Inner ear diseases such as inner ear deafness and various vestibular disorders, represented by Meniere's disease, have so far been studied morphologically and functionally from various aspects, and their entities are being elucidated by degree. However, much remains to be clarified as to their etiology and pathology. The possibility cannot be excluded that microcirculation disturbance in the labyrinth has a close bearing on the etiology of inner ear diseases. To the best of our knowledge, however, little progress has so far been made as concerns the microcirculation systems in the inner ear. Such being the case, the present study was designed to shed some light on this aspect.
In the present work, corrosion casts of blood vessels, prepared by a casting method using Mercox resin, were subjected to scanning electron microscopic examination.

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The capillary permeability of the inner ear was studied ultrastructually by tracer experiment with horseradish peroxidase (HRP) under normal and experimentalconditions. The results showed as follows: The capillary of the stria vascularis is of muscle capillary type, that of the cochlear plexus and endolymphatic sac of fenestrated capillary type and that of the vestibular regions of ordinary brain capillary type. The blood-labyrinth barrier consists of both blood-perilymph barrier and perilymph-endolymph barrier in the spiral ligament and vestibular organs, whereas it consists of only blood-endolymph barrier in the stria vascularis. The capillary permeability of the stria vascularis easily increased under acute hypertension, acute hypotension and 3-type allergic reaction.

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In order to discuss the permeability of the round window membrane, horseradish peroxidase (HRP) was injected into the middle ear cavity. The normal round window membrane resisted HRP penetration from the middle ear side, but when it became pathological after repeated injections, its permeability increased. In such specimens, HRP reaction products were traced in the cochlea and vestibular sensory cells and in the endolymphatic sac. Although the stria vascularis is impermeable to HRP, the vestivular dark cells were accessible, suggesting that the metabolic activity of the dark cells can be more readily controlled by drug applications through the middle ear cavity. This study showed that drugs and other large molecules injected into the middle ear cavity may reach the endolymphatic sac and alter it functionally.

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Hydrostatic pressure of the endo- and perilymph in the guinea pig cochlea was studied with a servo-controlled micropipet system. Both endo- and perilymphatic pressure increased in a linear manner without any time lag or dampening as the CSF was pressurized over a range of 0 to 50mmHg. Following the instillation of saturated NaCl solution (0.1ml) into the middle ear, labyrinthine pressure measurement showed gradual declines in endolymph and perilymph with a minimum pressure at 15 to 20min and slow recovery during the next 20 min. Systemic administrations of osmotic agents (glycerol, urea and mannitol) produced a substantial pressure reduction in both endo- and perilymph with a maximum reduction at 20 to 30 min. Difference in pattern of the pressure changes between the labyrinthine fluids and CSF indicastes a possible dehydration effect in the inner ear for glycerol and urea, but and ambiguous effect for mannitol.

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Glycerol, an osmotic agent, was administered intravenously and orally to guinea pigs, and samples of serum, CSF, scala tympani perilymph, scala vestibuli perilymph and cochlear endolymph were collected. The concentrations of Na and K in these fluids were then assessed using microflame photometry (I. L. 443 Instrumentation Lab.).
1. Both Na and K concentrations in the CSF, scala tympani perilymph and scala vestibuli perilymph showed characteristic and individual distribution.
2. In order to clarify when the characteristic distribution establish we collected from fetuses and neonates. By the 49th day of gestation Na and K levels in the cochlear endolymph were already established. The K level in the scala tympani perilymph in the fetus was high and almost the same as that in the scala vestibuli perilymph.
3. When Reserpine was administered, the Na concentration in CSF, both perilymphs was markedly depressed and then observed almost the same level. This is thought to be attributable to the indirect blockage of energy producing systems in membrane transport of inner ear by the reserpine-induced depletion of catecholamines.
4. Glycerol (I. V.) increased the Na level in the CSF and scala tympani perilymph. K level was increased only in the scala vestibuli perilymph.
5. Glycerol (P. O.) increased the Na level in the CSF, scala tympani perilymph, scala vestibuli perilymph and scala media endolymph. K level was increased only in the scala tympani perilymph.
The increase of Na was considered to be the result of dehydration caused by the osmotic effect of glycerol. This action is slower and stronger after oral administration than after intravenous injection.
The present study suggests that the inner ear fluids in endolymphatic hydrops might be concentrated by the administration of diuretics, and that in the treatment of Meniere's disease, oral administration is more effective than intravenous injection.

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The history of the in vitro perfusion techniques of the cochlea was briefly described and the advantages of our methods (vascular perfusion technique and the high-flow perilymphatic perfusion technique) were discussed with some representative experiments.

