Shigetaro Shibata

Two Modes of Auditory Hair Cell Loss following Acoustic Overstimulation in the Avian Inner Ear

To determine the type of cell death occurring and how the removal of damaged cells proceeds following overstimulation, we examined chick basilar papillae using an in situ DNA nick end labeling method and transmission electron microscopy. Two distinct modes of hair cell loss were identified. First, hair cells which had not progressed into typical cell death processes, apoptosis or necrosis, were deleted by extrusion from the epithelium just after sound exposure. Second, hair cells manifested degeneration through the process of apoptosis, then further deterioration within the epithelium after the beginning of the process of hair cell regeneration. The latter mode may contribute to the following repair processes.

Apoptotic cell death in Kikuchi's disease : A TEM study

The pathogenesis of Kikuchis disease (Kikuchi-Fujimoto disease, histiocytic necrotizing lymphadenitis) remains unclear. However, some previous studies have suggested that a disorder in cellular immunity is responsible for this disease, and apoptotic cell death appears to be the principal finding in the histogenesis of this disease. In the present study, a lymph node from a female patient with Kikuchis disease was examined by transmission electron microscopy (TEM). TEM revealed specific morphological features of apoptotic cells, such as nuclear chromatin condensation and fragmentation along the nuclear membrane with intact organelles, and the presence of histiocytes phagocytosing karyorrhectic debris (apoptotic bodies) in areas affected by Kikuchis disease. Although neither the role nor the trigger of apoptosis in Kikuchis disease has been clearly determined, our findings show that apoptotic cell death clearly plays a role in the pathogenesis of Kikuchis disease.

Study of tuberculosis in the field of otorhinolaryngology in the past 10 years.

Over the 10 years from 1988 to 1997, a total of 18 cases of tuberculosis of the head and neck (8 of the cervical lymph node, 5 of the larynx, 2 of the salivary glands, and 1 each of the hypopharynx, tongue and middle ear) were diagnosed in our department. Four cases were diagnosed in 1996 and 8 in 1997, indicating a rapid increase over these 2 years. Definitive diagnosis was based on histopathological examination in 14 cases, detection of M. tuberculosis in 1 case and exclusion diagnosis, mainly using the tuberculin skin test, in 3 cases. The length of time from the first examination to obtaining a definitive diagnosis was 14.2 +/- 6.5 days in five subjects in whom tuberculosis was suspected from the beginning or in whom tuberculosis was suggested by the physician in making a differential diagnosis, 25.3 +/- 17.2 days in the group in whom malignant tumour was suspected and 64.3 +/- 57.3 days in other cases. Although no significant difference was found (p = 0.077), the length of time until diagnosis tended to be shorter in the cases that were suspected of having tuberculosis from the beginning. Family history and past history of tuberculosis, contact with a tuberculous patient and abnormal findings at chest X-ray were examined as background factors, and some of these were present in nine out of the 18 cases. In the five cases diagnosed in the early stage, some of the background factors were found in four cases. However, no significant difference in the time until definitive diagnosis was found between the groups with or without background factors in all cases (p = 0.675). These results suggest the importance of considering tuberculosis when performing differential diagnoses.

Epithelial-Myoepithelial Carcinoma:An Unusual Tumor of the Paranasal Sinus

A 65-year old woman presented with nasal obstruction and on examination was found to have a huge mass in the maxillary sinus. This was removed, and histological examination revealed a mixture of trabecular structures consisting of inner dark cells, outer clear cells and solid structures consisting of only clear cells. Immunohistochemical examination showed the clear cells to be positive for α-smooth muscle actin. Ultrastructural examination confirmed the myoepithelial cell origin. The characteristic morphological, immunohistochemical and ultrastructural features aided in the diagnosis of epithelial-myoepithelial carcinoma.

Imprints of the tectorial membrane following acoustic overstimulation and kanamycin treatment.

Normal imprints in guinea pigs were examined mainly using a scanning electron microscope (SEM). Morphological changes in imprints after loading of the conditions mentioned below were also investigated. Imprints observed on the normal tectorial membrane were composed of small concavities lined on a W-shaped line and the W-type was of a V-type in proportion to ascend to the upper turn. The imprints occurred at the rate of one for every outer hair cell and in 3-4 lines corresponding to the outer hair cell hairs. With exposure to a high intensity of sound, small concavities of the imprints were deformed and remnant sensory hairs were observed, sporadically. Another line of new imprints was sometimes evident in the lower turn of the cochlea, in addition to the original imprints. In case of inner ear disturbances due to kanamycin (KM) loading, the imprints were little deformed, but the remnant sensory hairs were numerous and their time-course revealed a gradual decrease and the disposal required a longer time than seen in the degenerated cell fractions on the reticular membrane. With exposure to high intensity sound after KM loading, KM-type changes or high intensity sound-type changes were observed and depended on severity of the KM-induced disturbance. The imprints existed for a considerably long time even when the lower region sensory cells degenerated or disappeared in the presence of various conditions.

