Issei Ichimiya

Immunolocalization of Na+, K(+)-ATPase, Ca(++)-ATPase, calcium-binding proteins, and carbonic anhydrase in the guinea pig inner ear.

The distribution of Na+, K(+)-ATPase, Ca(++)-ATPase, carbonic anhydrase, and calcium-binding proteins were investigated immunohistochemically in paraffin sections of guinea pig inner ears. Marginal cells of the stria vascularis, type II fibrocytes of the spiral ligament, and cells in supralimbal and suprastrial regions, were positive for Na+, K(+)-ATPase. Type I fibrocytes of the spiral ligament were positive for Ca(++)-ATPase, carbonic anhydrase, calmodulin and osteopontin. In the vestibular system, dark cells were positive for Na+, K(+)-ATPase. However, these cells and subepithelial fibrocytes were negative for Ca(++)-ATPase, carbonic anhydrase, and the calcium-binding proteins. In the endolymphatic sac, epithelial cells in intermediate and distal portions were positive for Na+, K(+)-ATPase, but the reaction was less than that in the stria. The same endolymphatic sac cells that were positive for Na+, K(+)-ATPase were also positive for Ca(++)-ATPase and calcium-binding proteins, but negative for carbonic anhydrase. The presence of Ca(++)-ATPase and calcium-binding proteins in the type I fibrocytes of the spiral ligament suggests that these cells are involved in mediating Ca++ regulation. Lower levels of Na+, K(+)-ATPase and the co-existence of Ca(++)-ATPase and calcium-binding proteins in the epithelial cells of the endolymphatic sac indicate that these cells have a distinctive role in ion transport that is different from that of the cells of the stria vascularis and vestibular dark cells.

Significance of spiral ligament fibrocytes with cochlear inflammation

It has been recently suggested that the spiral ligament fibrocytes, which interconnect with the basal cells of the stria vascularis via gap junctions, may be critical in maintaining cochlear homeostasis. In animal models of pathological conditions such as labyrinthitis and otitis media, reduced immunostaining for gap junction protein connexin 26 is observed in the spiral ligament. This suggests that disruption of the spiral ligament fibrocytes could be among the causes of cochlear dysfunction due to cochlear inflammation. Cultured spiral ligament fibrocytes have been shown to secrete chemokines and other mediators after stimulation of proinflammatory cytokine TNF-alpha or IL-1beta. Each of these mediators might induce inflammatory cell movement, which would prolong the inflammatory response. It is reasonable that such enhanced biological defense ability could be the cause of spiral ligament fibrocyte damage.

Effect of proinflammatory cytokines on cultured spiral ligament fibrocytes

To clarify the effect of proinflammatory cytokines on spiral ligament (SL) fibrocytes, in vitro studies were performed using secondary cell cultures. Cultures from murine SL fibrocytes were stimulated by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha, and secretion of various mediators was measured by enzyme-linked immunosorbent assay. After stimulation with the proinflammatory cytokines, IL-6, TNF-alpha, monocyte chemoattractant protein-1, KC, macrophage inflammatory protein-2, soluble intercellular adhesion molecule-1, and vascular endothelial growth factor levels were elevated. Secretion of these chemokines and other mediators could induce inflammatory cell movement, which would prolong the inflammatory response, leading to fibrocyte damage. Given that SL fibrocytes may play a role in cochlear fluid and ion homeostasis, such fibrocyte disruption could cause cochlear malfunction.

Changes in immunostaining of cochleas with experimentally induced endolymphatic hydrops.

Cochleas with experimentally induced endolymphatic hydrops were immunostained for Na+,K+-ATPase, intracellular Ca++-ATPase, carbonic anhydrase, aldehyde dehydrogenase, calcium-binding proteins, vimentin, and the gap junction protein, connexin 26. No changes in immunostaining of hydropic ears were observed 1 week after blockage of the endolymphatic duct. Two weeks to 1 month after the operation, immunostaining of type I fibrocytes in the spiral ligament, which are positive for all but Na+,K+-ATPase, was slightly decreased on the operated side. These changes became more pronounced 3 months after the operation. However, staining for Na+,K+-ATPase of the stria vascularis and of type II fibrocytes of the spiral ligament was not reduced until 6 months postoperative. The reduction of enzymes and other cell constituents that may be involved in ion balance of cochlear fluids indicates that cells in the spiral ligament play an important role in cochlear homeostasis and that they merit further study in animal and human otopathology.

Age-related changes in the murine cochlear lateral wall

Cochleas from C57BL/6 mice were investigated electrophysiologically and histochemically to evaluate the pathology of presbycusis. The average auditory brainstem response thresholds from 6-week-old mice were significantly lower than those of 6-month-old mice and those of 1-year-old mice. Histologic observation revealed changes in the cochlea after age 6 months. Conventional hematoxylin and eosin (H&E) staining showed disorganization of the organ of Corti, a decrease in the number of spiral ganglion cells, and atrophy of the stria vascularis. Although H&E staining and type II collagen immunolabeling did not show obvious changes in the spiral ligament (SL), the density of connexin 26 staining was reduced in this region. Sodium-potassium-adenosinetriphosphatase immunolabeling was increased in the SL, whereas its average density was not significantly altered in the stria vascularis. These results suggest that the SL could be among the regions responsible for cochlear malfunction with aging.

