and consisted of a thick reticular network that contained lymphocytes, fibroblasts, and erythrocytes. From some of the lymphatic nodules, there were sinusoids that contained lymphocytes and extended to the epithelial surface, whereas from others there was a lymphocytic infiltration of the surrounding lamina propria.

Kinetics of the ascension of NTHi from the nasopharynx to the middle ear coincident with adenovirus-induced compromise in the chinchilla.

To determine the kinetics of ascension of the eustachian tube (ET) by non-typeable Haemophilus influenzae (NTHi) in situ from the nasopharynx to the middle ear using an experimental model of otitis media (OM), we examined snap-frozen sections of chinchilla ET and middle ear mucosa for adherent bacteria over a 14 day time period. Via fluorescent- and transmission electron-microscopy, we found that NTHi preferentially adhered not to the epithelial cells but to the mucus in the ET and gradually ascended this tubal organ, reaching the middle ear approximately 10 days after intranasal inoculation of adenovirus-infected animals. The number of NTHi adherent to mucus at the pharyngeal portion of the ET increased significantly in the first 4 days after inoculation of the nares whereas the number of adherent bacteria in both the mid and tympanic portions of the ET increased more gradually over time. NTHi were not observed in the middle ear until approximately 7-10 days after inoculation of the nares which was coincident with the onset of clinical signs of OM. These data confirmed our earlier in vitro investigation which suggested that adherence to and growth within stagnant mucus within a ET compromised by adenovirus was a possible mechanism by which NTHi, resident in the nasopharynx, might gain access to the middle ear and induce OM.

Roxythromycin prevents endotoxin-induced otitis media with effusion in the guinea pig.

Pharmacological agents that can normalize or enhance the ciliary and mucociliary activity of the tubotympanum should also be able to break the vicious circle of chronic otitis media with effusion (OME). Roxythromycin (RXM) has been shown to enhance the ciliary activity in vitro and also stimulate the mucociliary activity in vivo and may therefore, when clinically applied, prevent not only occurrence but also recurrence of clinical OME. The present study was designed to discuss the possible preventive effect of RXM on endotoxin-induced OME in the guinea pig. A total of 120 guinea pigs were used. The normal control group was treated with intratympanic injection of 0.1 ml of physiologic saline solution. The saline-control group was treated with oral administration of physiologic saline solution for 14 successive days. The low-dosage group and the high-dosage group were treated with oral administration of 5 and 50 mg/kg of sairei-to for 14 successive days, respectively. Then, the saline-control group, the low-dosage group and the high-dosage group were treated with intratympanic injection of 0.1 ml of lipopolysaccharide solution (100 micrograms/ml) derived from Klebsiella pneumoniae. All 10 animals in the four groups were sacrificed 1, 3, and 7 days after the intratympanic injection, to examine ciliary activity, mucociliary clearance time, and mucosal pathology of the tubotympanum. The saline-control group exhibited middle ear effusions and pathologies similar to human OME. The incidence of middle ear effusions was significantly reduced in the low-dosage and high-dosage groups. Throughout the observation period, the ciliary activity in the tubotympanum was significantly reduced in the saline-control group as compared with that of the normal control group. By contrast, the ciliary activity in the low-dosage and high-dosage groups was not so reduced in the Eustachian tube and the middle ear close to the orifice. Mucociliary clearance time in the low-dosage and high-dosage groups was not different from that in the normal control group throughout the period. The tubotympanum in the saline-control group exhibited mucosal pathologies characteristic of OME in human. By contrast, the low-dosage and high-dosage groups exhibited much milder pathological changes in the tubotympanum than those in the saline-control group. In conclusion, clinical application of RXM could be an effective measure to prevent the occurrence of OME and also the recurrence of the disease, especially in OME-prone individuals.

Ultrastructure of the nasopharyngeal orifice epithelium of the eustachian tube in otitis media with effusion.

Epithelial changes in nasopharyngeal orifice of eustachian tube in 15 patients with otitis media with effusion (OME) were studied. Ultrastructural examination of the epithelium revealed distinct alterations in the ciliated cells, intermediate cells and in the columnar cells with microvilli. The ciliated cells were the predominant cell type in the epithelium and were characterized by compound cilia and apical cytoplasmic bulgings with fine granular content. The intermediate cells showed more prominent lateral cytoplasmic bulgings. Cytoplasmic bulgings of both cell types eventually pinched off and set free as cytoplasmic bodies, similar to the cytoplasmic bodies derived from lymphocytes. As a result of epithelial destruction, the lumen of nasopharyngeal orifice was occupied by epithelial cellular debris among which leucocytic cells and cytoplasmic bodies with fine granular content. This accumulation in the lumen probably developed as a result of defective mucociliary activity which is due to compound cilia formation in the ciliated cells. Moreover, ultrastructural resemblance of cytoplasmic bodies derived from ciliated cells, intermediate cells and leucocytic cells indicates the possible role of these cells in common immune defence mechanisms in chronic otitis media with effusion.

Ultrastructure of eustachian tube mucosa in chronic otitis media with cholesteatoma.

