The only way to study all of the structures in the human ear as a whole is to evaluate histopathological specimens embedded in celloidin and stained by hematoxylin and eosin. Nevertheless, because it is difficult to evaluate spatial relations between anatomic structures in two-dimensional histological slides, we developed a computer program and method to visualize the anatomy and patology of the temporal bone in three dimenions, using two-dimensional histological slides of the specimen. We have also created computer software to measure these structures in three dimensions.
The computer-aided technique we developed is the only one we know of that permits three-dimensional measurement as well as reconstruction of structures in histological specimens. It gives accurate results regardless of the angle at which the specimen was cut, which has enabled us to define the true shapes, locations, dimensions, and reletionships between many structures in the middle and inner ears. Some of the entities that we have evaluated in this way are the cochlea, vestibular endorgans, semicircular canals, round window and its surrounding structures, and facial nerve.
The information we have gained using this new technique has proved essential to the investigation of physiology and pathophysiology of the ear. In addition, it has contributed much to the understanding of three dimensional surgical anatomy, the foundation of otologic surgery.

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The purpose of this study was to gain anatomical informations of the eustachian tube and its acceesory structures, such as the internal carotid canal, the tensor tympanic muscle and the middle cranial fossa in human temporal bones from 12 adults, each anatomical relation amoung those structures was observed at three different section showing the orifice of the tympanum, the bony portion and the isthmus of the tube. In each case, although some individual variations were seen in anatomical relations of the eustachian tube and its accesory structures, common findings were summarized as follows: In many cases, the carotid canal passed inferiorlly or inframedially for the tympanic orifice to the bony portion of eustachian tube. And then the carotid canal passed medially for the isthmus region of eustachian tube.
In many cases, the carotid canal was very close to the bony portion (0.39±0.41mm) and also close to the isthmus (0.70±0.52mm). There was only a thin bony wall (0.15±0.15mm) of the carotid canal in bony portion and also a thin wall (0.50±0.56mm) in the isthmus. In one case, absence of bony plate between the eustachian tube and the carotid canal was seen.
There was wide individual variation in the distance between the roof of eustachian tube and the middle cranial fossa, while there was no remarkable difference amoung each potion of the eustachian tube. Its distance was from 1.1 to 6.3mm.
The tensor tympanic muscle was seen on the supramedial part of the eustachian tube in the region of the tympanic orifice, but on superior part from the bony portion to isthmus of the eustachian tube in many cases.

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The relation between chronic middle ear inflammation and pneumatization is still controversial. Previous experiments using pigs, whose tympanic bullae closely resemble the human mastoid air cell system, we reported that chronic middle ear inflammation occurring in the early stages of life inhibits the development of the middle ear air cell system, and the later the occurrence of otitis media, the lesser the degree of inhibition of pneumatization.
In addition to time of onset, the number of incidents of inflammation is considered to be one of the important factors that affect the degree of pneumatization. Therefore, in this experiment, relation between the number of incidents of inflammation and pneumatization was investigated.
A total of four pigs were selected for this experiment. Bilateral ear drums were perforated, and glycerin was injected into the middle ear clefts to induce chronic middle ear inflammation. The four pigs were divided into four groups of one pig respectively according to the number of the treatment. Group I: treated once, 4 weeks after birth. Group II: treated twice, 4 and 5 weeks after birth. Group III: treated three times, 4, 5, and 6 weeks after birth. Group IV: treated four times, 4, 5, 6, and 7 weeks after birth. These treatments were performed under general anesthesia by intramuscular injection of ketamin hydrochloride and xylazine hydrochloride. The tympanic bullae of all animals were removed 6 months after birth to determine and compare the degree of pneumatization.
In the ears of group I, the development of the air cell system was excellent and revealed the same findings as normal bullae, apart from a slight inflammatory change in the air cells observed in one case. A slight degree of inhibition of pneumatization and some inflammatory change was detected in two ears of group II, and more severe inhibition and increasing inflammation was observed in group III. In group IV, somewhat stronger inhibition than that of group III was noted.
In the cases in which pneumatization was inhibited, the number of osteoclasts at the inner edge of the cortex facing the pneumatized cavities had decreased, as we already reported. Notably, few osteoclasts were detected in group III and IV.
As the number of injections of glycerin increased from group I to IV, the inhibition of pneumatization became increasingly more severe. The injection of glycerin did not always produce middle ear inflammation, as there were some cases in which little inflammatory change was noted as in group I. Accordingly, the number of dosages does not necessarily correlate with the number of incidents of inflammation, however, it was apparent that as the inflammatory stimulus increased, pneumatization became more severely inhibited. From these findings, it was considered that the number of incidents of inflammation is an important factor affecting the degree of pneumatization.
In the ears where pneumatization was inhibited, the number of osteoclasts had decreased, indicating impairment in bone resorption activity. The devlopmental process of the air cell system is disturbed owing to impaired bone resorption activity, and finally inhibition of pneumatization occurs. It was concluded that the degree of impairment of bone resorption activity becomes more serious as the number of incidents of inflammation increases, and as a result of this, increasingly severe inhibition of pneumatization occurs.

