The lateral wall of the cochlear duct dissected from gerbils or rats was studied by confocal laser microscopy. Specific structures in tissue preparations were stained with fluorescent dyes to clarify the following points: i) developmental changes in the capillary network in the lateral wall of the cochlear duct, ii) intercellular connection by gap junctions, iii) distribution of elongated cells connecting capillaries, and iv) three dimensional distribution of intermediate cells in the stria vascularis. These studies revealed structural characteristics related to specific functions of the stria vascularis.
FM1-43 is a fluorescent styryl dye to image optically the cycling of synaptic vesicles. The dye inserts into membranes but cannot cross them, and fluorescence is stimulated by contact with the membrane lipid. Thus, if endocytosis occurs in the presence of dye, dye is trapped in recycled synaptic vesicles or other endocytic organelles. Further exocytosis of the synapse causes loss of dye owing to release of dye-loaded synaptic vesicles. We demonstrate the recycling of vesicles in the inner hair cells isolated from guinea-pig cochlea. By applying FM1-43 extracellularly, the dye first appeared in the intracellular compartment around the neck region below the cuticular plate and spread towards the cell base. This result suggested that spontaneous endocytosis and exocytosis occurred in the cochlear inner hair cell. To exclude imaging artifacts arising from out of plane fluorescence, the distribution of FM1-43 was studied with greater resolution using the confocal microscopy. The dye first appeared in the apex of the cell and moved towards the base as observed by the conventional microscopy. We used whole organ of Corti in order to avoid the damage by cell isolation. Dye application demonstrated the trafficking of vesicles from the neck towards the basal area as shown in the isolated cells. External Ca2+-free solution inhibited dye uptake. In high-K solution, no uptake of FM1-43 was observed.
We investigated optical imaging of the evoked responses in chick embryo and mouse the cochlear and vestibular nucleus in the brainstem slices by electrical stimulation, of the vestibulocochlear nerve using a multiple-site optical recording system and an absorption voltage-sensitive dye, NK2761 and RH155. The spatiotemporal patterns of excitatory propagation in the cochlear and vestibular nucleus were shown with optical imaging. These optical signals were wavelength dependent and consisted of two components: spike-like fast signal and long-lasting slow signal. All responses were abolished by tetrodotoxin. The slow signals were eliminated under bath-applied Ca2+-free solution. The effect of Ca2+-free was reversible. Synaptic fatigue was observed when repetitive stimulation was applied to the vestibulocochlear nerve. These results suggest that the neural activities through the sodium channels gave rise to the fast responses and the slow signals corresponded to a postsynaptic potential. The present study indicated the feasibility of optical recording for revealing visually the synaptic transmission in the vestibular nucleus and cochlear nucleus in the brainstem with high spatiotemporal resolution.
The light is an ideal tool for biomedical researches, because it can interact with biological molecules without much perturbing them. Use of video systems including video cameras greatly improved a detection capability of light microscopes, so that even molecular images are now observable within the limit of resolving power. The confocal microscopy is useful for constructing high resolution 3D-images of cells and tissues. The evanescent field formed by the total internal reflection of laser light is also very useful for fluorescence observations of physiological responses on the cell membrane. Especially, an ultra high NA lens we developed helps create bright images of fluorescent molecules. Today, micro imaging at a molecular level is evolving rapidly and continuously.
Afferent dendrite swelling below inner hair cells (IHCs) caused by exposure to loud noise is very similar to that seen after administration of the glutamate into cochlea, so it was suggested that noise-induced hearing loss may be involved glutamate excitotoxicity. However, no previous studies directly examined that excess release of glutamate from (IHCs) during sound exposure and which relates to hearing impariment because powerful glutamate uptake systems remove the released glutamate in the synaptic clafts. GLAST is a glutamate transporter highly expressed in the cochiea. We now report that after acoustic overstimulation, GLAST-deficient mice exhibit increased accumulation of glutamate in perilymphs, resulting in exacerbation of hearing loss. These findings suggest that GLAST provides an important contribution to keep the nontoxic level of glutamate in the perilymph during acoustic overstimulation.