PURPOSE: Acquaintance with the severity of inflammatory condition and mucociliary destruction of the protympanic eustachian tube (ET) mucosa may provide some insight for management during surgery for cholesteatoma. MATERIALS AND METHODS: Protympanic eustachian mucosa was obtained from 25 patients undergoing ear surgery for cholesteatoma and from 5 cadavers undergoing dissection for a gross anatomy course. The specimens were processed for both scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: According to SEM, decreased ciliated cells were noted. Residual ciliated cells and numerous white blood cells (WBC) were noted in the protympanic mucosa of the eustachian orifice. Bacilli were commonly noted at the mucosal surface. Cilia might be collapsed and stagnated with mucus secretion. This finding was consistent with those of the TEM: that cilia of varied directions were embedded in mucus. Mucociliary function was impaired under such conditions. The mucosal alteration was severe in some children; there was no ciliated cell visible. According to the TEM, the mucosal cells contained numerous secretory granules and mitochondria. Polymorphous nuclear cells were noted in the submucosal area that was edematous. Compound cilia were rather common. Mucus blanket covered mucosal cells with widening intercellular spaces. In addition, neither cilia nor microvilli were visible in severe mucosal alteration. CONCLUSIONS: These occurrences indicated the presence of inflammatory reaction and impairment of clearance function of eustachian mucosa in cholesteatomatous ears. Thorough irrigation during surgery and antimicrobial therapy after surgery are warranted.

[The ultrastructure of the Eustachian tube].

To observe the ultrastructure of mucous membrane of Eustachian tube in human being, ninteen human's Eustachian tube from various ages, were divided into tympanic segment, isthmus and pharyngeal segment and observed under SEM. The results showed that the epithelium of Eustachian tube consisted of ciliated cells, nonciliated cells and goblet cells. At the isthmus, the bone and cartilage junction, the cells got crowded, and there were more and more goblet cells from tympanic side to pharyngeal side, as well as the cilia of ciliated cell leaned towards the pharyngeal side. The upper one third part of Eustachian tube was covered mostly by nonciliated cells, the floor and lower parts were ciliated cells. It was concluded that the upper parts of Eustachian tube's wall played an important role in maintaining equation of pressure in middle ear, and the floor and lower parts in providing an active mucociliary mechnism. The defence system of the Eustachian tube mucous membrane had developed during the fetal stage and was well established at birth.

Effect of oxatomide on otitis media with effusion--an experimental study.

The therapeutic effect of oxatomide on experimental otitis media with effusion (OME) induced by secondary immune response was investigated in chinchillas. Oxatomide was administered daily in three different doses (5, 10, 30 mg/kg b.w.t.) for a period of 2 weeks, starting 2 days after the instillation of keyhole limpet hemocyanin (KLH) into the tympanic cavity. The severity of OME was assessed by observing the eardrum under a surgical microscope, by tympanography, and by histological findings. The concentrations of leukotriene C4 and LTD4 in middle ear effusion (MEE) were also measured, 7 days after the KLH instillation. It was found that oral administration of oxatomide in dosages of 10 and 30 mg/kg alleviated the experimental OME. The mean LTD4 concentration measured in MEE from chinchillas given 30 mg oxatomide was significantly lower than that measured in the control group. The radioactivity of 14C-labelled oxatomide in MEE and the autoradiographic findings revealed that orally administered oxatomide was rapidly absorbed from the digestive tract and transported to the middle ear via blood circulation. The findings suggest that the effect of oxatomide can be anticipated in patients with OME.

[Ultracytochemical observation of secretory cells and surfactant-like lamellar bodies in mucosa of eustachian tube].

The distribution of secretory cells and surfactant-like lamellar bodies in mucosa of the guinea pig eustachian tube were studied ultracytochemically. Classified by means of their morphologic characteristics, three types of secretory cells were identified. The dark granulated cells were predominant in the tympanic orifice, the mixed cells were predominant in the isthmus portion, while the light cells were predominant in the pharyngeal orifice. The distribution of the secretory cells in different part of the eustachian tube might play a role in the pathogenesis of middle ear effusions. Surfactant-like lamellar bodies were found in ciliated cells, nonciliated cells and secretory cells of the eustachian tube mucosa. Surfactant-like lamellar bodies (surfactant-like substance) are important in maintaining the physiological function of the eustachian tube, their behavior in normal and pathological states should be further studied.

Temporal bone pathology of leukemia and malignant lymphoma with middle ear effusion.

This report describes the histopathological findings of the middle ear in patients with leukemia and malignant lymphoma whose middle ear effusion (MEE) was observed from temporal bone specimens. There were 19 patients (35 ears) with leukemia and 8 patients (16 ears) with malignant lymphoma. MEE was observed in 8 of 35 ears with leukemia, and 5 of 16 ears with malignant lymphoma. In 8 ears with leukemia with MEE, pathological changes included effusion containing floating tumor cells in 4 and tumor infiltration around the Eustachian tube in 3. In 1 of 5 ears with malignant lymphoma with MEE, MEE was accompanied by tumor infiltration. Infection was present in 13 ears of patients with leukemia and malignant lymphoma. It is suggested that there are various causes, including infection, obstruction of the Eustachian tube with tumor infiltration and a tendency to bleed, of MEE in leukemia and malignant lymphoma.