A new device for high-pressure freezing of cultured cell monolayer using 10-microm-thin stainless discs as both culture plate and specimen carrier.

High-pressure freezing (HPF) has been generally accepted as the most reliable method for cryofixation of biological samples, yielding a deep vitreous freezing. In recent cell biology, mammalian cultured cells are widely used, but HPF of cultured cell monolayer has not reached its full potential. In this study, we developed a new reliable device for HPF of cultured cell monolayer by using a 10-microm-thin stainless disc both as culture plate and specimen carrier. We describe the practical procedure, and demonstrate fine structures of HeLa cells cultured and cryofixed on the stainless discs as results.

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid.

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Exfoliation of gastric pit-parietal cells into the gastric lumen associated with a stimulation of isolated rat gastric mucosa in vitro: a morphological study by the application of cryotechniques.

It is clinicopathologically important to elucidate the cell kinetics for the maintenance of normal gastric epithelium. In a rat gastric mucosa isolated after stimulation, a number of cells were exfoliated into the gastric lumen of the pit region. The present study was undertaken to clarify the origin of exfoliated cells and their histochemical profiles by taking the advantages of cryotechniques. As results, most of the exfoliated cells were identified as pit-parietal cells labeled with both peanut-lectin and anti-H+/K+-ATPase antibody. Quantitative analysis verified a time-dependent increase in the number of exfoliated cells in the gastric mucosa isolated after stimulation. The exfoliated cells exhibited a diffuse intracellular staining for E-cadherin, suggesting a dissociation of the adhesion molecule prior to the cell exfoliation. It should be noted that most of the exfoliated cells were negative to the apoptotic markers (TUNEL staining and caspase-3). Ultrastructurally, autophagosome-like structures consisting of H+/K+-ATPase positive membranes were frequently seen in the exfoliated pit-parietal cells. In addition, the pit-parietal cell exfoliation was accompanied by sealing of their basal portion with the cytoplasmic processes of adjacent surface mucous cells. The present morphological findings provide a new insight into the cell kinetics in the gastric epithelium in vitro.

Mucosubstance histochemistry and pathogenesis of acquired cholesteatoma.

Mucosubstance histochemical study of 33 cholesteatoma tissues was performed to clarify the distribution and character of mucin in the perimatrix. Mean density of glandular cysts was 0.18 per mm(2). Mean frequency of ruptured cysts was 0.16 per cyst. Glandular cysts as well as hollow spaces in the perimatrix were filled with sulfomucin and sialomucin. Fragments of mucin were found in some macrophages and multinucleated giant cells. Since phagocytosis is host defenses attempt, the process indicates that mucin in the perimatrix is a cause of inflammation. Sialomucin infiltrated in the subepidermis where the epidermis formed papillary proliferation without an apparent sign of inflammation. Six glandular cysts were found in the matrix and the debris. They may have been eliminated from the perimatrix as a sequel to cholesteatoma growth. These findings suggest that embedded mucosa in the perimatrix may play a crucial role in pathogenesis of acquired cholesteatoma.

Ultrastructural transformation of gastric parietal cells reverting from the active to the resting state of acid secretion revealed in isolated rat gastric mucosa model processed by high-pressure freezing.

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H(+)/K(+)-ATPase demonstrated a progressive translocation of H(+)/K(+)-ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H(+)/K(+)-ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.

Three-dimensional regular arrangement of the annular ligament of the rat stapediovestibular joint.

The stapes footplate articulates with the vestibular window through the annular ligament. This articulation is known as the stapediovestibular joint (SVJ). We investigated the ultrastructure of adult rat SVJ and report here on the characteristic ultrastructure of the corresponding annular ligament. Transmission electron microscopy showed that this annular ligament comprises thick ligament fibers consisting of a peripheral mantle of microfibrils and an electron-lucent central amorphous substance that is regularly arranged in a linear fashion, forming laminated structures parallel to the horizontal plane of the SVJ. Scanning electron microscopy revealed that transverse microfibrils cross the thick ligament fibers, showing a lattice-like structure. The annular ligament was vividly stained with elastica van Gieson's stain and the Verhoeff's iron hematoxylin method. Staining of the electron-lucent central amorphous substance of the thick ligament fibers by the tannate-metal salt method revealed an intense electron density. These results indicate that the annular ligament of the SVJ is mainly composed of mature elastic fibers.

Confirmation of mucin in lymphatic vessels of acquired cholesteatoma.

