Three-dimensional observation of mouse tongue muscles using micro-computed tomography.

The aim of this study is to obtain information about the mouse tongue muscle rendered using micro-computed tomography (µCT) at low, middle, and high magnifications. Three-dimensional (3D) µCT is used in various fields. Most µCT observations are restricted to hard tissue in biomaterial samples. Recently, with the use of osmium tetroxide, µCT has been effectively employed to observe soft tissue; it is now believed that µCT observation of soft tissue is feasible. On the other hand, the structure of the tongue muscle has been well studied, but cross-sectional imaging enhanced by 3D rendering is lacking. We chose the mouse tongue as a soft tissue case study for µCT and generated cross-sectional images of the tongue enhanced by 3-D image rendering with histological resolution. During this observation, we developed new methods of low-magnification observation to show the relation between the tongue muscles and surrounding tissues. We also applied high-resolution µCT in high-magnification observation of muscle fiber fascicles. Our methodological techniques give the following results: (1) For low-magnification observation (field of view: 12,000 µm), pretreatment with decalcification and freeze drying is suitable for observing the area between the muscle of the tongue and the bone around the tongue using µCT. (2) For middle-magnification observation (Field of view: 3,500 µm), the use of osmium tetroxide to observe the muscle arrangement of the tongue by µCT is suitable. (3) For high-magnification observation (Field of view: 450 µm), high-resolution µCT is suitable for observation of the transversus muscle fiber fascicles.

Localization of type III collagen in the lingual mucosa of rats during the morphogenesis of circumvallate papillae.

In an effort to identify a possible role for type III collagen in the morphogenesis of circumvallate papillae on the surface of the rat tongue, we examined its appearance by fluorescent immunostaining, in conjunction with differential interference contrast images and images obtained, after staining with toluidine blue, in the transmission mode by laser-scanning microscopy. We analyzed semi-ultrathin sections of epoxy resin-embedded samples of the lingual mucosa of embryonic and juvenile rats, 13 days after conception (E13) to day 21 after birth (P21). Immunoreactivity specific for type III collagen was recognized first in the mesenchymal connective tissue just beneath the circumvallate papilla placode in fetuses on E13. At this stage, most of the lingual epithelium with the exception of the circumvallate papilla placode was pseudostratified epithelium composed of one or two layers of cuboidal cells. However, the epithelium of the circumvallate papilla placode was composed of several layers of cuboidal cells. Immunoreactivity specific for type III collagen was detected mainly on the lamina propria just beneath the lingual epithelium of the rudiment of the circumvallate papilla and the developing circumvallate papilla in fetuses on E15 and E17, and slight immunostaining was detected on the lamina propria around the rudiment. In fetuses on E19, immunoreactivity specific for type III collagen was widely and densely distributed on the connective tissue around the developing circumvallate papillae and, also, on the connective tissue that surrounded the lingual muscle. However, the immunoreactivity specific for type III collagen was sparsely distributed on the lamina propria of each central papillar structure. After birth, from P0 to P14, morphogenesis of the circumvallate papillae advanced gradually with the increase in the total volume of the tongue. At these postnatal stages, the intensity of the fluorescence due to immunoreactivity specific for type III collagen was distinctively distributed on the lamina propria around each circumvallate papilla, on each central bulge and on the connective tissue that surrounded the lingual muscle. However, immunofluorescence was less distinct on the connective tissue that surrounded the lingual muscle. Thus, type III collagen appeared in conjunction with the morphogenesis of the circumvallate papillae, as well as in the connective tissue that surrounded the lingual muscle during myogenesis of the rat tongue.

Three-dimensional observation of the mouse embryo by micro-computed tomography: Meckel's cartilage, otocyst, and/or muscle of tongue.

Three-dimensional observation during embryogenesis is possible with micro-computed tomography, but there are no observations of organ size. In this paper, three examples of three-dimensional observation of organs by micro-CT are tried. At 13.0 days post-coitum, mouse embryos were fixed in 4% paraformaldehyde for 24 h and stained enbloc by osmium tetroxide overnight. The embryos were then embedded in paraffin using standard methods for 24 h. Specimens were analyzed by micro-computed tomography and image processing was performed. The entire Meckel's cartilage and its relation in the mandible, as well as the complex structure of the otocyst, are easily visualized. Although it is difficult to extract detailed structures of the tongue muscles, it is possible to identify the inner and external tongue muscles. Relation among the organs and other are easily visualized. Three-dimensional observation by micro-computed tomography is an important technology for visualization of embryogenesis and could be used in organ culture.