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Hair cells are the primary receptors of auditory and vestibular systems, which produce mechanoelectrical transduction to afferent neurons. The biophysical basis of mechanoelectrical transduction was investigated by a whole-cell-clamp technique using the “Gigaseal”. Enzymatically isolated hair cells of the bull frog saccules were used. Voltage-clamp method resulted in biophasic currents consisting of initial inward and following outward currents. The membrane voltage was found to change like an action potential in current-clamp experiments.

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We have been studying the three-dimensional structures of some organelles in both the hair cell and the supporting cell using rapid-freeze, deep-etch, rotary-replication techniques. In this article, we have reviewed and have summarized in detail the technical procedures of this freeze-etch method and have demonstrated some photographs of the deep-etched replicas of the guinea pig inner ear. We have advocated the advantages in using this method for morphological and immunocytochemical studies of the inner ear and have discussed the futureprospects of this method in the inner ear research.

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The guinea pigs were passively sensitized with a serum containing 72 hr passive cutaneous anaphylaxis (PCA) antibody against dinitrophenyl (DNP), and challenged with DNP-bovine serum albumin into the facial nerve through the stylomastoid foramen. Horizontal nystagmus and deviation were observed in 31 of the 35 animals of the specifically challenged group about 15 minutes after the injection. Electrocochleograms exhibited large ampulitude of negative summating potential 15 hours after the injection in 8 out of the 10 animals of the challenged group. In 3 of the 4 animals sacrificed 30 minutes after the injection, cresent shaped degranulation of mast cells, edema and infiltration of eosinophils of the perisaccular connective tissue in the endolymphatic sac were examined electron microscopically. Edema in the tissue in 28 of 31 animals were observed, and the cresent shaped degranulation in 26 of 31 animals were observed. These results indicate that Type I allergy in the endolymphatic sac is the cause of tissue edema, resulting in endolymphatic hydrops. It may be concluded that the animal model with Type I allergy reported in this paper represents very similar figure to Menier's disease in man.

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In recent years, there have been several experimental data indicating that Type I or III allergy or autoimmune reaction may cause inner ear disorders. However, the inner ear immune system is not fully understood. In the present study, we investigated the origin of immunoglobulins in Perilymph. Chinchillas were introduced antigens into the inner ear through the facial nerve canal and subarachinoid space. Arrival of the antigens at the perilymph was confirmed. Results of present study would deny the local antibody production in the inner ear, particularly in the perilymphatic space. The transfer of immunoglobulin from cerebrospinal fluid through the cochlear aqueduct and filtration of immunoglobulins from blood vessels surrounding the perilymphatic space could be considered as candidates of the origin of perilymph immunoglobulins.

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Quantitative histochemistry was carried out by two different methods, cytofluorometry for densitometry and image analysis for morphometry. We described here the results of DNA cytofluorometry and image analysis of PAS reactions in cochlea.
Feulgen-DNA cytofluorometry on the component cells was carried out on serial sections of the human cochlea. The result obtained from 20 cases revealed that all the hair cells exhibited a diploid G₁ pattern. However, some supporting cells included tetraploid and octaploid cells. This polyploidization may be involved in the compensatory mechanism for the damaged organ of Corti.
Morphometric analysis of the PAS reactions in the outer hair cells of guinea pigs was performed by color image analyzer. The mean PAS reactions value and standard deviation for each outer hair cells in 1st, 2nd, 3rd and 4th turn were 29.2 ± 8.3, 46.1 ± 3.8, 56.9 ± 7.1 and 79.2 ± 8.0 × 10^-2μm²/μm², respectively. These results indicate that the hair cells of upper turn have more glycogen than that of lower turn.
Quantitative histochemical methods, cytofluorometry and image analysis, enable us to perform measurement in comparison with morphological information.

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The purpose of the study was to investigate the anatomy of the epitympanic cavity. For this study, serial horizontal sections of 50 normal temporal bones were used. Graphic reconstruction of the epitympanic cavity and the supratubal space was performed. The findings of this study revealed the following information. 1) There are two types of temporal bone about anatomical relationship between the epitympanic cavity and the supratubal space. One type of temporal bone shows developed supratubal space, while the other poorly developed supratubal space, although the epitympanic cavity is developed. 2) There is the tympanic mucosal fold separating the epitympanic cavity from the supratubal space. However, in 14 (28%) of the 50 specimens the defect in the mucosal fold communicating those two spaces exists.

