Morphological changes of the sensory hairs due to transtympanic infusion of fluids with different osmotic pressure were scrutinized using color guinea pigs. Animals were sacrificed after four days fluid injections and examined by scanning E. M. Transtympanic infusion of distilled water (0 mosmol), physiological saline solution (285) and 5% (148), 10% (315) saccharose solutions demonstrated no abnormality on the sensory hairs. 20% saccharose solution (720) and 2.4%NaCl solution (743) revealed moderately change of twistning, adhesion and fusion of the sensory hairs. 30% saccharose (1224) and 4%NaCl solution (1248) demonstrated ovious changes the sensory hairs.

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Pathologic change of the round window membrane (RWM) in guinea pigs induced by the endotoxin from E. coli were observed by the electron microscopy and the effect of the endotoxin on the permeability of the RWM was examined by a tracer using horseradish perioxidase (HRP).
Twenty four hours after injection of the endotoxin (Sigma, 100 μg/ml) into the bulla, RWM invariably demonstrated inflammatory changes remniscent to those of middle ear effusion: The subepithelial space was characterized by marked edema, where a large number of neutrophils and red blood cells and a few mononuclear cells appeared. These infiltrating cells were seen often migrating in scala tympani but no definite damages to the organ of Corti present.
When exposed to HRP, these pathologic RWM demonstrated normal uptake of the tracer into the epithelial cells as well as into some of the fibrous cells of the middle layer. However, we could not confirm increase of permeability of RWM, as evaluated by the amount of intracellular uptake of the tracer.

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Seven cochleas from 5 rats with spontaneous otitis media and 32 cochleas from 16 rats of similar age without otitis media, were examined by scanning electron microscopy to quantify cochlear hair cell loss. Loss of hair cell stereocilia was found in 3 cochleas with otitis media and was significantly increased when compared with uninvolved ears from animals of similar age. The hair cell loss associated with otitis media appeared to be most marked in the upper turns of the cochlea and increased in severity with the inflammation of the middle ear. The greater damage to hair at the basal turn suggests that this may be the result of influence of the middle ear inflammation on the cochlea through the cochlear window.

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Perilymph and endoltmphatic pressures were measured simultaneously using two sets of a servo-nulling system in guinea pigs, and the pressure difference between them associated with subarachnoid infusion was studied. Subarachnoid infusion of saline caused pressure increase in both the perilymph and the endolymph equally in normal guinea pigs. Similar results were obtained in the guinea pigs with surgical obstruction of endolymphatic sac and duct. Reissner's membrane may not have stiffness to hold the pressure difference between them.

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Pressure transmission from the external ear to the inner ear was studied in guinea pigs after closure of the Eustachean tube. The pressure was slowly applied to the ear canal to reach a peak of 1000 or -1000 mmH₂O. When the positive pressure was applied, pressures of the middle ear and inner ear increased up to around 400 mmH₂O. and 200 mmH₂O, respectively. When the negative pressure was applied, they decreased to -100 mmH₂O and -50 mmH₂O, respectively. This means that positive pressure load to the external ear is transmitted more than negative one. The decrease in the inner ear pressure during pressure loading is due to patency of the cochlear aqueduct. Rupture of the round window membrane occurred by the pressure load over 1000 mmH₂O.

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To study auditory and vestibular disorders following chronic perilymphatic fistula, we inserted a small tube into the scala tympani of the basal turn of the cochlea of guinea pigs. For observation of nystagmus in the guinea pig, specially fashioned Frenzel's glasses were used. Histopathological examination was done one month after the perilymphatic fistula was made. Our results showed the existence of directional preponderance of post rotatory nystagmus with corresponding endolymphatic hydrops, hair cell damage and partial injury to the vas spirale around that hair sell damage. We suspect that some reduction in the blood supply resulted in hair cell destruction.

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The cochlear potentials (EP, CM and AP) were monitored during an experimental labyrinthectomy in the guinea pig. The potentials were recorded with a glass microelectrode inserted in the scala media of the first or second cochlear turns.
The potentials suffered little change during and after drilling of the lateral semicircular canal. Although subsequent drilling of the vestibular labyrinth caused decline of potentials, the EP was quite stable before the vestibule was opened. The recovery of EP was observed in one ear of the three whose vestibule were packed with gelatin sponge.