Three-dimensional observation of the mouse embryo by micro-computed tomography: composition of the trigeminal ganglion.

The purpose of this study was to demonstrate a micro-computed tomography (CT) method for observations of the mouse embryo. At 13.0 days post-coitum, mouse embryos were fixed in 4% paraformaldehyde for 24 h and stained en bloc by osmium tetroxide overnight. The embryos were then embedded in paraffin using standard methods for 24 h. Specimens were analyzed by micro-CT and image processing was performed. Organs containing nervous and blood systems could be viewed as a result of different osmium-staining densities. The trigeminal ganglion was imaged using three-dimensional techniques. Observation of the embryo was possible by micro-CT with osmium tetroxide staining.

Immunohistochemical expression of type II collagen in the lingual mucosa of rats during organogenesis of the tongue.

OBJECTIVES: We examined the timing of the appearance and distribution of type II collagen as a possible component of the extracellular matrix that is involved in the morphogenesis of the rat tongue. METHODS: We examined the immunofluorescence of type II collagen, differential interference contrast (DIC) images, and images recorded in transmission mode after toluidine blue staining by laser-scanning microscopy (LSM) during the morphogenesis of filiform papillae and the keratinization of the lingual epithelium of rats on semi-ultrathin sections of epoxy resin-embedded samples. RESULTS: Immunoreactivity specific for type II collagen was scattered on cells over a wide area of the mesenchymal connective tissue of the fetal tongue on day 15 after conception (E15), when the lingual epithelium was composed of one or two layers of cuboidal cells. Immunoreactivity specific for type II collagen was recognisable on cells of the lamina propria of the lingual mucosa and around the developing lingual muscle of fetuses at E17 and E19. On E19, the epithelium was clearly of the stratified squamous type. At postnatal stages after birth (P0), immunoreactivity became more and more significant in the connective tissue of the lamina propria with the advancing of morphogenesis of the filiform papillae. In addition, immunoreactivity was widely distributed in the connective tissue around the lingual muscle, as myogenesis in the tongue advanced. The lingual epithelium was composed of stratified squamous cells, and keratinization of the lingual epithelium proceeded gradually as morphogenesis of filiform papillae continued during postnatal development. CONCLUSION: Type II collagen appeared not only in the connective tissue of the lamina propria as the morphogenesis of filiform papillae occurred and the lingual epithelium became keratinized but also in the endomysium and perimysium around the lingual muscle after myogenesis of the tongue is complete at P0.

Newly developed technique for dual localization of keratins 13 and 14 by fluorescence immunohistochemistry.

It is difficult to visualize histological details on semi-ultrathin sections by light microscopy after immunohistochemical labeling because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize the immunoreactivity of keratins 13 (K13) and 14 (K14), we used a newly developed technique for dual localization of antigens by fluorescence immunohistochemistry and confocal laser-scanning microscopy in transmission mode, after staining specimens with toluidine blue. Using this approach, we examined the immunolocalization of K13 and K14 on the lingual epithelium of fetal and juvenile rats by immunofluorescence while monitoring morphological changes in the filiform papillae by laser-scanning microscopy, in transmission mode, of the same sections. No K13 and K14 immunoreactivity was detected on the lingual epithelium of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of a few layers of cuboidal cells. K14 immunoreactivity was first detected on the lingual epithelium of fetuses on E17 and K13 immunoreactivity on E19. The number of layers of cuboidal cells in the lingual epithelium also increased from E17 to E19. K13 and K14 immunoreactivity was distinct at all postnatal stages examined. Although the respective patterns of K13 and K14 immunoreactivity differed as the filiform papillae developed, K13 immunoreactivity was generally evident in the suprabasal cells of the interpapillary cell columns and K14 immunoreactivity was detected in the basal and suprabasal cells of the papillary and interpapillary cell columns. Our newly developed technique for dual localization of antigens should be useful for investigations of very small specimens, such as fetal tissues and organs.

Long-term effect of silver powder in vivo.

