Yukio Katori

Increased von Willebrand Factor in the Endocardium as a Local Predisposing Factor for Thrombogenesis in Overloaded Human Atrial Appendage

OBJECTIVES We investigated immunoreactive von Willebrand factor (vWF), a platelet adhesion molecule, in the endocardial endothelium and its relationship to thrombogenesis in the human atrial appendage. BACKGROUND Intra-atrial thrombogenesis is generally thought to be induced by blood stasis in the atrial appendage involved with atrial fibrillation (AF). Little attention has been paid to alterations of the endocardial endothelium on which the thrombus develops. METHODS Atrial appendage tissue was obtained at heart surgery or at autopsy from AF and non-AF cardiac patients and from noncardiac patients. Immunohistochemistry for endothelial cell markers including vWF, CD31, CD34 and endothelial nitric oxide synthase (eNOS) and platelet glycoprotein Ib/IX or IIb/IIIa was performed and semiquantitatively graded. RESULTS In contrast to the apparent immunostaining for CD31, CD34 and eNOS, only focal or little immunoreactive vWF was seen in the endocardium of noncardiac patients. Immunoreactive vWF in the endocardial endothelium was increased in most cardiac patients, particularly in the left, but not in the right, atrial appendage of patients with mitral valvular disease, irrespective of whether AF was present. Platelet adhesion/thrombus formation in the endocardium was found in limited sites in which the overlying endothelium was deficient in eNOS and CD34. When warfarin-treated cases were excluded, there was a significant correlation between the immunohistochemical grade for vWF and the degree of platelet adhesion/thrombus formation in the endocardium. CONCLUSIONS Immunoreactive vWF in the endocardial endothelium was increased in overloaded human atrial appendage, which may be a local predisposing factor for intraatrial thrombogenesis.

Expression of connexin 31 in the developing mouse cochlea.

Connexin 31 (C×31) mutations cause an autosomal dominant form of high-frequency hearing loss. The immunohistochemical localization of C×31 in mouse cochlea was studied at different ages between 0 and 60 days after birth (DAB). C×31-like immunoreactivity was detected in fibrocytes of spiral ligament and spiral limbus at 12 DAB, gradually enhanced with the increase of age and reached the adult pattern on 60 DAB. Immunoreactivity decreased gradually from the basal to apical turn in all developmental stages. The mRNA of C×31 was also identified by RT-PCR. The distribution of C×31 and connexin 26 were obviously different in the developing mouse cochlea. The expression and distribution of C×31 in the development may explain the progressive hearing loss in human C×31 mutations.

Density of motility-related charge in the outer hair cell of the guinea pig is inversely related to best frequency

Whole cell voltage clamp and freeze fracture were used to study the electrophysiological and ultrastructural correlates of the outer hair cell (OHC) lateral membrane molecular motors. We find that specific voltage-dependent capacitance, which derives from motility-related charge movement, increases as cell length decreases. This increasing non-linear charge density predicts a corresponding increase in sensor-motor density. However, while OHC lateral membrane particle density increases, a quantitative correspondence is absent. Thus, the presumed equivalence of particle and motor is questionable. The data more importantly indicate that whereas the voltage driving OHC motility, i.e. the receptor potential, may decrease with frequency due to the OHCs low-pass membrane filter, the electrical energy (Q x V) supplied to the lateral membrane will tend to remain stable. This conservation of energy delivery is likely crucial for the function of the cochlear amplifier at high frequencies.

Expression of connexin 26 and Na,K-ATPase in the developing mouse cochlear lateral wall: functional implications

The immunohistochemical localization of connexin 26 (a gap junction protein) and Na,K-ATPase in the mouse cochlear lateral wall was studied at different ages between 0 and 30 days after birth (DAB). Connexin 26-like immunoreactivity was sparsely distributed among the connective tissue cells just lateral to the future marginal cells of the stria vascularis on 0 DAB. In the mice of 3-6 DAB, connexin 26 was observed in the strial basal cell area, and was increased in its distribution density on 10 DAB. Connexin 26 was sparsely distributed among the fibrocytes in the spiral ligament and the suprastrial zone on 10 DAB, and its distribution density increased rapidly in the mouse on 12 DAB. The immunohistochemical distribution reached the adult pattern in the cochlear lateral wall on 15 DAB. Weak Na, K-ATPase-like immunoreactivity was observed in the epithelial cells, corresponding to the future strial marginal cells, on 0 DAB. Its staining intensity was enhanced with the increase of age, and reached the adult pattern on 10 DAB. In contrast, Na,K-ATPase-like immunoreactivity in the type II fibrocytes and suprastrial fibrocytes was first detected on 12 DAB, and reached the mature level on 15 DAB. It is well known that the endolymphatic potential (EP) reaches the adult level 2 weeks after birth. The expression patterns of connexin 26 and Na,K-ATPase in the fibrocytes of the spiral ligament and the suprastrial zone coincided with the rapid growth and maturation of EP. These findings may suggest a role for the gap junctional communications and Na,K-ATPase activity of the fibrocytes within the cochlear lateral wall in the generation and maturation of EP.

The usefulness of computed tomography in the diagnosis of impacted fish bones in the oesophagus

The usefulness of computed tomography (CT) in the diagnosis of fish bone impaction in the oesophagus was evaluated. Thirty-two patients were examined by plain X-ray followed by direct oesophagoscopy for suspected fish bone impaction. Among 25 cases in which fish bones were actually removed, foreign bodies were not clearly demonstrated by plain X-ray in 14 cases (56 per cent). Eleven cases underwent CT prior to the oesophagoscopic examination. Fish bones were clearly demonstrated by CT in all patients. CT also clearly visualized secondarily-induced inflammatory changes in the neighbouring structures. In order to confirm this result, we made a simulation model of oesophageal fish bone impaction, using fish bones of three different species surrounded by a water bag. In comparison with plain X-ray, CT depicted a superior image of fine fish bones and provides extremely useful information for the management of impacted fish bones in the oesophagus.