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The influence of round window membrane (RWM) rupture on the cochlear potentials was investigated with special reference to the EP. The RWM of the guinea pig was incised with a needle or a glass micropipette and the EP as well as the CM and AP of second cochlear turn were recorded before and after the operation.
Although the EP suffered considerable decline in five of 11 animals, it showed little change in the remaining six animals. Changes of the CM and AP seemed parallel to that of the EP. This study suggests that hearing loss can be caused by the tear of the RWM, however, it does not necessarily happen even in the presence of the obvious rupture.

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Studies on the patency of the cochlear aqueduct were made on 330 temporal bones histopathologically, and clinical significances of cochlear aqueduct patency were discussed. Large patent cochlear aqueduct of 0.2mm and over in diameter at its narrowest point in the otic capsule was found in 25 out of 330 temporal bones, i. e., 7.6% of bones. Patency of cochlear aqueduct renders possible round window membrane rupture through rises in cerebrospinal fluid pressure, perilymph oozer, and a two-way spread of infection between the perilymph and cerebrospinal fluid.

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We have reported the concentrations of furosemide (FU) and penicillin G (PG) in the scala tympani (ST) and in the scala vestibuli (SV) of the guinea pig cochleae to clarify the transportaion mechanism of organic anions from plasma to perilymph. In this study, the concentrations of salicylate (SA) and aspirin (ASA) in sequential samples from ST and SV perilymph of the guinea pig after intravenous injection of 100mg/kg SA or ASA were measured by high-performance liquid chromatography. The SA kinetics in the ST perilymph are very similar to those in the SV perilymph. These results are not analogous to our FU or PG experiments suggesting the pronounced difference of anion transportation mechanism between the ST and the SV. The different transport mechanism between SA and FU is suggested in the ST of the guinea pig.

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Glycerol concentrations in CSF, serum and perilymph after intravenous administration (3.0g/kg) were measured by fluorometric direct assay. Less than 2μl of perilymph (scala tympani) sample was taken from the third turn of the guinea pig cochlea after blocking the cochlear aqueduct to prevent the CSF contamination. The concentratoins of glycerol in CSF and perilymph lagged behind the serum increase. The concentration of glycerol in CSF reached the maximum value within 30 minutes after administration. A peak concentration was obtained in perilymph at 60 minutes with a subsequent slow decline. The concentrations in perilymph were lower than those in CSF at any time after injection of glycerol. These results suggest the hydrostatic pressure in perilymph is reduced more effectively than in CSF through the injection of an osmotic agent (glycerol) into systemic circulation.

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The effects of glycerol and mannitol on oxygen tension of the cochlear perilymph were studies using a polarographic technique in the guinea pig. Following a transient fall immediately after the intravenous administration of each drug, the oxygen tention increased to reach a peak 5 to 10 minutes later, and then decreased gradually. The magnitude and duration of the oxygen tention increase were larger after the administration of glycerol than that of mannitol. These results were in good agreement with the clinical data that glycerol was more effective for producing hearing gain in Meniere's disease patients than mannitol. The increase of oxygen tension in the cochlea, therefore, is considered to be one of the possible contributory factors in the improvement of hearing in patients with Meniere's disease after glycerol administration.

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The amino acids and their conversions of the perilymph in the scala tympani were measured by using two types of High Performance Liquid Chromatography. The perilymph was collected into the micro-glass pipette via the round window. The samples were pretreated and were derivertized with OPA or PITC, and were analyzed to clarify the kinds and the concentrations. All kinds of amino acids, Tau, GLN, ASN and ORN were detected in the normal perilymph. The rate of Taurine was 2.79% and the content in the perilymph was about 28μmol/1, while it was increased with the contamination of the serum (130μmol/1).

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The amino acid content of perilymph in the scala tympani of guinea pig was measured by using High Performance Liquid Chromatography. After ligation of one-side anterior inferior cerebellar artery by using ventral surgical approach, the perilymph was collected in both-side via the round window. Animals were divided into three experimental groups; immidiately, one hour later and three hours later after ligation. Both CSF And arterial plasma were collected severally. All the samples were derivertized to the PITC-amino acid and analized the concentrations. Aspartic acid one hour later, and glutamic acid three hours later increased in the ligated side.