Although glucocorticoids are widely measured in serum and urine using high performance liquid chromatography (HPLC) and there are several reports on their measurement by HPLC in the organs, such as liver, kidney, spinal cord, and others, the measurement of glucocorticoids in the cochlea has never been carried out. We tried to measure the standard solution of metylprednisolone and prednisolone, though metylprednisolone solution was difficult to measure. Prednisolone solution could be clearly measured by HPLC method in the cochlea. At 1 hour after intravenous injection of prednisolone sodium succinate (100mg/kg), the whole cochlear and labyrinthine tissue were dessected and homogenized with water. The homogenate was centrifuged and its supernatant was subsequently subjected to steroid extraction. The supernatant was injected to the HPLC system and measured. We also tried to measure the concentration in perilymph. The prednisolone in the cochlea was measurable with HPLC method, i.e. 35.3 ng/cochlea (mean) after injection of 100mg/kg of prednisolone, whereas prednisolone in perilymph was not detectable in 4 of 5 animals. Therefore, the method of prednisolone detection adopted in the present study is useful for further studies to elucidate the mechanism of steroids in the cochlea.
In inferior vertebrate animals, active hair bundle motion links to the mechano-electric transducer current in auditory hair cells. In order to investigate whether the mammalian hair cells possess the property of active hair bundle movement, we measured the current change that was linked to the active movement in isolated inner hair cells of guinea-pig cochlea. In free-standing hair bundle, depolarization positive to +80 mV evoked a stable outward current attributable to the opening of transducer channels. Repolarization to-80mV evoked a transient inward current indicating the opening of transducer channels and fast adaptation followed. Time constant of adaptation increased as the membrane potential depolarized after repolarization. Extracellular application of 0.2mM dihydrostreptomycin diminished in both the outward and inward currents.
The growth of the air cell system with age was evaluated mainly by the planimetric method or the rectangular approximation based on the ear roentgenograms. On the contrary, our technique based on CT image proved to be much more useful because of higher accuracy and capability of 3D assessment. The detailed analyses has been obtained only through the postmortem histological study. Our method is, therefore, of high signicance because it allows to assess the antemortem developmental changes in infants. The study covered 43 males and 37 females, aged 1 to 18 years. The temporal bone was imaged by slicing at 2-mm thickness through the high resolution CT so that the entire air cell system was included, and the resultant data were analyzed by the image processing technique. It was confirmed that the volume of the infant's air cell system tended to increase with age. While the mean value of the air cell system volume in the adult is 5.97 ml, 80% cases of infants of 9-10 years, the system seemed to have grown already to this size. The 3D display of CT image allowed to study various morphological features of the system. The present report will describe our findings on the measurement of the mastoid air cell system of infant and its developmental changes with age.
Rapid progress of medical technology has contributed greatly to improve imaging ofthe temporal bone. The ability of high resolution computerized tomography (HRCT) provided exquisite contrast and spatial resolution. As a result, HRCT has been the current method of choice for the delineation of fine structures of the temporal bone. Helical scanning is one of the techniques in HRCT, and it has attracted a great deal of attention as an innovative diagnostic imaging technique for X-ray CT. The helical CT system is superior to conventional CT system in requiring a much shorter examination time, and enables us to get many more continuous images. We have been using this system since 1992. Since 1999 we have been able to use the new helical CT system. This system is able to perform the multislice scanning, and to obtain the thin slice thickness images as compared with the conventional helical CT system.
We conducted a retrospective survey of 371 ears operated on over a ten-year period from 1989 to 1998. All of the surgical procedures were performed by the same surgeon. Data included 260 ears with chronic otitis media (OMC) and 111 ears with middle ear cholesteatoma (CHOLE). The success rate of the surgery was analyzed using the three criteria suggested by the Japan Clinical Otology Committee in 1999. The procedure was classified as successful when the hearing level at 6 months after surgery met at least one of these three conditions: 1) a hearing level above 30dB, 2) a gain of more than 15dB, or 3) an air-bone gap of less than 15 dB. We used the autograft cartilage for a columella for ossicular reconstruction. Staged operation was not included in this study. In case of OMC, 209 ears recieved type I tympanoplasty, 45 ears modified type III, 6 ears modified type IV. In case of CHOLE, 31 ears recieved type I tympanoplasty and 67 ears modified type III, 13 ears modified type IV. In case of OMC the success rates were 83.7% for type I, 75.6% for modified type III and 16.7% for modified type IV tympanoplasty. In case of CHOLE the success rates were 87.1% for type I, 73.1% for modified type III and 46.2% for modified type IV tympanoplasty. The characteristics of the unsuccessful cases were examined. The causes of the unsuccessfull results in hearing improvement were the following, severe sensorineural deafness, the stapes adhesion, ossicular destruction due to a large cholesteatoma and severe inflammation around the stapes.