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The purpose of this study was the comparison of morphological changes in the inflammatory and the normal middle ear mucosa in pigs, which have an air cell system similar to that in human beings, by the electron microscopy.
Otitis media was effected in the each right ear of 4 pigs by the transcanal injection of glycerin into the middle ear cleft at 1 and 2 weeks after birth. The left ears remained normal. Two of the pigs were sacrificed respectively at each of 1 and 6 months after birth. Middle ear mucosal samples for scanning (SEM) and transmission electron microscopy (TEM) were obtained from the Eustachian tube, middle ear cleft, and air cell system. The specimens were fixed in 2.5% glutaraldehyde within 3 hours after sacrifice, buffered in 0. 1 M cacodylate at pH 7.2 and postfixed with 1% osmium tetroxicide for 2 hours. After being dehydrated through ascending grades of alcohol, the samples for SEM were dried to a critical point in liquid carbon dioxide, mounted on aluminium stabs, spattercoated with gold and viewed by scanning electron microscopy Hitachi S430. The samples for TEM were embedded in Epok 812, sectioned by microtome and stained with lead citrate and uranyl acetate. Grids were viewed and photographed with transmission electron microscopy Hitachi HS9 and H800.
In the normal ears, the epithelial cells consisted of ciliated cells, non-ciliated cells, secretory cells and basal cells. Each ciliated cell had 80 to 100 cilia which were about 5 to 8μ in length. There were 3 types of secretory cells: one with only light secretory granules, one with only dark secretory granules and the other with both light and dark secretory granules. Non-ciliated cells had no cilia or secretory granules but had microvilli on their flat or mildly bulging surfaces. The ciliated cells were distributed most densely in the Eustachian tube and tubal orifice of the middle ear cleft, decreasing in number towards the air cell system. In the Eustachian tube, a high density of cilia was noted to occur regulary near the pharyngeal orifice. The secretory cells were distributed in the same way as the ciliated cells. However the non-ciliated cells were distributed in a reverse pattern. In the air cell system, the epithelial cells consisted of only flat non-ciliated cells and there were no ciliated or secretory cells. The features at 1 month after birth were the same as those 6 months after birth.
In the inflamed ears, the construction and distribution of epithelialcells were the same as in normal ears. However the cilia were deformed or missing and were irregularly detached from all epithelial cells in some places of the Eustachian tube and middle ear cleft at 1 month after birth. On the other hand ciliated cells, bulging secretory cells and columnar cells were increased in numbers in the Eustachian tube and middle ear cleft at 1 month after birth. It was observed that these features had returned to normal by 6 months after birth. In the air cell system, SEM revealed hardly any changes at 1 and 6 months after birth compared with the normal cases, whereas any TEM showed subepithelial thickening in the air cell system persisting even at 6 month after birth.
Although it was functioning actively in the normal Eustachian tube and middle ear cleft, the muco-ciliary system seemed stimulated in the inflamed cases-seen by the increase of ciliated cells, bulging non-ciliated cells and columnar cells. The inflammatory changes in the Eustachian tube and middle ear cleft which appeared at 1 month after birth returned to normal at 6 month after birth. However in the air cell system without epithelial changes, the subepithelial thickening remained at 6 month after birth. These results suggest that inflammation which effects the air cell system where there is no muco-ciliary system is prolonged more easily than in the Eustachian tube or middle ear cleft which has a muco-ciliary system.