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The perilymphatic space was perfused with artificial perilymph containing 5×10^-3M potassium canrenoate, measuring the EP, K⁺ and Na⁺ activity in the scala media with double barrelled K⁺ or Na⁺ selective microelectrode. Following the onset of the perfusion, the EP gradually declined and was stable after about 20 minutes. K⁺ activity also declined gradually but Na⁺ activity unchanged. When the EP became stable, the artificial ventilation was stopped, the EP dropped to a large negative potential and K⁺ activity decreased gradually but Na⁺ activity increased by degrees. The same results were observed in the normal animals when the ventilation was stopped. There are no pathological changes in the TEM view of the stria vascularis. These results suggest that this drug especially affect K⁺ conductunce of the stria vascularis.

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The first irritative and then the second paralytic nystagmus are observed after introducing KCl solusion to the tympanic cavity of ginea pigs. This is thought to be induced by the change of K⁺ consentration delivered through the round window to the labyrinth. By measuring the time course of the K⁺ activity change in the perilymph using the K⁺ specific microelectrade method, the following result was obtained: K⁺ activity of perilymph increased so rapidly to higher degree in 20 minutes after introducing KCl solusion and then decreased gradually to the half of the peak in 120 minues.

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After the application of saturated NaCl solution to the round window membrane, the K⁺ or Na⁺ activity was recorded in perilymph or endolymph of the basal turn with the double barrelled K⁺ or Na⁺ selective microelectrode for 60 minutes. The K⁺ and Na⁺ activities in the scala tympani rapidly increased and then decreased. The K⁺ activity in the scala media also rapidly insreased and then decrased, but the Na⁺ activity continued to increase during 60 minutes. After the application the K⁺ activity in the scala vestibuli did not change, the Na⁺ activity slightly increased. These results suggest that the K⁺ and Na⁺ clearances are higher in the scala vestibuli than in scala tympani, and Na⁺ clearance is lower than K⁺ clearance in the scala media.

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Guinea Pig Specific Cytomegalovirus was inoculated to the heart, cochlea and endolymphatic sac obliterated animals. Posterior modiolar vein (PMV) was histologically observed to evaluate its importance in viral load. Viral antigen was found in PMV in intracardiac and intrasaccular inoculated animals. The inflammatory cells were found in and around PMV and these cells were pouring from this vein to the scala tympani in other group. These finding indicate this vein play an important roll to communicate systemic circulation to the cochlea in viral lord.

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Guinea Pig Specific Cytomegalovirus (GPCMV) was injected into the scala tympani (ST) of 24 guinea pigs, they were sacrificed on days 2, 4, 6 and 8 after the viral inoculation. The threshold of compound AP was measured on day 0 and on the days they were sacrificed. The histological investigation of their cochleae was also performed.
The compound AP threshold of seronegative animals elevated gradually, and it showed no reaction on day 8. While that of seropositive snimals kept its own level.
Exemination of the cochleae revealed hemorrhage and mild cell infiltration in the ST and in the lower part of the spiral ligament on day 4; moderate cell infiltration including macrophages and giant cell in the ST on day 6; and the typical cytomegalic inclusion cell, fusion cells and degeneration of the spiral ganglia and organs of Corti on day 8. The sequent elevation of the compound AP threshold seemed to correlate well with histological damages of the cochleae.

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Inoculation of herpes simplex virus type I (2×10³pfu/2μl) into the cochlea of guinea pigs through the round window membrane produced viral labyrinthitis. Marked fibrosis filled the scala tympani with partial ossification. Endolymphatic hydrops with atrophy of the stria vascularis was also a common finding throughout the whole cochlea. The organ of Corti took on a strange appearance. Loss of the outer hair cells was the first step of its degeneration process. Its outer surface markedly ballooned into the cochlear duct, severing the heads of pillar cells, and the cells eventually disappeared. These hydropic changes in the organ of Corti are discussed.

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BSA sensitized cochleae in secondary immne response (GR-I) and immune complex reaction (GR-2) were studied morphologically. Vacuolar degeneration, swelling of intermediate cells of stria vascularis, protrusion of marginal cells into endolymphatic spase were seen (GR-I). Slight hydrops was present mainly in the basal turn (GR-2). No IgG deposits were found in either group. It appears that the possibility of immune complex deposits in CSS causing direst injury to the vessels of the cochlea is small. These dissimilarities between renal and cochlear tissue is because of differences in regional blood flow, capillary endthelial cells and the existence of the blood-labyrinth barrier.

