Yusuke Esaki

Ciliary activity in the in vitro tubotympanum.

SummaryWe have introduced a tissue culture technique in which the ciliary activity of the cultured tubotympanum can be studied. We have found that ciliated cells of the eustachian tube and middle ear lining continued a beating activity during 168 h of observation. The mean loss of activity of the ciliated cells from the eustachian tube or mucosal sites proximal to the tube was 20% or less, while that of mucosal sites distal to the tube was about 40% even after 168 h of cultivation in our culture medium of RPMI 1640. Our technique of tissue culture is available for studying the long-term effects of various therapeutic agents or adverse factors on the ciliary activity in the tubotympanum.

Mucociliary Disease of the Middle Ear during Experimental Otitis Media with Effusion Induced by Bacterial Endotoxin

Lipopolysaccharide (10 μg/mL) derived from Klebsiella pneumoniae was injected into the middle ear of guinea pigs. The animals were killed painlessly on days 1, 3, and 7 after inoculation, and the mucosal samples from two sites within the tympanic cavity, close to the tympanic orifice and distal to the orifice, were examined for ciliary activity and epithelial morphology. At day 1 and day 3 serous effusion was observed and deterioration of ciliary activity and morphologic changes were observed. No effusion was recognized at day 7, when the ciliary activity in the distal mucosa was still diminished and that in the proximal mucosa had recovered to a normal level. Our data have shown that lipopolysaccharide extracted from K pneumoniae can produce otitis media with effusion in laboratory animals, and dysfunction of cilia due to lipopolysaccharide probably is responsible for the accumulation of middle ear effusion. The mucociliary system is indeed an important defense system and failure of such a system, especially in the mucosa close to the tympanic orifice, can cause the buildup of effusions.

Influenza a Virus-induced Otitis Media and Mucociliary Dysfunction in the Guinea Pig

There is much epidemiologic, clinical and laboratory evidence that viral infection is involved in otitis media with effusion (OME). However, few studies have demonstrated any direct influence of viruses on the tubotympanum. The purpose of this study was to establish the effect of influenza A virus having invaded the tubotympanum and so elucidate the possible mechanism by which this virus contributes to the pathogenesis of OME. Eighty guinea pigs with normal otoscopic findings were inoculated with 0.2 ml suspension of influenza A (3.3 x 10(8) PFU/ml) into the tympanic cavity through the tympanic membrane. To serve as controls, the same number of guinea pigs were injected with 0.2 ml of physiologic saline solution into the tympanic cavity. At 3, 7, 14, and 28 days postinoculation, they were used for examination of the mucociliary function. Middle ear effusions as well as mucociliary dysfunction were observed only in the animals inoculated with the virus. The ciliary activity in the bulla was declined at any time examined. On the other hand, the ciliary activity in the Eustachian tube and the tympanic orifice was slightly lowered between 7 and 14 days, but the level was not different from that of the controls. However, the number of active ciliated cells (showing more than 500 beats/min) was significantly smaller than that of the controls. The mucociliary clearance time of the tubotympanum was more prolonged than that of the controls at 3, 7, and 14 days, and returned to the control level at 28 days. A variety of morphological changes were observed in the tubotympanum treated with the virus. Major pathologies observed included general inflammatory cell infiltration, vacuolation and other degeneration of ciliated cells, and vascular damage and increased vascular permeability. Regeneration of cilia or ciliated cells followed the degeneration, which included an increased number of basal cells and newly formed centrioles. However, the viral infection also affected the epithelial cells with new centrioles. Our study demonstrates that viral infection can evoke mucociliary dysfunction of the tubotympanum and create increased susceptibility to bacteria. Therefore, viral infection may enhance bacterial infectious processes in the tubotympanu thereby contributing to the occurrence of OME.

Effects of Bacterial Endotoxin on the Ciliary Activity in the in Vitro Middle Ear Mucosa

In the present study, we have examined the hitherto unknown effects of long-term exposure to lipopolysaccharide (LPS), possessing the major parts of biological activity of endotoxin, on ciliary activity in the middle ear. Our results show that LPS can affect the ciliary activity in a dose-response fashion: 1) LPS does not impair the ciliary activity up to 168 h if the concentration is 1 ng/ml or less; 2) 10 ng/ml of LPS does not impair ciliary activity in the middle ear close to the tympanic orifice up to 168 h, but can cause reduced activity distal to the orifice after extended exposure (more than 96 h); 3) 100 ng/ml or more of LPS can cause dysfunction of cilia in the tympanic cavity, and in particular 1 microgram/ml or more of LPS can quickly disable the ciliary activity in the middle ear distal to the orifice.