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The response of normal tubotympanic cilia from the guinea pig was investigated in an in vitro experimental system to evaluate ciliary responsiveness without the influence of secretions. Mucosal samples were obtained from three different tubotympanic locations (eustachian tube, and middle ear close to the tube [proximal site] and more distal to the tube [distal site]). Each mucosal sample was cultured in 2 ml of RPMI1640 and 1 ml of heparinized venous blood from a non-allergic human subject, a patient with ragweedsensitive allergy, or 3 patients with D. farinae-sensitive nasal allergy. 1μg of D. farinae extracts were added in each chamber to evoke an in vitro allergic reaction, and the ciliary activity of the most active cell in each chamber was monitered up to 120 min.
When D. farinae extracts were added into the chamber containing blood from subjects without sensitivity to the antigen, no changes were induced in ciliary activity through the examination. When the antigen was added into the chamber containing blood from D. farinae-sensitive subjects, ciliary exitation was induced in the culture from the eustachian tube and the proximal site. The peak was observed 30min. post-reaction and a gradual slowdown to the baseline was observed. No ciliary depression was induced by an allergic reaction. Such ciliostimulatory effects were correlated with D. farinae induced percent histamine release from whole blood. In the culture from the distal site, a gradual ciliodepression followed the peak of ciliary exitation.
In conclusion, an in vitro allergic reaction affected the ciliary activity in the distal site, but such ciliary dysfunction was not in the eustachian tube or the proximal site during such a reaction.

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The fine structure of the synapse at a small efferent nerve ending on the lateral wall of the outer hair cell and its course were studied by means of transmission electron microscopy using the serial ultrathin section technique. An efferent fiber formed its first varicosity at the bottom of the cell having axodendritic synapses with two efferent endings and then formed the second varicosity without having synapse at the level of top of nucleus. It went further upward and ended as a small terminal at the mid-part of the cell with a synapse. At this synapse, the outermost layer of subsurface cisternae was flattened like the subsynaptic cisterna in the ordinary efferent synapse. The outer part of inner layers was interrupted at the synaptic zone whereas inner part was invaginated to form cone shape. Many vesicles were seen inside the invagination. These findings suggest that this type of ending has some function probably on hair cell motility. It is also suggested that the outermost layer of subsurface cisternae is closely related to efferent transmission and has a different property from inner layers.

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Vascular smooth muscle cells and angioarchitecture of the inner ear arterial system in guinea pig were examined by SEM using combined techniques of resin injection and HCI hydrolysis. Overlapping stringshaped muscle cells are associated with the arteries from the anterior inferior cerbellar artery to the spiral artery. Overlapping spindle-shaped muscle cells surround the vestibulo-cochlear artery, the anterior vestibular artery and the primary main branches of the spiral modiolar artery. A single layer of spindle-shaped cells of the coiled arterioles is gradually replaced by ring-shaped cells as the arterioles straighten out before entering the membranous labyrinth. Unique small ramifications with a single layer of ring-shaped muscle cells directly branch out from the arterial trunk within the internal auditory canal and the modiolus. Further analysis of the arterial smooth muscle cells without resin-injection technique has reveals morphological variation: fairly smooth adventitial surface of the cells with wide intercellular spaces in dilatation and highly rugged surface in contraction. Morphological evidence suggests that the major regulation of inner ear blood flow is accomplished by each arterial trunk with strongly contractile musle cells and to a lesser degree by the farther arteriolar branches. Nevertheless, the characteristic morphological features of the inner ear arteriolar segments such as coiled angioarchitecture and the presence of a bony covering may function to supply the membranous labyrinth with blood flow under minimal pulsation.

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In order to examine cell kinetics in the stria vascularis, nuclei synthesizing DNA were discriminated and traced by prolongation of the survival time up to 14 days by ³H-thymidine autoradiography with cumulative flash labelling of ³H-thymidine. Labelled nuclei, which indicate the evidence of nuclear DNA synthesis, were found not in marginal and basal cells, but in some intermediate cells. Although labelling index peaked at 3 days after the last injection of ³H-thymidine and thereafter decreased, the portion of labelled intermediate cell seemed to move towards marginal side and basal side. The intermediate cell seems to play an important role for cell kinetics of the stria vascularis in mature cochlea.