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Two weeks following direct antigen (KLH) secondary challenge of the endolymphatic sac (e. sac), caloric responses were significantly suppressed in the KLH challenged ears as compared to the unchallenged ears. Perilymph antibody levels to KLH were found to be significantly elevated in the KLH challenged ears. The local accumulation of plasma cells and lymphocytes was seen in the perisaccular space and the e. sac lumen. Haemorrhage in the vestibule, vacuolization of hair cells in both of the macula and the lateral crista and endolympahtic hydrops in both of the saccule and the cochlea were seen.

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The immunocytochemical distribution of γ-aminobutyric acid (GABA), GABA synthesizing enzyme; glutamic acid decarboxylase (GAD) and degradative enzyme; GABA transaminase (GABA-T) in the guinea pig organ of Corti was investigated.
Although GABA-like immunoreactivity was found predominantly in the third and fourth turn, GAD and GABA-T-like immunoreactivity was present throughout all turns of the cochlea. These findings indicate GABA system may play an important role not only in the apical turn but also in the lower turn.

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Immunohistochemical methods were used to inyestigate the development of inner ear immunocompetent cells in BALB/c mice over a 3-week period as a result of secondary inner ear response against keyhole limpet hemocyanin. Macrophages and granulocytes were observed at 6 hours post. challenge in the endolymphatic sac (ES) and cochlea, and rapidly increased in both sites. T cells gradually increased in the ES and cochlea after challenge. The predominant immunoglobulin-bearing cell in the ES was IgG, followed by IgM, with IgA seen late in the response. Suppressor T cells were detectable in the ES and cochlea by 3 weeks post. challenge. We conclude that the inner ear has the capacity to mount as immune response through the accumulation of needed immunocompetent cells.

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The effect of direct antigen (KLH) challenge of endolymphatic sac (e. sac) on caloric responses was investigated in guinea pigs. Neither caloric responses nor inner ear endorgan was found to be significantly damaged in the primary KLH challenged ears as compared to the unchallenged contralateral ears. Seven days following secondary KLH challenge of the e. sac, caloric responses were significantly suppressed in the challenged ears as compared to the unchallenged contralateral ears. Caloric responses were distinguished into three types which were a) mild-reversible, b) severe-reversible and c) severe-irreversible.
Vacuolization of the sensory hair cells of the lateral crista was seen in the severe-irreversible response type.

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Subpopulations of lymphocytes existing to rat endolymphatic sac (ELsac) were examined after inner ear challenge of KLH (keyhole limpet hemocyanin) following systemic sensitization, employing immunostaining with ABC methods. A large number of lymphocytes was seen in perisaccular space (PSS) of challenged side. B cells bearing IgG were predominantly seen, but IgM or IgA bearing B cells were fewly seen. T cells as well as B cells were mobilized to the perisaccular space, expressing phenotype markers of helper/inducer or cytotoxic/suppressor on their cell surface. These findings indicate that T-B interaction takes place at endolymphatic sac, mounting and modulating the local immune response in the inner ear.

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Keratin distribution in cochlear cells has been studied immunoelectronmicroscopically by both pre-and post-embedding methods. Keratin immunoreactivity in guinea pig cochlea was distinctly demonstrated in: Hensen's cell, reticular laminae surrounding both outer and inner hair cells, outer and inner pillar cells, Claudius' cell, inner and external sulcus cells, interdental cell of spiral limbus, Reissner's membrane, border cell, inner phalangeal cell, Deiters' cell and spiral prominence cell. Keratin expression at ultrastructural level showed a non-filamentous keratin system in the cytoplasm of cochlear supporting cells. It may be an interesting approach for further study.

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5 mg of lipopolysaccharide derived from E. coli which was dissolved into 1 ml of water was injected through the femoral vein. 2 hours after the injection of endotoxin, horseradish peroxidase (HRP) was injected through the femoral vein. 2 minutes after the injection of HRP both cochleas were infused with a fixing solution of 2% glutaraldehyde through the previously opened bullas. 5 minutes after infusion of the fixative, guinea pig was decapitated. Ultrathin cross sections of each turn of cochlea were taken at several locations and observed through an electron microscope. Blood sludging was observed in many strial capillaries in upper turns. In third and forth turns, HRP reaction product was not observed in 10% of the strial capillaries, which meant the strial blood flow was completely blocked.