Between January 1990 and September 1999, 125 cases with middle ear cholesteatoma were operated in Mie University Hospital. Ninety-three cases (74.4%) were operated on by canal wall down tympanoplasty with canal reconstruction, 17 cases by canal wall down tympanoplasty, 13 cases by canal wall up tympanoplasty, and 2 cases by radical mastoidectomy. For the canal reconstruction, cortical bone was used for attic reconstruction in 59 cases, and auricular cartilage was used in 30 cases. Postoperative attic retraction was found in 40 of 59 cases (67.8%) with cortical bone reconstruction and 15 cases developed retraction cholesteatoma. In 32 of 40 cases (80%), attic retraction was found within 1 year after the surgery, and all retraction cholesteatoma occurred within 5 years. Attic retraction was not found in 28 of 30 cases (93.3%) with auricular cartilage reconstruction, and recurrent cholesteatoma did not occur. Revision tympanoplasty was performed in 14 of 19 cases with recurrent cholesteatoma: seven cases by auricular cartilage reconstruction, four cases by cortical bone reconstruction, and three cases by radical mastoidectomy. Attic retraction was not found after the revision surgery with auricular cartilage reconstruction, while all four cases with cortical bone reconstruction developed attic retraction again. Auricular cartilage is useful for attic reconstruction to prevent retraction cholesteatoma.
Eight to nine small epithelial pearls were found on the mastoid antral wall of the right ear of a 57-yearold woman. Hearing test revealed 17dB conductive hearing loss in the right ear. CT showed poor mastoid air cell development, an aerated small mastoid antrum and cloudy attic with the almost intact malleus and incus. The authors diagnosed the patient as having attic cholesteatoma, with the cholesteatoma matrix possibly split and disseminated by severe acute middle ear infection during childhood. The multiple epithelial pearls formed from the split matrices during a rather quiet recovery period, and remained unchanged since. The reparative process after acute middle ear infection may sometimes produce osteoplastic changes around the attic cholesteatoma, with the ear becoming sclerotic as in the present case. In such cases an epithelial split is also occasionally observed. Changes often encountered include a second cholesteatoma isolated from the attic cholesteatoma, or a disfigured cholesteatoma hidden behind newly formed bony structures but still connected to the attic cholesteatoma. We speculated that a similar process, but with only a slight bony ingrowth, might have occurred in the present case of multiple cholesteatoma.
Delayed endolymphatic hydrops (DEH), a disease entity that can be differentiated from Meniere's disease, develops in patients who have experienced a profound hearing loss. Kamei, Nadol and Wolfson first reported this condition. Schuknecht classified DEH into three types, ipsilateral, contralateral, bilateral. DEH becomes gradually established as a clinical concept though the diagnostic criteria seems to be incomplete. In this study, we considered the criteria and made a few alterations to them. Further, we reported clinical features of 50 cases of DEH. Twenty-one cases were ipsilateral type, 15 contralateral type and 14 bilateral type. Causes of hearing loss were sudden deafness in 18 cases, juvenile unilateral hearing loss in 9 cases, otitis media in 5 cases, mumps in 3 cases, noise trauma in 2 cases, head trauma in 2 cases, rubella in 1 case, ototoxic drug in 1 case, unknown in 9 cases. Ipsilateral type was predominant in the cases of sudden deafness and contralateral type was in the cases of juvenile unilateral hearing loss. The latency between early history and late history ranged from 3 months to 72 years and averaged 19.4 years. In the cases of sudden deafness was short (average 8.2 years), but in juvenile unilateral hearing loss, the latency was much longer (average 38.3 years).
Cl-channels, which are expressed in a wide variety of cell membranes, are known to play important roles in the resting potential formation, excitability modulation and fluid transport. Recently, it has become evident that Cl-channels have other important functions as well. This review describes three of them: the pathways for release of autocrine/paracrine signal ATP and for volume-regulatory Cl-efflux involved in the regulatory volume decrease and the apoptotic volume decrease.