Abundant inflammatory cells infiltrate in the advancing front of the cholesteatoma perimatrix towards the middle ear mucosa. However, the cause of inflammation is not yet clear. The middle ear mucosa is often embedded in the cholesteatoma perimatrix. We hypothesized that the embedded mucosa is the cause of inflammation. Surgical specimens obtained from 20 cases of acquired cholesteatoma were used for the study. Lymphatic vessels were stained by the immunohistochemical method using the antibody against podoplanin. Mucin was simultaneously stained by alcian blue. The results of our examination were the following: (1) the presence of infiltrating mucin in the perimatrix; (2) the degeneration and reduction of lymphatic vessels; (3) the accumulation of inflammatory cells around morbid lymphatic vessels. Based on our findings, cholesteatoma can be defined as an inflammation affected by infiltrating mucin that escaped from embedded mucosa in the perimatrix.

The cryofixation of isolated rat gastric mucosa provides new insights into the functional transformation of gastric parietal cells: an in vitro experimental model study.

Cryofixation is currently accepted as the best initial fixation step to preserve not only the fine structure but also the antigenicity of biological samples. To elucidate the functional transformation of gastric parietal cells, we have newly developed an in vitro experimental model, named the isolated gastric mucosa. In this study, acid secretion of the parietal cell was stimulated with histamine or inhibited with cimetidine, and the samples were cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, the organization of glandular cells was well-preserved and quite similar to freshly excised rat gastric mucosa for at least 2 h after isolation. Immunohistochemistry of H+/K+-ATPase demonstrated a translocation of H+/K+-ATPase from the cytoplasm to the apical membrane associated with histamine-stimulation. In cimetidine-treated mucosa, most of the parietal cells were morphologically in the resting state, showing numerous tubulovesicles in their cytoplasm. In contrast, histamine-stimulated parietal cells exhibited well-developed intracellular canaliculi lined with long microvilli. To the best of our knowledge, the present study is first to demonstrate an electron micrograph that strongly suggests a membrane fusion between the tubulovescile and the apical membrane. Moreover, a stimulation-associated translocation of ezrin was clearly shown from the cytoplasm to the apical region, corresponding to apical microvilli development in the isolated gastric mucosa model. We here describe the preparation of the isolated rat gastric mucosa model, which provides new insights into the functional transformation of parietal cells by the application of cryotechniques.

Application of 10-microm thin stainless foil to a new assembly of the specimen carrier in high-pressure freezing.

High-pressure freezing (HPF) has been accepted generally as the most reliable method for cryoimmobilization of biological samples. However, the depth of vitreous freezing in biological samples was less than expected, probably because of the poor thermal conductivity with high water contents. In this study, we introduce a new assembly of the specimen carrier using a 10-microm thin stainless foil for the specimen chamber cover in the HPF technique and describe the fine structure of rat gastric glands processed with the assembly. A low-magnification view of the gastric surface region showed a well-preserved morphology in which the vitreous freezing reached deeper than 100-microm from the freezing face. The present results prove that the 10-microm thin stainless foil is useful in HPF, providing deep vitrification in biological samples.

Cryofixation processing is an excellent method to improve the retention of adrenomedullin antigenicity.

We reappraised the precise immunohistochemical localization of adrenomedullin (AM) by means of the combined use of the catalyzed signal amplification (CSA) system and plunge freezing (PF)/freeze substitution (FS) for light microscopy or high-pressure freezing (HPF)/FS for electron microscopy, focusing on the rat adrenal gland and heart. In the case of adrenal glands, the PF processing showed that almost all medullary cells were intensively immunoreactive, while the cortical cells showed weak immunoreaction. In the heart, almost all cardiac muscle cells of the atria were also vividly stained with the PF/FS and the CSA enhancement. On the contrary, traces of immunoreactions were seen in most of the ventricular cells. These results are consistent with the previous reports of AM radioimmunoassays and the expression of AM mRNA. However, the chemical fixation processing revealed heterogeneous immunostaining in the atrial and ventricular myocardium as well as the adrenal medulla. Intensity of the immunostaining in the chemically fixed tissues was not likely to correspond with that of AM radioimmunoassays. The HPF/FS processing clearly demonstrated the immunogold labeling on secretory granules of adrenal medullary cells as well as cardiac muscle cells of the right auricles. Immunogold labeling intensity of the cryofixed specimens was 3- to 25-fold higher than that of the chemically fixed ones.