The influence of pneumococcal otitis media on the cochlear lateral wall.

The cochlear influence of otitis media was investigated in order to identify damaged regions causing cochlear malfunction. BALB/c mice were challenged with viable Streptococcus pneumoniae into the middle ear cavity and were killed 1 day to 1 month later for immunohistochemical analysis. Otitis media was induced in all of the animals, and some showed inflammatory cells in the cochlea. Although other changes were not obvious by hematoxylin and eosin staining, immunohistochemistry showed the presence of fibrinogen in the cochlea, mainly in the lower portion of the spiral ligament and in the spiral limbus. Immunostaining for connexin 26 was decreased in the spiral ligament, accompanied by marked fibrinogen staining. Immunostaining for sodium-potassium-adenosine triphosphatase in the stria vascularis and in the type II fibrocytes of the spiral ligament was not affected obviously. The presence of fibrinogen in the cochlea suggests disruption of the blood-labyrinth barrier caused by the middle ear inflammation. Changes in connexin 26 staining suggest the possibility that the spiral ligament could be among the regions responsible for the cochlear malfunction.

Localization of Glucocorticoid Receptors in the Murine Inner Ear

We performed an immunohistochemical investigation of the distribution of glucocorticoid receptors (GRs) in the murine inner ear and found that GRs were expressed extensively, but with various degrees of immunoreactivity in different regions. We observed the strongest GR expression in the type III fibrocytes of the spiral ligament. Although the immunoreactivity of the cochlear hair cells and of the vestibular sensory epithelia was weak, the neighboring cochlear supporting cells and the subepithelial regions of the vestibular sensory epithelia were immunostained. Staining for GRs was also positive in the spiral ganglia and vestibular ganglia, as well as in the endolymphatic sac. The role of GRs in the inner ear is discussed.

Functional architecture of the nasopharyngeal tonsil

The exact architecture of the normal nasopharyngeal tonsil remains obscure because most histopathologic investigations have been based on surgically removed adenoids. We compared enlarged adenoids and normal nasopharyngeal tonsils under both light and electron microscopes. The marked features of clinically enlarged adenoids were a large extension of the reticular epithelium and increased germinal centers. A tendency toward increased stratified squamous epithelium and decreased ciliated epithelium was apparent in enlarged adenoids, possibly due to inflammatory conditions. One type of nonciliated cell seemed to transport foreign material into underlying lymphocytes, as do the M cells of gut-associated lymphoid tissue. This type of nonciliated cell was rarely found in the extended reticular epithelium of enlarged adenoids. These findings suggest a disturbance of the antigen-trapping system and surface protections in adenoidal enlargement.

Dexamethasone inhibits tumor necrosis factor-α-induced cytokine secretion from spiral ligament fibrocytes

To investigate the effect of proinflammatory cytokines on spiral ligament (SL) fibrocytes and regulation of cytokines by dexamethasone (Dex), in vitro studies were performed in murine secondary cell cultures. Cultured SL fibrocytes were stimulated with tumor necrosis factor-alpha (TNF-alpha), and the secretion of various mediators was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcribed-polymerase chain reaction (RT-PCR). After stimulation with TNF-alpha, levels of keratinocyte-derived cytokine (KC), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) were elevated in the culture supernatant, and their corresponding messenger RNAs were detected in the cultured fibrocytes. When the cultures were incubated with both TNF-alpha and Dex, the levels of KC, MCP-1, MIP-2 and IL-6 were significantly lower than those in cultures treated with TNF-alpha alone. The data suggest that Dex suppresses the inflammatory response in SL fibrocytes. Given that SL fibrocytes play a role in cochlear fluid and ion homeostasis, glucocorticoids may suppress the cochlear malfunction caused by SL inflammation.

Distribution of immunocompetent cells in normal nasal mucosa : comparisons among germ-free, specific pathogen-free, and conventional mice

To better understand the role of immunocompetent cells in the defense mechanism of the upper respiratory tract against microbial invasions, the distribution patterns of those cells were investigated in nasal mucosa of mice maintained in three different conditions: Germ-free (GF), specific pathogen-free (SPF), and conventional (CV) conditions. Immunostaining by the indirect peroxidase method and toluidine blue staining were employed for the detection of immunocompetent cells and mast cells. For immunostaining, anti-IgG, -IgA, and -IgM polyclonal antibodies and anti-Lyt-1, -Lyt-2, and -Mac-1 monoclonal antibodies were used as primary antibodies. In nasal mucosa of CV mice, Mac-1+ cells, mast cells, and all cell types of lymphocyte subsets were present. In nasal mucosa of SPF mice, all cell types were also positive, but fewer in number than those of CV mice. In nasal mucosa of GF mice, IgG+, IgA+, and Lyt-2+ cells were rare, although IgM+ and Lyt-1+ cells were present in small numbers. An electron microscopic study revealed that follicle-like lymphocyte aggregates with high endothelial venules were present in nasal mucosa close to the mucosal epithelia. These findings suggest that lymphocytes are mobilized to nasal mucosa, responding to continuous antigenic stimuli, and play an important role in the local defense mechanism of the upper respiratory tract.