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Calmodulin (CAL) is an ubiquitous calcium binding protein which regulares calcium-mediated cellular reactions. Using immunohistochemical techniques Slepecky et al [8] demostrated a selective accumulation of CAL in the inner and outer hair cells of the organ of Corti (OC). In this study we tried to quantitate the CAL levels in OC and its substructures for the purpose of estimating a possible role of CAL as a regulatory protein using quantitative polyacrylamide gel electrophoresis.
Tissues of the inner ear and other organs were obtained from pigmented guinea pigs and chinchillas. The temporal bones were quickly removed from the skull after decapitation under general anesthesia, followed by freeze-drying under vacuum for 96 hours at-40°C. Specimens of various inner ear structures including OC substructures were then dissected stereomicroscopically at 20°C and a relative humidity of 40% or less, and weighed on quartz fiber balances. Freeze-dried specimens ranging from approxiately 1μg to 7μEg dry weight and CAL standard series were subjected to two dimensional polyacrylamide gel electrophoresis (2-D PAGE) with isoelectric focusing in the first dimension and SDS gel electrophoresis in the second essentially by the method of O' Farrel [9]. These gels were followed by staining with silver by a modification of the method of Oakley [11]. The densities of identitied CAL spots were then measured with Laser densitometer and the CAL levels were calculated from the standard curve in each experiment.
The results of this study were summarized as follow. The CAL level in OC was estimated 2400 mg/kg wet weight, the highest among other inner ear structures or tissues analysed in guinea pig. This level reached as much as three times higher than that in the cerebral cortex or the cerebellum. The CAL level was very low in the stria vascularis and the Reisner's membrane. Among the substructures in OC of chinchilla it was found that the CAL level is by far the highest in the outer heir cells, approximately 6% of total protein. For comparison, the highest known accumulation of CAL (in the head of spermatosona) amounts to 12% [13]. CAL was cosiderably lower in the inner hair cells (about 1%), and was not detectable with usual protein loads in other cell types of OC. These results suggest that the calcium dependent intracellular response is performed actively in the outer hair cells of OC. The relative distribution of CAL in OC is very similar to that of glycogen, which reaches the exceedingly high level (600mmol/kg dry weight) in the outer hair cells [14]. This circumstantial evidence suggests that the activation of the glycogen phosphorylase kinase is an important role of CAL in the outer hair cells, whereby ultimate energy for contractile motility is mobilized from glycogen.
The mechanisms responsible for a slow sustained shortening in the outer hair cells are still not known, but the idea is supported in many laboratories that the actin-myosin interaction could induce the shortening in a manner similar to that which occurs in the smooth muscle and non-muscle cells. Consequently, CAL-induced activation of myosin light-chain kinase is likely to take place as a part of the contractile process.

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Cisplatin which is an anticancer drug exerts anticancer effect by damaging DNA. Its utility is well known. However, the use of cisplatin is complicated by a number of side-effects, especially nephrotoxicity, nausea and vomiting, peripheral neuropathy, myelosuppressipn and ototoxicity. A number of analogues, which not only maintain the useful antitumor properties but are less toxic, have been developed. 254-S and DWA2114R have been received clinical trials in Japan. In this study, four platinum anticancer drugs of cisplatin (CDDP), carboplatin (CBDCA), 254-S, DWA2114R (DWA) were administered to the guinea pigs. And the ototoxicities of these drugs were investigated functionally and morphologically.
Male albino guinea pigs, weighing from 350 to 450g and exhibiting good Preyer's reflex, were used. The dosage rate of these drugs were decided to be 1: 3: 10: 10 (CDDP: 254-S: CBDCA: DWA) on the basis of the acute LD₅₀ value in mice. CDDP was administered intraperitoneally to the guinea pigs in three groups 5 mg/kg for 3 successive days. As for the other drugs, 254-s was administred in a dose 3 times the; CDDP and CBDCA, and DWA were 10 times that. Animals of all 4 groups were anesthetized with pentobarbital 3 days after the final administration. The animals were underwent tracheostomy and were ventilated with respirator by room air. The cochlea was then exposed by submandibular approach and a needle electrode was placed near the round window on the bony surface of the cochlea. The N₁ threshold of compound action potentical (AP) was measured through the electric stimulation of 100 filtered clicks of the 4 kHz at intervals of 100msec. After measurement of the AP, the inner ear was removed, fixed in 2.5% glutaraldehyde, and postfixed in 2% osmium tetroxide. After critical point dry following dehydration, the cochlea was dissected to expose the surface of the organ of Corti and coated with gold. The surface of the organ of Corti was observed using a HITACHI S-450 scanning electron microscope (SEM). In the middle part of each turn of the cochlea, 50 outer hair cells (OHCs) were observed and their stereocilia examined in each of the three rows. Thus, of a total of 150 OHCs examined in each turn, only those having normal stereocilia were counted. These distribution were recorded in a cochleogram as a record of the degree and the extent of damage to the organ of Corti.
N1 threshuld values of APin the maximum dose of each drug were 50.7±11.9dB SPL (CDDP), 47.3±18.2dB SPL (254-S), 37.4±4.5dB SPL (CBDCA) and 34.9±7.0dB SPL (DWA). The thresholds were increased by about 20dB for CDDP and 254-S as compared with the control group (31.3 ±4.2dB SPL), but the elevation of thresholds for CBDCA and DWA was slight. Comparing with the cochleograms of these four drugs, it can be seen that the surviving rate decreases with increased dosage in a dose-dependent fashion. In the cochleograms of the maximum dose of each drug, there was damage mainly in the basal to second turn. And in the basal turn, percent surviving of OHCs were 67.5±22.1% (CDDP), 71.5±31.4% (254-S), 90±11.5% (CBDCA) and 91.5±12.2% (DWA). Therefore, in the dosage rate of 1: 3: 10: 10, it was found functionally and morphologically that CDDPand 254-S caused strong damage of almost the same intensity, CBDCA and DWA caused mild damage to the organ of Corti.
The factors which affect ototoxicity are such as plasma half-life, binding rate of platinum to plasma protein, organ distribution, urinary excretion rate and the degree of nephrotoxicity. The plasma half-life of CDDP is longer and the percent of binding to plasma protein of CDDP is higher than other three compounds. Furthermore, 24-hrs urinary CDDP excretion is lower than other drugs. As a result of these pharmacokinetics, CDDP remains for longer in body fluids and thereby it can be suspected that strong side-effects arise.