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The physiological and morphological changes in the inner ear of the mice induced by intravenous injection of M. chelonae were investigated. Evaluation of ABR were risen in liminal value, especially in high frequency. These changes began 3 days after injection. 14 days after injection, 35% of mice showed abnomal behavior, so-called “spinning disease”. Morphological damages in the cochlear and vestibular sensory cells were observed using SEM. In the organ of Corti, the inner and outer hair cell cilia had degenerated and disappeared. In the vestibular end organ, disappearance, fusion, and ballooning of the sensory hairs were observed and cross-links and rough nature of the sensory hair membrane have disappeared.

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Ultrastructural changes in vestibular endorgans folloing administration of CDDP (cisplatin) were investigated using guinea pigs. Various patterns of degeneration, such as swollen mitochondria, dilatation of vesicles of Golgi's apparatus, various size of vacuoles were found in the sensory epithelia. These findings were more remarkable in type I hair cells, and in the central part of the crista ampullaris.

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The study examined the distribution of CDDP in the inner ear and kidney of guinea pig. Therefore searching for distribution of platinum, sections of inner ear and kidney were not stained with U, Pb and Os were examined with TEM. In the kidney dense electron perticle were found in the cytoplasms of renal tubles. In the inner ear few perticle were found, apert from dense electron substance in cytoplasms of, the outer hair cell. The stronger hearing acuity was impaired, the higher the freqency of, these findings became.

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The Present study was designed to examine the effect of bismuth subnitrate (BSN) on the ototoxicity of cis-diammine dichloroplatinum (CDDP), using normal golden hamster. BSN was poured into the stomach during five days. On the 6th day, CDDP was injected intraperitoneally at 10mg/kg or l2mg/kg for once. After observation with scanning electron microscope, the remaining of outer hair cells were counted from apical turn to basal turn, and cytocochleograms were made. In the investigation of the cytocochleograms, the rate of hair cells remaining was slightly higher in the BSN-given groups than in the BSN-ungiven groups, at the distance of 3-4mm from apex. This result was considered to show the protective effect of BSN against nephrotoxicity, although the relationship between the cochlea and the effect of BSN was unknown.

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Four kinds of anticancer platinum drugs (Cisplatin, Carboplatin, 254-S and DWA2114R) were administrated to the guinea pigs intraperitoneally. Ototoxicities of these four drugs were investigated electrophysiologically and morphologically. The group of Cisplatin was divided into three groups of 5mg/kg for 3 consecutive days, 5mg/kg for 2 consecutive days and 1mg/kg for 5 consecutive days. The ratio of doses per each administration of these drugs was 1 (Cisplatin) to 3 (254-S) to 10 (Carboplatin and DWA2114R), which are based on LD₅₀. From the result of N₁, threshold of action potential, ototoxicity of DWA2114R was milder than the other three drugs. And from the survived outer hair cell numbers (%) counted by scanning electron microscopy, ototoxicities of Cisplatin and 254-S were stronger than those of Carboplatin and DWA2114R.

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Hair cell damage in chick cochlea following chronic treatment of kanamycin was examined at light or electron microscopic level. Four day-old chicks were given intramuscular injection of kanamycin at a dose of 200mg/kg for 10 days and sacrificed 3 days after the last injection. Obvious damage of hair cells was identified in the middle to distal region on the basilar papilla, especially at its inferior side, while hair cells in the proximal region were morphologically intact. These results support our behavioral data suggesting low frequency selective effects of kanamycin in the avian inner ear.

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Transplacental Kanamycin (KM) distribution to inner ear of 30 to 60 day-embryo was investigated by immunohistological technique.
1) KM easily penetrated placenta and localized in the cochlea in every embryonal stage. This means that placenta does not have so tight barrier function to KM.
2) Drug pathway to organ of Corti consists of 2 routes; spiral limbus-tectorial membrane pathway and spiral vessel, its derivative-hair cell pathway.
3) Immaturation of strial capillary contributes to littel accumulation of KM in stria vascularis.

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Effect of streptomycin sulfate on the live period of the cultured vestibular ganglion cells was studied using cell culture method. The tissue in the 20th and 21st gestational day rat embryos was used. Streptomycin sulfate at 10, 20, 30 mg per 1ml was instilled in and number of cultured cells was counted on the 5th day in vitro. On the 10th day in vitro, culture cells were fixed and counted. Decrease of the live cells from the 5th to the 10th day in vitro was estimated and expressed in percentage. These results suggested that large doses (more than 20 mg per 1ml) had a marked effect on the survival rate of the cultured vestibular ganglion cells, but not in the 10 mg per 1ml.