The cytotoxicity of silver is a known property of this metal. Interestingly, in the cases of argyria and tattoos, Ag remains in the tissue for a long time without causing harm to the host except pigmentation. To understand these contradictions, pure silver implantation by an original subcutaneous injection method was performed. Two sizes of silver powder particles were implanted subcutaneously: 100 nm (P-silver) and the maximum 45 microm (G-silver). The sulfuration of silver and histopathologic changes were observed for a year. Results were as follows: silver affected the host in the case of P-silver to a greater extent than in G-silver, especially on the 7th day and after 2-4 weeks. Nonetheless, the effect of silver weakened at 12 months after implantation. The presence of P-silver caused various histological reactions, while the decline of silver effect on the host was correlated with an increase in the sulfuration of silver.

Immunohistochemical detection of epidermal growth factor and epidermal growth factor receptor in the lingual mucosa of rats during the morphogenesis of filiform papillae.

We examined the immunofluorescence labelling epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR), as well as differential interference contrast (DIC) images, during the morphogenesis of filiform papillae and the keratinization of the lingual epithelium of rats on semi-ultrathin sections of epoxy resin-embedded samples using laser-scanning microscopy. We also examined semi-ultrathin sections of epoxy resin-embedded, toluidine blue-stained samples by light microscopy to obtain details of cell histology and morphology. No immunoreactivity specific for EGF and EGFR was detected on the lingual epithelium of fetuses on days 12 and 16 after conception (E12 and E16), during which time the number of layers of cuboidal cells in the lingual epithelium increased from one to several. Immunoreactivity specific for EGF and EGFR was first detected on the lingual epithelium of fetuses at birth or on postnatal day 0 (P0). Immunoreactivity specific both for EGF and EGFR appeared in the connective tissue and the basal cells of the papillary and interpapillary cell columns. The lingual epithelium was composed of stratified squamous cells. The rudiments of filiform papillae were compactly arranged and interpapillary cell columns were very narrow. Immunoreactivity specific for EGF and EGFR was distinct on the cell membrane of basal cells of the papillary cell column and weakly positive on the cell membrane of basal cells of the interpapillary cell column on postnatal day 21 (P21). Thus, the patterns of immunoreactivity of EGF and EGFR differed as the filiform papillae developed. Filiform papillae developed gradually from P0 to P21. The width of interpapillary spaces also increased during this period. These observations indicate a possibility that EGF might affect the expression of keratins in the lingual epithelium via epithelium-mesenchymal interactions.

Immunohistochemical expression of keratins 13 and 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae.

We examined the immunofluorescence of keratins 13 (K13) and 14 (K14) and differential interference contrast (DIC) images during the morphogenesis of filiform papillae and the keratinization of the lingual epithelium of rats on semi-ultrathin sections of epoxy resin-embedded samples by laser-scanning microscopy. We also examined semi-ultrathin sections of epoxy resin embedded, toluidine blue stained samples by light microscopy to obtain details of cell histology and morphology. No immunoreactivity specific for K13 and K14 was detected on the lingual epithelium of foetuses on days 13, 15 and 17 after conception (E13, E15 and E17), during which time the number of layers of cuboidal cells in the lingual epithelium increased from one to several. Immunoreactivity specific for K13 and K14 was first detected on the lingual epithelium of foetuses on E19. The immunoreactivity specific for K13 appeared in the suprabasal cells of the papillary and interpapillary cell columns and immunoreactivity specific for K14 was detected in the basal and suprabasal cells of the papillary and interpapillary cell columns. The lingual epithelium was composed of stratified squamous cells. The rudiments of filiform papillae were compactly arranged and interpapillary cell columns were very narrow. Filiform papillae developed gradually from postnatal day 0 (PO) to 21 (P21). The width of interpapillary spaces also increased during this period. Immunoreactivity specific for K13 and K14 was distinct at all postnatal stages examined. Thus, the patterns of immunoreactivity of K13 and K14 differed as the filiform papillae developed.

Immunohistochemical detection of the expression of keratin 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae.