Acute effects of irradiation on middle ear mucosa

Single field, fixed irradiation of bilateral tympanic cavities using 200-kV x-rays was administered to five guinea pigs. The irradiation dose was 30 Gy. They were killed immediately after irradiation, and bilateral middle ear mucosa was examined for ciliary activity and epithelial structure. Significant deterioration of the ciliary activity in the middle ear mucosa was observed, proximal as well as distal to the eustachian tube. Electron microscopy showed various changes in the irradiated middle ear mucosa. The most conspicuous findings were hyperreactivity in secretion, vacuolation of ciliated cells, and stomal edema.

Functional Disorder of Eustachian Tube in Experimental Otitis Media with Effusion following Inoculation of Bacterial Endotoxin

A 10-μg/mL solution of lipopolysaccharide derived from Klebsiella pneumoniae was inoculated into the middle ears of guinea pigs. The animals were killed painlessly on the first, third, or seventh day after inoculation, and the mucosal samples from the bony portion of the eustachian tube were examined for ciliary activity and epithelial morphology. On the first and third days, when middle ear effusions were present, deterioration of ciliary activity and morphologic changes in the mucociliary system were observed. On the seventh day, when middle ear effusions were absent, the ciliary activity had recovered to normal. Our data show that endotoxin extracted from K pneumoniae can produce otitis media with effusion and that dysfunction of cilia caused by endotoxin is a factor responsible for the manifestation of otitis media.

Ciliary activity on adenoids of patients with otitis media with effusion

Ciliary activity of the adenoidal surface in patients with otitis media with effusion (OME) was evaluated by a photoelectric method. Three groups of patients were compared: a non-OME group whose members had experienced no episode of OME; a middle ear effusion (MEE)-negative OME group whose members had an episode of recurrent OME but no MEE during the month preceding the operation; and an MEE-positive OME group whose members were experiencing an episode of recurrent OME with MEEs when tissue was taken for examination. To serve as a comparison, the ciliary activity in the central pharynx of normal guinea pigs was evaluated using the same technique. The ciliary activity in all patients was significantly lower than that in normal guinea pigs. No significant difference in ciliary activity was apparent between the non-OME group and the MEE-negative OME group. However, both were significantly higher than the ciliary activity of the MEE-positive OME patients. The following findings are drawn from our data: ciliary activity on the surface of hypertrophic adenoids is depressed, and patients with OME have reduced ciliary activity compared with those who have no otitis media or those who have otitis media without effusion.

Acute effects of sulfur dioxide exposure on the middle ear mucosa

A variety of atmospheric pollutants are known to depress mucociliary function in the respiratory system. Since the mucociliary function in the middle ear is similar, and the middle ear may be invaded by atmospheric pollutants, we decided to investigate the possible contribution of sulfur dioxide to middle ear effusion. Guinea pigs were exposed for 24 hours to 300 ppm of sulfur dioxide or air. Immediately after exposure, ciliary activity and epithelial structure were examined close to the tympanic orifice (proximal site) and more distal to it (distal site). In the animals exposed to sulfur dioxide, no effusion was found in the tympanic cavity. Ciliary activity was reduced only in the distal site. Electron microscopy demonstrated hypersecretion in the proximal site and severe pathologic changes in the distal site. Although the normally functioning cilia in the proximal site may prevent retention of surplus secretions in the ear, sulfur dioxide may promote middle ear effusion when combined with other detrimental factors, because it stimulates mucus secretion in the proximal site and impairs ciliary function in the distal site.

Effects of Bacterial Endotoxin on Ciliary Activity in the Tubotympanum

The discovery of endotoxin in effusion of otitis media with effusion (OME) has suggested the possibility that bacterial endotoxin may be involved in the pathogenesis and development of OME. In this study, we investigated the direct effects of lipopolysaccharide (LPS), possessing the major part of endotoxin activity, on the ciliary activity in the tubotympanum. The study shows that LPS deteriorates ciliary activity in a dose-response fashion and that even low levels can, with extended exposure, cause dysfunction of cilia. It can be postulated that endotoxin in middle ear effusions aggravates the condition of mucociliary dysfunction, thus leading to chronic OME.

Mucosal pathology of the eustachian tube after exposure to sulfur dioxide.

SummaryWe studied the mucosal pathology present in the eustachian tubes of guinea pigs following exposure to sulfur dioxide. Secretory sthenia but with slight deterioration of ciliary activity was observed in the mucosal samples examined. Our data have shown that 24-h exposure to 300 ppm sulfur dioxide does not cause otitis media with effusion in the guinea pig since active ciliary function will prevent any secretions produced from stagnating in the tympanic cavity.