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The wall of the tympanic cavity was made of connective tissue without bony tympanic bulla. The malleus showed unique shape and fused with annular tympanic bone, while the incus and stapes was similar to those of other laboratory animals. Eustachian tube of 1.5mm length was composed of the dilated tube ensheathed by semicylindrical cartilage (cartilaginous part) and the diverticulum with narrow lumen. Epithelium of the tympanic cavity was simple squamous except a part near the tubal orifice, of which epithelium was simple columnar or cuboidal one, intermingling some goblet cells. Lumenal linings of the cartilaginous part and diverticulum were stratified squamous and pseudostratified columnar epithelia, respectively. Some intermediary cells were found in the latter, throughout the length of the tube. Simple cuboidal epithelium intervened between above mentioned two parts. Large lymphatic tissue and mixed glands were found around the diverticulum.

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To study the evolution of connective tissue components in wound healing of perforated tympanic membrane, light and electron microscopic observations were made of experimentally perforated tympanic membranes of 86 guinea pigs. Regenerating fiber bundles in early stage were thinner than those of control animals and their arrangement were less compact and irregular. Regenerating fiber bundles were composed of three kinds of fibrils: fibrils of quadrangular appearance in transverse section, collagen fibrils and elastic fibers. Small amount of collagen fibrils and elastic fibers also found in fiber bundles of control animals. Collagen fibrils were formed in early stage and fibrils of the quadrangular appearance in transverse section and elastic fibers were formed later. The results indicates that a regenerating process of connective tissue components in tympanic membrane of ginea pigs after experimental perforation was composed of not only forming granulation and scar, but also forming usual fibrils of original tympanic membrane and interfibrillar matrix.

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We observed the ultrastructure of the Incudo-stapedial joint of adults with TEM and SEM, and the changes resulting from aging. We obtained the following results.
1) Capsule of the Incudo-stapedial joint was chiefly composed of collagenous fibers and elastic fibers.
2) By aging, changes were identified in the cartilage and the capsule of the Incudo-stapedial joint, for example; i) Disturbance in the arrangement of the collagenous fibers and reduction of the elastic fibers. ii) Apperances of fissures and unevenness on the surface of the articular cartilage.

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Because pathogenesis of the eustachian tube dysfunction is still unclear, its treatment is not established. We used pig normal eustachian tube in order to clarify its physiological function in this study. Opening and closing pressure of the tube were measured before and after tubal washing. The tubal lavage was analyzed with its phospholipid and protein composition. Surface tension measurement was also accomplished.
1. After washing the eustachian tube with physiological saline, opening and closing pressure were increased than that of before washing.
2. The eustachian tubal lavage was analyzed. Surface tension of the lavage appeared to be stronger than that of serum and saliva. Approximately 51% of total phospholipid of the lavage was phosphatidylcholine (PC). In fatty acid composition of PC, the ratio of palmitic acid was 36.3%. The ratio of dipalmitoly phosphatidylcholine in total PC was 11.3%. Protein analysis was accomplished with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). There were five bands in the gel which was not exist in serum. One of the five bands indicated molecular weight 35K which is the same molecular weight as apoprotein of the pulmonary surfactant.

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The purpose of this study is to observe the mucociliary movement of the eustachian tube mucosa in normal guinea pigs and to compare with that of the other mucosal linings of the upper respiratory system, such as tracheal or nasal mucosa, using the photo-electric method, with which ciliary activity of the mucosal linings can be observed directly and quantitatively. A total of ten healthy albino guinea pigs weighing approximately 300g were used. Ciliary beating frequencies in the mucosal lining of the eustachian tube were recorded to range from 640 ciliary beats per minite (cbm) to 356 cbm, with a mean of 516±85 cbm. Those in that of the tracheal mucosa were recorded to range from 622 cbm to 363 cbm, with a mean of 476 ±81 cbm. Although it appears to be more active in the ciliary activity of the eustachian tube mucosa than that of the tracheal mucosa, statistically significant differences in over-all ciliary frequencies could not be found between the eustachian tube and tracheal mucosa.

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The purpose of this study was to know the influence of negative middle ear pressure on the clearance of fluid from the tympanum via the eustachian tube. Twenty-five adult cats were used for this study. Experiment 1: Mucociliary clearance of fluid under negative middle ear pressure. Middle ear pressure was decreased through the tube connected to the tympanic bulla and a colored fluid was placed in the tympanum. The interval between the placement and beginning of discharge of the fluid from the pharyngeal orifice of eustachian tube was measured. It was revealed that the clearance time at the tympanic pressure level of-300mmH₂O and-400mmH₂O was not elongated. Experiment 2: Muscular clearance of fluid under negative middle ear pressure. To simulate swallowing, the tensor veli palatini muscle was stimulated electrically. It was found that massive discharge of fluid by muscle contraction occurs only when the negative middle ear pressure was low. From these results we concluded that negative middle ear pressure did not disturb mucociliaty clearance but muscular one.