An immunofluorescence study of the expression of keratin 14 (K14) during the formation of filiform papillae was performed and the progress of keratinization of the epithelium of the rat tongue was monitored on semi-ultrathin sections by laser-scanning microscopy. Differential interference contrast (DIC) images were also examined to provide details of histology and cell morphology. No cells with immunoreactivity specific for K14 were detected on the lingual epithelium of foetuses on embryonic days 12 and 16 (E12 and E16), when the lingual epithelium was composed of a single layer or several layers of cuboidal cells. Immunoreactivity specific for K14 was detected first on basal and suprabasal keratinocytes of the dorsal epithelium of the tongue of new-borns on postnatal day 0 (P0) and was conspicuous in juveniles on P14. The immunoreactivity was particularly strong on the basal and suprabasal keratinocytes along the connective tissue papillae. The immunoreactivity extended over the entire cytoplasm but was not detected in the nucleus. The lingual epithelium was composed of stratified squamous cells and the rounded rudiments of filiform papillae were compactly arranged at equal intervals, for the most part, and the spaces between them were narrow and indistinct. Immunostaining of K14 was distinct on basal and suprabasal keratinocytes of the filiform papillar area of tongues of juveniles on P21, when the filiform papillae were conical. The spaces between them were relatively wide and, as a result, interpapillar cell columns were clearly visible. Immunoreactivity specific for K14 in the basal and suprabasal keratinocytes of the interpapillar cell columns was recognizable but was weaker than that in cells of papillar cell columns. The thickness of the epithelium in papillar and interpapillar areas increased gradually with the development of filiform papillae. However, sizes of basal and suprabasal keratinocytes remained almost unchanged during this process. These results suggest that the basal and suprabasal keratinocytes of the filiform papillar area proliferate with the initiation of the morphogenesis of filiform papillae and the keratinization of the epithelium. In addition, it appears that, after P14, the basal and suprabasal keratinocytes of the interpapillar area proliferate to supply the keratinocytes of the expanding interpapillar regions.

Long-term effects of Ag-containing alloys on mucous tissue present in biopsy samples.

The aim of this study was to investigate the long-term effects of alloys containing silver (mainly Ag-Sn alloy) on oral mucous tissue. We observed biopsy tissue specimens from patients diagnosed as having amalgam tattoo and/or metal pigmentation by light and electron microscopy and electron-probe microanalysis (EPMA). In most cases, Ag-Sn alloy was present in the tissue but it could not be confirmed if the alloy originated from amalgam. Distributions of both Ag-S and Ag-Sn have typical patterns. Most Ag forms Ag2S and is stably deposited in three patterns along the collagen, basement membrane, and fibrous cells without inducing any host reaction. On the other hand, Sn forms large granules that contain Ag, S, C, N, P, and Ca, and is in soft state in the tissue. Tissue reactions to the alloy become weaker as time passes.

Patterns of immunoreactivity specific for gustducin and for NCAM differ in developing rat circumvallate papillae and their taste buds.

α-Gustducin and neural cell adhesion molecule (NCAM) are molecules previously found to be expressed in different cell types of mammalian taste buds. We examined the expression of α-gustducin and NCAM during the morphogenesis of circumvallate papillae and the formation of their taste buds by immunofluorescence staining and laser-scanning microscopy of semi-ultrathin sections of fetal and juvenile rat tongues. Images obtained by confocal laser scanning microscopy in transmission mode were also examined to provide outlines of histology and cell morphology. Morphogenesis of circumvallate papillae had already started on embryonic day 13 (E13) and was evident as the formation of placode. By contrast, taste buds in the circumvallate papillae started to appear between postnatal day 0 (P0) and P7. Although no cells with immunoreactivity specific for α-gustducin were detected in fetuses from E13 to E19, cells with NCAM-specific immunoreactivity were clearly apparent in the entire epithelium of the circumvallate papillary placode, the rudiment of each circumvallate papilla and the developing circumvallate papilla itself from E13 to E19. However, postnatally, both α-gustducin and NCAM became concentrated within taste cells as the formation of taste buds advanced. After P14, neither NCAM nor α-gustducin was detectable in the epithelium around the taste buds. In conclusion, α-gustducin appeared in the cytoplasm of taste cells during their formation after birth, while NCAM appeared in the epithelium of the circumvallate papilla-forming area. However, these two markers of taste cells were similarly distributed within mature taste cells.

Fluorescence immunohistochemistry in combination with differential interference contrast microscopy for studies of semi-ultrathin specimens of epoxy resin-embedded samples.