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The ototoxicity of gentamicin (GM) and Kanamicin (KM) was analyzed using an organ culture system. Inner ear of mouse 16 day embryos were cultured for 5 days with or without GM or KM. The damage of crista ampullaris and macula utriculi of cultured inner ear was evaluated according to our ototoxicity score based on the morphological change by a light microscopic observation of serial sections of the samples. We gave one grade damage for each of the following criteria; 1) damage of apical surface of the hair cells, 2) debris in the endolymph space, 3) disappearance of the hair cells, 4) degeneration of the supporting cells. Using this system we showed that the effect of GM and KM was dose dependent and that GM was more toxic than KM at the concentration of more than 100 μg/ml. This organ culture system can serve as a useful and convenient model system of ototoxic evaluation.

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In the previous report, we demonstrated that cerebroside sulfate (CS) and ganglioside GM3 are major acidic glycosphingolipids in rat cochlea. These two glycosphingolipids are also major acidic glycosphingolipids in kidney and furthermore, the both lipids have negative charges. Therefore, the binding activity of one of aminoglycosides, tobramycin to these lipids was studied using [³H] tobramycin. On the thin layer chromatography, tobramycin seemed to bind cerebroside sulfate. On the column chromatograpy using cerebroside sulfate-octyl-Sepharose 4B or GM3 acid-Sepharose, adsorbed tobramycin was eluted with increacing ionic strength in elution buffer. It was assumed that the binding between acidic glycosphingolipids and tobramycin was. of weak ionic bond.

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Our previous reports showed that the cochlear spontaneous discharge rate was decreased following the injection of 5mg/kg of lidocaine to the aspirine-treated guinea pigs. This study is aimed to know whether or not changes of cochlear spontaneous discharge rate occur after the the cochlea is impaired by injection of kanamycin.
Guinea pigs were treated with intramuscular injections of kanamycin at a dose of 400mg/kg for 8 or 12 consecutive days, until the hearing loss appeared.
The cochlear spontaneous dischage rate was decreased significantly (p<0.05) following the injection of 5mg/kg lidocaine. Thethreshold to the tone stimuli was not changed.
It is suggested that the tinnitus originated from abnormal cochlear spontaneous discharges may be relieved by the injection of lidocaine.

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The effect of streptomycin sulfate (SM) on the sensory epithelia of the inner ear of guinea pigs were studied morphologically by light microscpoe and electron microscope. The guinea pigs, in which the right endolymphatic duct were oblierated surgically, were parenterally treated with SM 250mg/kg 15 times in one month.
The study revealed that SM caused more remarkable damage on the vestibular sensory cells of the operated side compared with that of the opposite. Degenerations of the vestibular endoorgans were (1) loss of sensory hairs of the cristae ampullares and maculae, (2) flattening of cristae ampullares. Those were the most remarkable in the lateral ampulla. The organ of Corti remained intact on both sides.

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Protective effect of cepharanthine against ototoxicity induced by Kanamycin was investigated in monkey.
KM was injected intramusculary at 200mg/kg continuously for 28 days, and cepharanthine was injected intramusculary at 1mg/kg and 5mg/kg simultaneously with KM.
Cochlear damage was studied by ABR and SEM. The cochlear damage of monkey administered both KM and cepharanthine was more severe than that of monkey administered KM only.
But presuming by the changes of body weight, the result might be strongly affected by indivisual difference of nephrotoxicity.

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Ethacrynic acid (EA: 30mg/kg, not effective itself for threshold change of guinea pig's cochlea) potentiated markedly the ototoxicity of gentamicin (GM: 150mg/kg) injected 90 min before the treatment of EA. GM was detected mainly in the outer hair cells (OHC) of the cochlea 3 to 24h after EA injection with a gradient of concentration toward basal turn. When put into a sound proof room immediately after EA injection, animals showed no significant threshold shift at all frequencies until 24h, although GM appeared in the basal OHCs even at that time. Furthermore, we found the initial GM uptake around the basal body of OHC. These findings suggest that GM uptake is potentiated by the hair cell activation due to sound stimulation.