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Some patients of otitis media with effusion have sensorineural hearing loss. In many causes being assumed, the influence of middle ear pressure was examined in the present study. Cochlear microphonics (CM) was recorded as an indicator of the inner ear function using guinea pigs. Positive or negative pressure was given to the tympanic cavity and external auditory canal at the same time. Pure tone was given to drive CM to the external auditory canal. CM amplitude went down under both positive and negative pressures. The decrease of CM amplitude was greater under negative pressure than under positive pressure.
CM went down directly and became stable immediately under negative pressure. On the other hand, CM went down at first and then went up gradually under positive pressure. Although perilymphatic pressure of scala tympani measured by a servocotrolled micropipet system went down directly and became stable soon, it went up at first and then gradually went down under positive pressure. This phenomenon would have some relationship to the CM change.

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Vibration modes of the ossicles and the leverage function were studied in human cadaver temporal bones with intact cochlea. After placing tiny steel spheres on the ossicles, ossicular vibration to a constant sound pressure was measured by observing the displacements of spheres under microscope with strobe illumination and by utilizing a video measuring system. The average lever ratio obtained by comparing the displacement of the umbo and that of the stapedial head was 1.9 at 0.6kHz (minimum), which gradually increased over 1.2kHz. This relatively high lever ratio was considered to be caused by loading of the cochlear fluid on the ossicular system.

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Pentobarbital sodium has been reported to have a depressive effect on the acoustical stapedial reflex in man, squirell monkey, cat and rabbit. The acting site of this anesthetic agent is thought to be the reticular formation, which includes facilitating route of the acoustical stapedial reflex. On the other hand, ketamine hydrochloride, which stimulates the reticular formation, had been prospected to have no effect on the acoustical stapedial reflex.
The purposes of this experiment are 1) to confirm whether pentobarbital sodium has the depressive effect on the acoustical stapedial reflex in guinea pig, 2) to examine whether ketamine hydrochloride stimulates or depresses or does nothing. We found that both anesthetic agents had the depressive effect on the acoustical stapedial reflex. There seemes to be neuroanatomical links to the final common pathway of the stapedial reflex which facilitate the stapedial reflex, and the links seem to be located in the reticular formation and originate from at least the midbrain level.

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A new simultaneous measuring system of sound pressure levels, pressure levels of the ear canal and nasopharyngeal pressures was developed using the sonotubometric method. The sound (115dBSPL) of white noise was applied to the nostril with a horn driver. The sound level change of the external ear canal was monitered while the positive or negative pressure was applied to the ear canal of intact or perforated ear drum, and was compared to the nasopharyngeal pressure monitered in the opposite nostril. In a case of floppy tube, the tuba opened easily when the positive pressure (+100 mm H₂O) was applied to the ear canal. However, the negative pressure (-200 mm H₂O) applied to the ear canal induced a decrease of the sound pressure level of the ear canal and caused no opening of the tuba by swallowing. This means that the negative pressure in the bony portion of tuba through perforated ear drum locked the pharyngeal portion of tuba and then changed the acoustic transmission characteristics. This device can detect the abnormal status of sound transmission in the middle ear - tuba - nasopharyx, especially the pharyngeal portion of tuba.

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A new method for measuring middle ear pressure was reported. Under general anesthesia without N₂O, a micro-tip catheter pressure transducer (Mikro-tip^®) with diameter of 1mm was inserted into tympanic cavity through the Eustachain tube. Measurement of three normal ears, one ear of tubal dysfunction and six ears with otitis media with effusion (OME) were carried out. The normal ears showed the middle ear pressure of 45, -10, 15mm H₂O. Three ears with OME showed negative pressure of -40--50mm H₂O, -50--60mm H₂O and -100--120mm H₂O, while two ears with OME showed positive pressure of 0-20mm H₂O and 30-50mm H₂O, and one ear showed 0mm H₂O.

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It is generally believed that a cause-and-effect relationship exists between chronic inflammatory condition and suppressed growth of the pneumatized cellulae. But we had already expressed the suppressive process of the pneumatization in experimental studies using pigs and it has been clear that the middle ear inflammatory condition suppressed the bone metabolic layer beneath the epithelium of the pneumatized cavities and suppressed the expansion of the cellulae. So this time we investigated about the human fetal middle ear and it was found that there were two processes of the bone metabolism of pneumatization, one was passive bone metabolism with only absorbing the osteoid tissues by osteoclasts and the other was active bone metabolism with absorbing the osteoid tissues and changing the osteoid tissues to the trabecular bone by osteoclasts and osteoblasts. This active process of pneumatization was observed in the mastoid cavities, corresponding to the inner side of mastoid process and it was same as the process of pneumatization of pigs. So it is naturally considered that the suppressive process of the pneumatization by the continuous middle ear inflammatory condition will also occur in the pneumatized cellulae of human temporal bone.

