Masafumi Sakagami

Fine structure and permeability of capillaries in the stria vascularis and spiral ligament of the inner ear of the guinea pig

SummaryThe blood capillaries in the stria vascularis and the spiral ligament of guinea pigs were studied by electron microscopy with freeze-fracture and thin section methods, including tracer experiments with horseradish peroxidase (HRP) and microperoxidase (MP). The endothelial cells of the capillaries of both tissues are connected by tight junctions, and contain about the same number of micropinocytotic vesicles. In cases of intravascular administration before fixation, both of the tracers stained the perivascular space and almost all endothelial vesicles in the stria vascularis. On the other hand, the perivascular space and many vesicles in the spiral ligament were unstained. The endothelial tight junctions in the stria vascularis prevented the penetration of HRP, but sometimes allowed the penetration of MP. Those of the spiral ligament were impermeable to both tracers. In cases of tracer administration after fixation, leakage spots of HRP from capillaries were sparsely located all over the stria vascularis. Transendothelial channels and isolated fenestrae formed by micropinocytotic vesicles were detected. It is concluded that the capillaries of the stria vascularis are similar to the muscle capillaries and to the capillaries of the elasmobranch brain, whereas those in the spiral ligament are similar to the brain capillaries of higher vertebrates.

Localization of osteopontin in the otoconial organs of adult rats.

Although it is known that mammalian otoconia consist of calcium bicarbonate and organic materials, none of the protein components have been identified in mammals at the molecular level, and the mechanisms of morphogenesis and calcification of the otoconia is still unclear. In the present study, we demonstrated the presence of osteopontin (OPN) in rat otoconia by using immunohistochemistry, and detected OPN mRNA in the sensory hair cells by a non-radioisotopic in situ hybridization technique. These results indicate that OPN is one of the protein components in rat otoconia and suggest that sensory hair cells are involved in the production of otoconia.

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.

An immunohistochemical and electrophysiological study on Isk protein in the stria vascularis of the guinea pig

SummaryThe reaction product against rat Isk protein antibody was demonstrated on the endolymphatic surface of the marginal cell of the guinea pig, using an immunohistochemical method. Clofilium, a potassium channel blocker which has been previously found to block rat kidney Isk channels, decreased the endocochlear potential (EP) when applied by perilymphatic perfusion and iontophoresis into endolymph. This finding of a decreased EP supports the presence of Isk protein on the endolymphatic surface of the marginal cell.

Comparison of summating potential in Menière's disease between trans- and extratympanic electrocochleography

The summating potential (SP) recorded concurrently with transtympanic (TT) and extratympanic (ET) electrocochleography in Menière ears was compared with respect to polarity and amplitude. There was a difference in SP polarity and amplitude between the two methods. The incidence of the increase in the ratio of -SP amplitude to the amplitude of the auditory nerve action potential (AP) (-SP/AP ratio) at 80 dB nHL as a response to a click was higher with the ET than with the TT method. Most of the ears exhibiting an increased +SP/AP ratio with the TT method showed an increased -SP/AP ratio with the ET method. The incidence of increased SP/AP ratios including -SP/AP and +SP/AP ratios was similar with both methods. A +SP was recorded in 14 of 25 ears upon stimulation with an 8-kHz tone burst with the TT method, whereas no +SP was recorded in 25 ears with the ET method. Tone bursts of 1 and 2 kHz produced a -SP and similar waveforms with both methods. Comparison of -SP amplitude recorded with both methods revealed that the ratio of -SP amplitude with both methods is different across the stimulus frequencies. The present study suggests that the -SP elicited by tone bursts of mid to low frequencies is more stable in the diagnosis of Menières disease than -SP elicited by a click because of the lesser influence of the electrode location and that the increase in the +SP recorded with the TT method may be of clinical significance in the diagnosis of Menières disease.

Strial Dysfunction in a Melanocyte Deficient Mutant Rat (Ws/Ws Rat)

A homozygous mutant rat at the white spotting (Ws) locus showing a deficiency of melanocytes has recently been found (4, 5). The function and morphology of the inner ear of the Ws/Ws rat were examined by auditory brainstem response (ABR), endocochlear DC potential (EP), and electron microscopy. The mean ABR threshold of the Ws/Ws rat was significantly higher than that of the control +/+ rat. Most Ws/Ws rats showed no or very little EP. In electron microscopy, the stria vascularis of the Ws/Ws rat proved to be very thin and flat with poor interdigitation of marginal cells, and absence of intermediate cells. The organ of Corti appeared to be intact in both the +/+ rat and the Ws/Ws rat. These electrophysiological and morphological findings suggest that the Ws/Ws rat suffered from severe hearing loss caused by strial dysfunction.