Assessment of Hair Cell Death Using the Dye Extrusion Method

We examined the usefulness of the dye extrusion method for assessing cochlear hair cell death in guinea pigs. Unilateral temporal bones were incubated in sterile Dulbecco modified Eagles medium for 1-6 h and then perfused with PBS/0.3% trypan blue to determine hair cell viability. Cells with nuclei stained with trypan blue were assumed to have died. The contralateral temporal bones of each animal were incubated with the same medium for 1-6 h and examined by transmission electron microscopy (TEM). This method revealed that hair cell viability under these conditions was maintained for at most 3 h. After incubation for more than 3 h, hair cell injury began and increased. After 6 h incubation, TEM revealed apoptotic death of outer hair cells. The results with this method agreed with the TEM findings. The dye extrusion method is simple, easy and useful for screening hair cell death in the cochlea under pathological conditions.

Cleavage Product from the NO Donor NOC-5 and Inner Ear Hair Cell Damage

Nitric oxide (NO) is a neurotoxic gas which causes neuronal cell death. NO ototoxicity has also been reported, 1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5), NO donor, was used for studying NO. In this in vitro study, we examined whether NOC-5 and the cleavage products arising from it after production of No have ototoxic effects on the cochlea. The temporal bones of 55 guinea pigs were dissected and incubated in Dulbecco Modified Eagles Medium (DMEM) with low glucose and 10% foetal bovine serum (FBS) perfused with various concentrations (100, 50, 10, 1 mM, 100 microM) of NOC-5 (pH 7.4) or the same concentrations of N-isopropyl 1-3 propanildiamine (R-NH2). Hair cell death was assessed using trypan blue staining. With NOC-5, percentages of outer hair cell death were 99.2, 98.0, 45.2, 40.5, and 20%, respectively, and percentages of inner hair cell death were 69.3, 70.2, 22.5. 11.1 and 0%, respectively. With R-NH2, percentages of outer hair cell death were 90.3, 81.1, 29.8, 21.0 and 19.3%, respectively, and percentages of inner hair cell death were 61.4, 48.1, 10.2, 5.4 and 0%, respectively. Ultramicroscopic examination showed that hair cell death was necrotic. Although NOC-5 is useful for study of NO, its cleavage products are also ototoxic.

Role of Amino Acids in Cochlear Degeneration: Morphological Changes in Cochlear Outer Hair Cells Following Glutamate Application

Glutamate is an excitatory neurotransmitter in the cochlea and has toxic effects on the organ of Corti in various pathological conditions. The toxic effects of glutamate have not been determined in detail. In this study, we examined morphological changes in the organ of Corti of guinea pigs following local application of glutamate. Morphological changes were noted in outer hair cells. Degeneration of outer hair cells was found 24 h after glutamate treatment. The extent of degeneration depended on exposure time. Inner hair cells did not exhibit any degeneration. In addition, no degenerative changes were detected in nerve endings attached to hair cells. These findings suggest that outer hair cells are the initial site of degeneration caused by application of excess glutamate to the inner ear.

Role of amino acids in cochlear degeneration: deprivation of cystine induces death of cochlear hair cells of guinea pigs in vitro.

Redox regulation reportedly plays a role in maintaining cochlear homeostasis. However, little is known about the roles of oxidation-reduction systems in the cochlea. We examined the role of the cystine/cysteine oxidation-reduction system in survival of cochlea hair cells in vitro. The survival of hair cells was evaluated in cochlea specimens following incubation with the medium supplemented with various concentrations of cystine. Dying hair cells were detected by the trypan blue extrusion method. The rates of cell death for both outer and inner hair cells increased significantly with a decrease in the concentration of cystine. In addition, the rate of cell death of IHCs tended to be higher than that of OHCs. These findings suggest that the cystine/cysteine system might be required for maintenance of homeostasis in cochlear hair cells, especially in IHCs.

Surgical Treatment of Cancer Extending from the Retromolar Fossa to the Masticator Space

Cancer extending from the retromolar fossa to the masticator space with neck metastasis (T4N2M0, Stage IV) was surgically removed en bloc by maxillectomy using a mandibular swing technique. This approach allowed a wide surgical view and was useful as handling the external and internal pterygoid muscles and upper neck dissection could be combined. Though there have been several surgical approaches to the masticator space, in the case of tumors which are commonly malignant there, this approach is useful and satisfactory.