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Guinea pigs were exposed to 400 ppm or 5, 500 ppm of isopropyl alcohol (IPA) for 24 successive hours, and then middle ear mucosal samples in both proximal and distal sites to the eustachian tube were obtained for functional and morphological studies. The ciliary activity was examined according to the photoelectric method, and the morphology of the middle ear lining was examined by transmission electron microscopy. The results showed that IPA at the allowable level of 400 ppm has an acute effect on the mucociliary system of the middle ear mucosa. Recovery from damage occurs within two weeks. At higher levels of exposure (more than ten times the allowable level) to IPA, moderate deterioration of ciliary activity and severe degeneration of ciliated mucosa were observed and recovery within two weeks was not seen. It is concluded that IPA at the allowable level of 400 ppm does not cause significant middle ear disorders, but workers exposed to higher levels of IPA should be given careful otolaryngological examinations and follow-up observations.

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A total number of thirty-two guinea pigs (250g) were exposed to 30 ppm of nitrogen dioxide or 300 ppm of sulfur dioxide for 24 successive hours. The middle ear mucosal samples were obtained immediately after the exposure period. The right middle ear mucosa was examined for ciliary activity according to the photoelectric method, and the left middle ear mucosa was offered for scanning and transmission electron microscopy. Serous effusion was found in the tympanic cavities of guinea pigs exposed to nitrogen dioxide. The middle ear mucosa after exposure to nitrogen dioxide showed deterioration of ciliary activity and morphological changes of mucociliary system and epithelial cells. On the other hand, little effusion was found in the tympanic cavities of guinea pigs exposed to sulfur dioxide although the mucosa showed secretory sthenia and good ciliary activity. It has been derived from the present study that ciliary activity at the middle ear mucosa around the tubal orfice has an important role in the menifestation of otitis media with effusion and that air pollution is involed in the menifestation of otitis media with effusion

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Twenty-four guinea pigs were sacrificed, and a mucosa at the bony portion of each eustachian tube and from the middle ear proximal and distal to the tube were sampled. The mucosal samples were irradiated with 200 kV_p X-rays discretely at a dosage level of 0.5-30.0 Gy, and induced changes in ciliary activity were expressed as a percent deviation from baseline levels before irradiation. The present study demonstrated that the cilia of the eustachian tube and middle ear mucosa show different reactions to irradiation according to their tympanic locations. In the meanwhile, the middle ear mucosa immediately after irradiation of 30 Gy showed some pathological changes including severe vacuolation of ciliated cells, secretory sthenia and sporadic drop-off of epithelial cells.

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In 18 cases of otosclerosis and 42 cases of chronic otitis media, anti-collagen antibody levels were determined by ELISA method. As the antigens, types I, II and III collagens separated and purified from human skin, articular cartilage and placenta were used. The secondary antibody used was goat anti-human IgG antibody. In sera from 4 cases of otosclerosis anti-type II collagen antibody of IgG class was detected. On the other hand, no such antibody was detected in sera of patients with chronic otitis media. In none of the cases anti-type I and type III collagen antibodies were detected.

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In the previous report, we have demonstrated that Sendai virus could induce viral lesion of the middle ear mucosa in guinea pigs. This study further revealed the histopathological aspect of the eustachian tube epithelia in these animals. The filamentous structure was found in some of the epithelial cells which was deemed as viral nucleocapsids based on immunolabeling with HRP. The lumen of the tube was often filled with the effusion of rather high electron density. The epithelial layer was flattened and ciliated cell population was markedly decreased. These features indicated disturbance of the mucociliary transport of the eustachian tube epithelia in acute otitis media induced by the virus.

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Otitis media was induced in rabbits by intrabullar inoculation of Streptococcus pneumoniae DP-2 and Staphylococcus aureus 209-D. The dose of bacteria injected varied at several degree. The temporal bone histopathology was studied at 30 days after inoculation.
Acute inflammation such as severe mucosal edema and diffuse infiltration with polymorphonuclear leukocytes was observed in 27% (3/11ears) of Streptococcus pneumoniae inoculated animals and in 33% (1/3ears) of Staphylococcus aureus group. The rest of animals showed scanty changes of chronic inflammation such as serous effusin, focal infiltration of lymphocytes and osteoneogenesis. There were little inflammatory changes in the inner ear.
The severity of inflammatory changes didn't correlate with the dose of inoculated bacteria.