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The endolymphatic sac of the guinea pig was observed by light and electron microscopy. The endolymphatic sac was divided into three portions by the morphological feature of the epithelium as previously described by Guild (1927); the proximal, intermediate and distal portion. The epithelial cells of the sac were classified into two types. One was characterized by numerous mitochondria, a large number of pinocytotic vesicles in the cytoplasm and well-developed microvilli at the apical surface (type 1), suggesting higher cellular activity of reabsorption of endolymph.
The other was characteristic for poorly developed cytoorganelles, fewer in number of microvilli and numerous intracellular filaments occupying the cytoplasmic matrix (type 2), suggesting supporting functions of the epithelium of the sac. Further, a few intermediate type cells between them are also present. The previous classification (i. e. light cell and dark cell, Lundqueist; 1965) is supposed to be owing to shrinkage of the cytoplasm by poor techniques of the specimen preparation. Neither type 1 cells or type 2 cells are so dark in their cytoplasms as dark cells reported in the previous papers. When the tissue materials are fixed by the perfusion method with glutaraldehyde solution, “dark cells” which have been reported in various organs are difficult to recognize. Similar opinions have been reported for various organs such as liver, exocrine pancreas, central nervous system, and so on.
In addition, some special regards were also given to the endocytotic activity of the endolymphatic sac. The luminal space of the sac was filled with India ink particles, 24 hours after the injection of the ink particles into the cochlear duct. Not only free floating cells in the luminal space of the endolymphatic sac, which were probably macrophages, but also both types of the epithelial cells took them up. Ink particles taken up are stored in large or small cytoplasmic vacuoles. The type 1 epithelial cells took them up better than the type 2 cells.

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The short-term influence of round window membrane (RWM) rupture on cochlear potentials was investigated with special reference to the endocochlear DC potential (EP). The RWM of the guinea pig was incised with a needle or a glass micropipette without imposing pressure in the perilymphatic space, and the EP as well as the cochlear microphonics (CM) and compound action potentials (AP) of second cochlear turn were recorded before, during and after the operation.
While the EP suffered a moderate decline (about 36 mV in two hours) in five of ten animals, it showed little change in the remaining five animals. Changes of the CM and AP seemed parallel to that of the EP.
This study suggests that hearing loss can be caused by a tear of the RWM; however, such loss does not necessarily occur even in the presence of an obvious rupture. The reduction of the EP is likely to be responsible for the hearing loss in some cases of fresh RWM rupture. However, this hearing loss may not be severe, as demonstrated by the relatively small degree of reduction of cochlear potentials in this study.

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Two fast and slow-components are seen frequently in records of stimulated otoacoustic emissions at the 20ms after stimulation. The fast and slow components were examined in ears with normal hearing and those of sudden deafness. Acoustic stimuli were tonze bursts. In sudden deafness, the incidence of fast components was 100% in normal hearing ears at middle frequencies, 96.3% in 27.5-54.5dB hearing loss ears and 68.4% in over 55dB hearing loss ears. The incidence of slow components was 50.6% in normal hearing ears, 14.8% in 27.5-54.5dB hearing loss ears and 0% in over 55dB hearing loss ears. The incidence, especially of the slow components decreased according to the grade of hearing loss. The mean psuedo-threshold of fast components was 16.3dBnHL in normal hearing ears with both co mponents and that of slow components was 3.8dBnHL.
The mean threshold of slow components was lower than that of fast components. Mean latency of fast components was 9.4ms and that of slow components was 13.6ms. The difference of the mean latency between the fast components and the slow components was c. a. 4ms. The gradient of input-output curves of the slow components was not so steep as that of the fast components. It was suggested that the slow components rather than the fast components might be related to more effective synchronization in cochlear negative damping.

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