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Fine structures of the inner ear vessels in the guinea pig fetuses were observed by scanning electron microscope with casting method using Mercox resin. In 32 days fetus, the vascular pattern of cochlea and vestibule differed in appearance from those in adult. With olders than 40 days, the vascular patterns closely resembled those of adult. The vestibule in guinea pig, as well as human, advanced in development of blood vessel earlier than cochlea. The fetus blood vessel pattern was denser than in adults. It was assumed that the loss of blood vessels occurs in the inner ear after birth. Imprints on the corrosion casts of blood vessel in fetus were observed more deeply than in adult. The present indicates those structures were beneficial for their. development.

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It is our subject why otic capsule is cartilage bone. Recently, we are considerate that it is difficult to solve the problems in biological and biophysical methods and discussions have been evolutionally attempted.
The results have an intetesting bearing on nature of ossicles. Stapes is derived from hyomandibula in teleosts, malleus from articular and incus from quadrate. Skull of codfish shown in figure consists of dermal bone and cartilage bone.
It is suspected that these cartilage bone of teleosts is derived from palatoquadrate and hyomanddibula and dermal bone replaces with brain case in shark.

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The Localization of Na⁺, K⁺-ATPase activity was demonstratesd in the developing tegmentum vasculosum of the chick cochlea. At stage 37 epithelial folds (Lamellae) were relatively well developed. Light and dark cells were identified ultratructurally. At stages 41 to 42, K⁺-NPPase activity showing Na⁺, K⁺-ATPase appeared in the folded cytoplasmic processes of the dark cell. At stages 45 to 1 day old the number of mitochondria in the dark cell increased. The convoluted basolateral infoldings were highly developed. K⁺-NPPase activity was limited to the cytolpasmic processes of the dark cell.

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Rat embryos were explanted at 12.0 day and cultured for 24 hours in rotating bottles. At the time of explantation they were exposed to culture medium by the opening yolk sac method. Differentiation of the embryos was monitored by counting somites, and protein determinations were made to measure growth. In addition, serial sections were observed with the light microscope to elucidate the inner ear development in-vitro. During the culture period the mean somite number of the cultured embryos increased 9 in number and the mean protein content made the increment of 1.4 mg. Inner ear of the cultured embryos showed nearly as good development as that of 13.0 day embryos.

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The ultrastructures of the lateral cochlear wall were examined using rapid-freeze, freeze-substitution method. The cytoplasms of marginal cells were more dense than those of conventional chemical-fixed samples, especially in the marginal cells, since the cryofixation method can preserve the soluble cytoplasmic matrix more than the chemical fixation method. The plasma and intracytoplasmic membranes were clearly observed as trilaminar smooth contour. In the basal infolding region of marginal cells, the width of adjacent cell processes was very narrow and sometimes they were fused at their outer membrane layers. Such membrane fusions and typical gap junctions were mixed in that region. This method allowed a good preservation of membrane elements and brought a new ultrastructural findings.

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Extracellular matrix of the inner ear such as spiral ligament, tectorial membrane, basilar membrane, limbus, and perilymphatic space of both vestibule and semicircular canal, is composed of fibroblasts and many fine fibrils (10-13nm in diameter). Longitudinal section of those fibrils is straight and unbranched and cross section of them is rectangular. We reconfirmed such structural appearance of the extracellular fibrils in the tectorial membane using thin section method. Moreover, we showed clear stranded pattern of them using positive staining method. This stranded pattern is similar to the typical collagen fibril.

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The inner and outer hair cells and their nerve endings in the basal and third turns of the guinea pig cochleae were three-dimensionally reconstructed by means of computer graphics. These cells were also morphometrically analysed and comparad. In the inner hair cells, the total number of nerve endings in the basal turn was higher than that in the third turn. The majority of afferent endings were accompanied by typecal presynaptic bodies. The other data were almost equal in the basal and third turns. Meanwhile, in the outer hair cells, there were big differences between the basal and third turns in every data. The results of this study may suggest that the outer hair cells mainly distinguish the frequency.

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A change barrier has been found for the first time in the glomerular basement membrane of the kindey and plays an important role in the filtration of solutes. In the guinea pig cochlea, Tomoda et al have reported the similar structures located in the strial capillary basement membrane. In the present study, we observed electronmicroscopically the anionic sites of the charge barrier in the healthy guinea pig endolymphatic sac. The tissues removed after the decapitation were immersed in the solution of cationized polyethyleneimine (PEI, MW 1800). The results showed an arrangement of PEI with two strata in the basement membrane of the epithelium and the capillary. These sites function as a charge barrier, and their locations in the endoymphatic sac may play important roles in the filtration system of water, ion and macromolecules.

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