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The relation between the degree of chronic middle ear inflammation and pneumatization was investigated in piglets, whose tympanic bullae closely resemble human mastoid air cell system.
A total of 8 piglets were selected for this experiment. Otitis media was made by the injection of glycerin into the middle ear clefts at 1 month after birth, and it was attempted to vary the degree of inflammation by the intramuscular injection of antibiotics and topical use of steroid solution. All pigs were sacrificed at 6 months after birth to examine the relation between the degree of chronic middle ear inflammation and pneumatization of the tympanic bullae.
Various degree of inflammation could be induced by the injection of antibiotics, but no effect of topical use of steroid solution was detected. With the increase in inflammation, the inhibition of pneumatization became severe. It was concluded that the degree of the inhibition was proportional to the severity of chronic middle ear inflammation.

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Otitis media with effusion (OME) was induced in guinea pigs by intratympanic instillation of lipid A, the lipid moiety of gram-negative bacterial lipopolysaccharide (LPS), from Salmonella minnesota Re595. Lipid A was chosen as an inducer because its similar composition among various bacterial species. Animals were killed from the first to I4th day after instillation of various concentrations (0.2, 2, 20, 200ug/ml) of lipid A in 0.5% triethylamine. By 3 days after instillation, all experimental animals developed serous middle ear effusion (MEE). The histological findings included hemorrhage, mucosal edema, capillary engorgement and migration of infiltrative cells including macrophages, polymorphonuclear neutrophils and lymphocytes. These findings were most prominent 3 days after instillation, and the recovery of the middle ear epithelium was observed within I4 days. Repeated instillation of lipid A (2ug/ml) at an interval of I4 days resulted in the enforcement of the local response accompanied by serous MEE. These findings indicate that lipid A can induce the inflammatory changes of MEE, and that lipid A plays an important role in the pathogenesis of OME.

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The biological activities of polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) in the 17 samples of MEEs from OME children were examined by means of Karnovsky's DAB reaction. Light microscopic examination revealed that peroxidase positive PMNs and MNCs were predominant in serous and muciod effusions, but less in number in glue. Electron microscopic study also confirmed that the electron dense DAB precipitates were generally localized at the intracytoplasmic granules and at the lysosomal membranes in both serous and mucoid effusion, whereas it was not obvious in the PMNs and MNCs of the glue effusion. The ground substance in the glue effusion, however, showed relatively high electron density, which seemed to indicate DAB positive reaction. The present observations seem to postulate that the serous and mucoid effusions still have chemotactic activities containing many active PMNs and MNCs, but the glue effusion do not. Further investigations are necessary to clarify the biological characteristics of the glue effusion.

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We have reported the biochemical analysis of acidic glycosaminoglycans (AGAG) in middle ear effusions (MEE), by use of two-dimentional electrophoresis using cellulose acetatic strips in order to know the nature of MEE. In the present study, radioautography on Dowex 1 × 2 column chromatography was carried out to analyze the composition of AGAG.
Results of the present study found, 1) that MEE contain dermatan sulfate besides hyalulonic acid, low sulfated chondroitin sulfate and heparan sulfate. And 2) that total AGAG content is not difference between mucoid MEE and serous MEE. By the radioautography, an unidentified slow component was found, which may concern the viscoelasticity of MEE.

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This study was designed to evaluate the biological property of light molecular weight immune complex, in comparison with that of heavy molecular weight immune complex. Soluble immune complexes were obtained by mixing horseradish peroxidase (HRP) with guinea pig antiserum to HRP and were devided into several fractions according to their molecular weight by a gel filtration. After the chemotactic activity of each fraction was examined in vitro, the lightest and the heaviest molecular weight immune complexes were injected into a guinea pig's left and right superior bulla, respectively. Although a middle ear effusion developed in both sides of the bulla, the histopathological feature of an experimentally induced otitis media with effusion varied with the molecular weight of the injected immune complex. The light molecular weight immune complex was proved to be a stronger inducer for a macrophage than the heavy molecular weight immune complex both in vitro and in vivo, and to have a tendency to persist in the tympanic cavity for a longer duration than the heavy molecular weight immune complex.

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As an approach to immunological onset mechanism of otitis media with effusion (OME), the authors measured concentrations of immunosuppressive acidic protein (IAP) and immunosuppressive substance (IS) in the middle ear effusion by the single radial immunodiffusion method.
In 34 ears of 34 patients, a mean IAP concentration in serous middle ear effusion (MEE) was 789.4±325.3 μg/ml and a mean IS concentration in MEE was 1082.8±327.9 μg/ml. In the patients, a mean serum IS level was 391.8±169.0μg/ml and a mean serum IS level was 694.7±224.1μg/ml. In 24 healthy people, a mean serum IAP level was 381.3±104.5μg/ml and a mean serum IS level was 509.7±104.4μg/ml. Thus, the mean IAP and IS concentrations in MEE were significantly higher (p<0.01). IAP and IS, immunosuppressive substances, seem to be involved in pathogenesis of serous OME.