Increased Capillary Permeability of the Stria Vascularis to HRP, Induced by Experimental Acute Hypotension in Rats

Acute hypotension was produced in rats by using two experimental techniques: intravenous infusion of a ganglion-blocking agent (Arfonad), and venesection of a femoral vein. Horseradish peroxidase (HRP) was injected intravenously after each procedure, and subsequently observed in the inner ear by light and electron microscopy. In both experimental models, a large amount of tracer spread into the intercellular spaces, but it was halted by tight junctions bordering the stria vascularis. The endothelium exhibited a high distribution density of labelled vesicles, which suggested increased vesicular transport. There was no extravasation of HRP from capillaries in the spiral ligament in spite of the presence of some labelled pinocytotic vesicles. The present study was concerned with the discovery of enhanced capillary permeability of the stria vascularis under acute hypotension, as in the case of acute hypertension (Sakagami et al., 1984).

Quantitative Evaluation of Pinocytosis of Capillaries of the Stria Vascularis under Normal and Experimental Conditions

In order to examine evidence of high activity of pinocytosis in capillaries of the stria vascularis, quantitative morphometry of pinocytotic vesicles was carried out ultrastructur-ally by the horseradish peroxidase (HRP) tracer method. In guinea pigs, there was a significant difference between the stria vascularis and spiral ligament in the number of vesicles per urn(2) (p<0.05) and labelled vesicles per urn(2) (p<0.001). In normotensive control rats, the number of labelled vesicles per u,m(2) in capillaries of the stria vascularis was significantly greater (p<0.001) than that of the spiral ligament. Results of acute hypertensive and acute hypotensive experiments both indicated that enhanced permeable capillaries of the stria vascularis showed no significant increase in the number of pinocytotic vesicles per urn(2), but that they showed a more significant increase in the number of labelled vesicles per um(2) than non-enhanced permeable capillaries of the stria vascularis (p<0.01) and capillaries of the spiral ligament (p<0.001). These findings provide ultrastruc-tural confirmation of our previous studies (4, 8, 9) that pinocytosis contributes to the high permeability of capillaries of the stria vascularis under normal and experimental acute hypertensive and acute hypotensive conditions.

Morphologic and Biochemical Study of Vascular Permeability of the Middle Ear Mucosa in Experimental Otitis Media

The vascular permeability of the middle ear mucosa of chinchillas was examined by light and electron microscopy using horseradish peroxidase (HRP). Two kinds of experimental otitis media were used: Serous otitis media (SOM) produced by eustachian tube obstruction and purulent otitis media (POM) produced by inoculation with Streptococcus pneumoniae type 7F. Normal mucosa showed no leakage of HRP. In SOM, vessels were dilated and tortuous, but they did not show HRP leakage. In POM, vessel leakage of HRP was extensive. Biochemically, we measured the middle ear effusion–to-serum ratio of both albumin and α2-macroglobulin by an immunodiffusion method. The ratios of the two marker proteins for vascular permeability were significantly higher in POM than in SOM. This suggests that POM increases vascular permeability to a higher degree than SOM, and morphologic findings in the middle ear mucosa correlate with the biochemical composition of the middle ear effusion.

Nystagmus Measured by ENG after Stapes Surgery

Much attention has been paid to hearing results after stapes surgery, but the risks of vestibular disturbance has not been extensively studied. Postoperative spontaneous nystagmus was measured daily at bedside by portable ENG in order to evaluate the vestibular damage from stapes surgery. Thirteen patients underwent primary stapedotomy or stapedectomy from August 1, 1992 to June 30, 1993. Nystagmus toward the operated ear was observed in 3 cases, that toward the opposite ear in 2 cases, that changing from toward the operated ear to toward the opposite ear in 2 cases, that changing from toward the opposite ear to toward the operated ear in 2 cases and no nystagmus in 4 cases. There was no relationship between duration of nystagmus and that of dizziness. Nystagmus was thought to be due to the following: i) inner ear damage by operation, ii) postoperative perilymphatic fistula, iii) floating footplate, and iv) stimulation of hair cells by high potassium ion in the perilymph due to blood flow into the inner ear.