Expression of connexin 30 in the developing mouse cochlea.

Mutations in the GJB6 gene encoding connexin 30 (Cx30) can cause dominant forms of nonsyndromic deafness. By studying immunohistochemical localization of Cx30 in the mouse cochlea at different ages from 0 to 30 days after birth, we found that the expression of Cx30 is nearly the same as that of Cx26. These findings suggest that as well as Cx26, Cx30 may also contribute to the generation and maturation of endocochlear potential.

Expression of the Sox10 gene during mouse inner ear development

Mutations in the SOX10 gene, encoding a cell-lineage specific transcription factor, are associated with congenital deafness. We analyzed the expression of Sox10 mRNA in developing mouse inner ear by in situ hybridization. Sox10 mRNA is expressed in the entire epithelia of the otic vesicle at embryonic day 11.5 (E11.5) and in the developing cochlea and vestibule at E13.5. In postnatal day 8 and adult cochleas, Sox10 expression is restricted to the supporting cells of the organ of Corti. These expression profiles suggest that Sox10 is important for development of the cochlea.

The dimensions and structural attachments of tip links in mammalian cochlear hair cells and the effects of exposure to different levels of extracellular calcium.

The tip links between stereocilia of acousticolateral hair cells have been suggested to contain cadherin 23 (CDH23) comprising an upper branched portion that is bound to a lower portion composed of protocadherin 15 (PCDH15). The molecular conformation of CDH23, its binding to PCDH15, the tip links, and mechanoelectrical transduction have all been shown previously to be sensitive to exposure to low levels of calcium. The aim of this study was to compare the characteristics of tip links in guinea-pig cochlear hair cells with reported features of the CDH23-PCDH15 complex. Tip links were examined using field emission scanning electron microscopy and transmission electron microscopy in conventional preparations and after treatment with the detergent Triton-X-100 or varying calcium concentrations in the extracellular solution. The results showed that tip links have a twisted double-stranded appearance with a branched upper region. They survived demembranation of the stereocilia by detergent suggesting that they have transmembrane domains at both ends. Their lengths, when fixed in the presence of 2 mM extracellular calcium, were approximately 150 nm. With prior exposure to 1 mM calcium their lengths were approximately 164 nm. The lengths in 50 microM calcium are similar ( approximately 185 nm) to those reported for CDH23-PCDH15 complexes in 100 microM calcium ( approximately 180 nm). Exposure to 1 microM calcium caused loss of tip links and an increased distance between the residual attachment sites. The data indicate that extracellular calcium concentration affects tip-link length. One model compatible with the recently proposed tip-link structure is that the CDH23 double strand undergoes calcium-dependent unfolding, changing the length of the links. The bundle may also tilt in the direction of the tallest row of stereocilia as the tip link lengthens and then is lost. Overall, our data are consistent with a tip link composed of complexes of CDH23 and PCDH15 but do not rule out other possibilities.

Differential distribution of β- and γ-actin in guinea-pig cochlear sensory and supporting cells

Abstract Sensory and supporting cells of the mammalian organ of Corti have cytoskeletons containing β- and γ-actin isoforms which have been described as having differing intracellular distributions in chick cochlear hair cells. Here, we have used post-embedding immunogold labelling for β- and γ-actin to investigate semiquantitatively how they are distributed in the guinea-pig cochlea and to compare different frequency locations. Amounts of β-actin decrease and γ-actin increase in the order, outer pillar cells, inner pillar cells, Deiters’ cells and hair cells. There is also more β-actin and less γ-actin in outer pillar cells in higher than lower frequency regions. In hair cells, β-actin is present in the cuticular plate but is more concentrated in the stereocilia, especially in the rootlets and towards the periphery of their shafts; labelling densities for γ-actin differ less between these locations and it is the predominant isoform of the hair-cell lateral wall. Alignments of immunogold particles suggest β-actin and γ-actin form homomeric filaments. These data confirm differential distribution of these actin isoforms in the mammalian cochlea and reveal systematic differences between sensory and supporting cells. Increased expression of β-actin in outer pillar cells towards the cochlear base may contribute to the greater stiffness of this region.

Late-onset hearing loss in a mouse model of DFN3 non-syndromic deafness: morphologic and immunohistochemical analyses

Recently, we reported that homozygous males and females of a mouse model of DFN3 non-syndromic deafness generated by the deletion of Brn-4 transcription factor showed profound deafness due to severe alterations in the cochlear spiral ligament fibrocytes from the age of 11 weeks, whereas no hearing loss was recognized in young female heterozygotes. It is known that a part of obligate female carriers of DFN3 showed progressive hearing loss. In the present study, we examined the late-onset effect of Brn-4 deficiency on the hearing organ of the mouse. About one third of heterozygous female mice revealed late-onset profound deafness at the age of 1 year. Furthermore, in these deafened heterozygotes, characteristic abnormalities in Reissners membrane attachment and type II fibrocytes in the suprastrial zone became evident under light microscope, similar to homozygous female mice. A significant reduction in the immunoreactivity of connexin 26 (Cx26), connexin 31 (Cx31), Na,K-ATPase and Na-K-Cl cotransporter in the spiral ligament fibrocytes was observed in aged heterozygotes showing late-onset profound deafness. The late-onset phenotype observed in heterozygous mutant mice, being consistent with the progressive deafness observed in human female heterozygotes, may be explained by alterations of the ion transport systems in the spiral ligament fibrocytes.