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In order to investigate OME from the systemic perspective, we have studied histological reactions of the mucous membrane in the bilateral middle ears in the sensitized animals. Histological changes of the middle ear mucosa in the sensitized ear were characterized by hypertrophy of mucosa with fibrosis. Numerous mononuclear cells also existed. In non-sensitized ear (opposite side), the changes were edema of the mucosa showing mild proliferation of fibroblast and vasodilation. Mainly mononuclear cells were scattered.
For the purpose of researching the mechanism of the histologic change of the opposite ear (non-sensitized ear), the immunohistologic examination and the measure of the circulating immune complex were performed. As a result, the mucosal response was based on the immune complex.

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To clarify the mechanism of bone destruction in chronic otitis media with cholesteatoma, bone chips of tympanic bulla of guinea pigs were soaked in the propionic acid solution and it's electric conductance, pH and concentration of Ca²⁺ & PO₄^3- were measured. The results indicated that neutralization and releasing ions from the bone proceeded simultaneously in the solution. And we could get the equation reflecting well actual bone destruction, suggesting that the rate of bone destruction depends on pH and bone surface area. It was also recognized that these ions such as Ca²⁺ and PO₄^3- in the solution derived mainly from hydroxyapatite in the bone. Besides, a mass of collagen type II or a bone chip of protein-part was soaked for two weeks in the solution of which conductance indicating bone destruction (demineralization) had reached equilibrium and then samples were observed using laser-Raman spectroscopy. From the results it may be concluded that the remineralization could occur on the surface of the materials like collagen with adsorptive activity in the saturated solution.

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The development of the middle ear cholesteatoma arises from the mitotic activity of epithelial cells induced by inflamation. We have established epithelial cell line from the cholesteatoma (CH-Ep), fibroblast like cell line from the cholesteatoma (CH-Fi), epithelial cell line from the human fetal tympanic menbrane (F-Ep) and fibroblast like cell line from human fetal tympanic membrane (F-Fi) in vitro. We are able to synthesis of DNA and protein in each cell by using either ³H-thymidine or ³H-proline isotopes. The synthesis of DNA and protein in either CH-Ep or F-Ep does not change in the presence of LPS (10^-4-10^-10g/ml). However, the synthesis of DNA and protein in CH-Fi increases in the presence of LPS (10^-6-5×10^-11g/ml). The conditioned medium of CH-Fi, stimulated by LPS (50μg/ml), increases the synthesis of DNA and protein in CH-Ep. Whereas, the conditioned medium of F-Fi, stimulated by LPS (50μg/ml), does not increase the synthesis of DNA and protein in F-Ep. In this experiment, no direct effect of LPS upon the mitotic activity of the epithelial cells is recognized. LPS stimulated CH-Fi, produces growth factor and stimulates the mitotic activity of CH-Ep.

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Demineralization seems to be the first step of bone resorption, followed by enzymatic degradation of bone protein. Therefore collagenase appears to be a major factor in the degradation process of organic matrix of bone. The clinical evidence has shown that chronic otitis media with cholesteatoma causes greater bone resorption than otitis media without cholesteatoma. What is the role of the epithelium and its products on the morbidity of cholesteatoma?
Recently we have studied the mechanism of the production of collagenase in cell culture of rat epithelial (skin) and mesenchymal cells (skin fibroblast like cells) under various conditions. Epithelial cells significantly induced mesenchymal cells collagenase production. It was concluded that cellular interaction between epithelial cells and fibroblast like cells to produce collagenase appears to be crucial for bone resorption in cholesteatoma.

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The purpose of this study is to establish a novel epithelial clonal strain of middle ear cholesteatoma. During the operation, a cholesteatomous matrix is obtained from a 34 years old female. Cultured cells at passage 7, is characterized by hyperkeratinization and heavy adhesion to each other because of well-developed desmosome. Fig1, 2 are cloning methods and materials used. Cultured cells grow in a mono layer like cobblestone. Fluorescent microscopic examination, EM examination are performed. Cytoplasma fluoresces similar to filaments and the outer edge of the cells fluoresce stronger. In other findings, keratin produced from the cells fluoresce and become membrane like. Golgi apparatus and desmosome are well-developed.

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Under light microscope, we observed the cholesteatoma which were taken at tympanoplasty. Compared with the clinical data, we examined histopathological characteristics of residual cholesteatoma, especially.
Results were as follows:
At second look operation of cholesteatoma, we found out two different types of residual cholesteatoma (pearl-like and non-pearl-like). Histopathologically, the pearl-like residual cholesteatoma showed thin epithelial cell layer with little inflammation in subepithelial layer. There seemed to be little difference between children and adults. The epithelial cell layers of cholesteatoma in antrum and tympanum were thicker than those in attic and of the ossicles.

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Two types of cholesteatoma, one has thick epithelial layer and another has thin one, were observed by electron microscope. The findings were as follows; 1) Basal cell and spinous cell of the thick epithelial layer were more active according to the morphological observation. 2) The thickness of the epithelial layer were concerned with proliferation of spinous cells which depend upon the function of basal cells.

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In our previous paper we induced cholesteatoma-like lesion experimentally in Mongolian gerbils by closing external auditory canal to elucidate the mechanism and a part of etiology of bone destruction in cholesteatoma of the tympanum. The present paper describes our histologic (light and electron microscopic) studies in Mongolian gerbils continued to be fed for a prolonged period (12 to 18 months). The findings after 12 to 18 months feeding period revealed that bone destruction further progressed and chiefly fibroblast-like cells were observed in the marginal area of bone destruction. Other than these, osteoclasts were also found. Since findings showing contact of fibroblast-like cells, neutrophils and epithelial cells were obtained, cellular interaction among these cells was suspected and considered to be involved in bone destruction in some way.

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To clarify whether or not sodium thiosulfate (STS) protects cisplatin (CDDP) ototoxicity, the following experiments were carried out. Fifty five guinea pigs were divided into four groups; group 1 was given 12.5mg/kg CDDP intraperitonealy, group 2 was given 12.5mg/kg CDDP and 530mg/kg STS after 5 minutes, group 3 was given the same dose of CDDP and STS after 2 hours, and group 4 had no treatment. Seven days after administration of these drugs, N₁ threshold increased and the hairs in the basal turn and second turn observed by SEM disappeared sporadically or continuously in group 1 and 3, but group 2 showed almost normal appearance. Therefore, there was protective effect by STS in group 2, but there was little effect in group 3. As the compound of CDDP and STS has no anticancer effect, it is considered that two-route infusion chemotherapy is clinically useful for the protection of CDDP ototoxicity.

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Large doses of aspirin are clinically known to cause transient tinnitus and hearing loss. The mechanism of hearing loss is still debatable. Our previous report showed that we can know cochlear nerve activity by means of recording the single nerve action potentials (spikes) evoked by electrical stimuli to the cochlea. Using this method we studied the effects of aspirin on the cochlear nerve activities. We recorded the spontaneous discharges of the cochlear nerve and minimum threshold to sound stimuli too. The elevation of the threshold of spikes evoked by electrical stimuli rose to about 20% of preinjection level at several minutes after injection of 300mg/kg. This suggested that intravenous injection of aspirin acts directly on the cochlear nerve. The threshold increased about 10 dB within 7 minutes. The spontaneous discharge rate decreased rapidly after injection of aspirin. When the recrding of spikes is good, the spike rate increased gradually and recovered. The changes of temporal pattern of spontaneous discharges (interspike interval) were classified into 3 types. 1. similar to histogram of the vestibular nerve but with large coefficient (1.2) of variation; 2. unimodal histogram; 3. bimodal histogram.

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Chronic methyl mercury intoxication was induced in rats by the oral administration of methylmercury (II) chloride, 2mg/Kg of body weight for 5 times per week with a total dosage of 48mg/Kg-92mg/Kg. Inner ear findings were studied by scanning electron microscope (SEM) and partially by light microscope (LM).
The change in the organ of Corti were a little in the outer hair cells by SEM. This findings were apparent in the lower 2nd turn, and it took the scattered pattern of hair cell damage only in the 1st low. Spiral ganglion was slightly atrophied by LM. However these cochlear change was not examined in some rats, so toxity of cochlear organ by methyl mercury was not clear. Vestibular system (saccule and utricle) and semisercular canals had little change.

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Cochlea damage of the Hamster induced by injection of Kanamycin into the middle ear cavity were studied using SEM.
The following findings were obtained.
1) The changes in the organ of Corti were apparent in the outer hair cells. The changes in the inner hair cells often slight.
2) The damage of outer hair cells in the lower turn were more severe than those of the upper turn.
3) The damage of 1st row was most severe in the middle turn and the lower portion of the apical turn.
4) The damage of inner hair cells were more severe in the basal turn and the apical turn.

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