We have developed a technique, using a combination of immunofluorescent staining of semi-ultrathin sections of epoxy resin-embedded samples and the corresponding differential interference contrast (DIC) images obtained by light microscopy that provides detailed information about the immuno-localization of histological and cellular structures. To demonstrate the effectiveness of our method, we examined the immunofluorescence of immuno-stained keratin 13 (K13) and type III collagen (CIII) and the corresponding DIC images during the morphogenesis of filiform papillae on the rat tongue. Immunoreactivity specific for K13 and CIII was detected on the lingual epithelium of juveniles on postnatal days 7 and 14 (P7 and P14). The immunoreactivity specific for K13 was clearly located in the intermediate-layer cells of the interpapillary cell columns, while that specific for CIII was also distinct in the connective-tissue fibers between the lingual epithelium and the lingual muscle. The DIC images revealed the keratinization of the stratified squamous cells of the lingual epithelium and, also, myogenesis beneath the connective tissue. In addition, immunoreactivity specific for CIII was also recognizable in the endomysium and perimysium around the lingual muscle. Thus, our method demonstrated changes in patterns of immunoreactivity of K13 and of CIII during the morphogenesis of the rat tongue.

Localization of keratins 13 and 14 in the lingual mucosa of rats during the morphogenesis of circumvallate papillae.

We used fluorescence immunohistochemistry, analysis of differential interference contrast (DIC) images and confocal laser-scanning microscopy in the transmission mode, after staining specimens with toluidine blue, to examine the localization of keratin 13 (K13) and keratin 14 (K14) in the lingual epithelium of fetal and juvenile Sprague-Dawley rats during the prenatal and postnatal morphogenesis of circumvallate papillae. No immunoreactivity specific for K13 and K14 was detected in the lingual epithelium of fetuses on day 15 after conception (E15), at which time the primitive rudiment of the circumvallate papillae was detectable by the thickening of several layers of cuboidal epithelial cells. On E17 and E19, the developing circumvallate papillae were clearly recognizable, consisting of a central papilla and the surrounding sulcus. No immunoreactivity specific for K13 and K14 was evident in the lingual epithelium around these structures at this time. K14-specific immunoreactivity was first detected in the basal layer of the epithelium of the circumvallate papillae on postnatal day 0 (P0) and K13-specific immunoreactivity was detected on P7. Morphogenesis of the circumvallate papillae progressed significantly from P0 to P14, and immunoreactivity specific for K13 and K14 was clearly recognizable after P7. The respective patterns of K13-specific and K14-specific immunoreactivity differed during the development of the circumvallate papillae: K13-specific immunoreactivity was generally evident in cells of the intermediate layer of the epithelium, while K14-specific immunoreactivity was detected in cells of the basal and suprabasal layers. The present results are discussed in the context of the previously determined localization of K13 and K14 in the dorsal epithelium of the anterior part of the rat tongue during its morphogenesis.

Immunohistochemical analysis of type III collagen expression in the lingual mucosa of rats during organogenesis of the tongue.

We examined the distribution of immunofluorescence due to immunostaining of type III collagen, differential interference contrast (DIC) images and images obtained in the transmission mode after toluidine blue staining by laser-scanning microscopy of semi-ultrathin sections of epoxy resin-embedded samples, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium, and myogenesis of the rat tongue. Immunoreactivity specific for type III collagen was distributed widely in the mesenchymal connective tissue in fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Immunoreactivity specific for type III collagen was clearly detected on the lamina propria in fetuses on E17 and E19, and it was relatively distinct just beneath the lingual epithelium. Immunoreactivity specific for type III collagen was sparsely distributed on the connective tissue around the developing lingual muscle. In fetuses on E19, the epithelium became clearly stratified and squamous. At postnatal stages from newborn (P0) to postnatal day 14 (P14), keratinization of the lingual epithelium advanced gradually with the development of filiform papillae. On P0, myogenesis of the tongue was almost completed. The intensity of the fluorescence immunoreactivity specific for type III collagen at postnatal stages was almost same as that on E19. The immunoreactivity around the fully mature muscle was relatively distinct between P0 and P14. Thus, type III collagen appeared in conjunction with the morphogenesis of filiform papillae and the keratinization of the lingual epithelium as well as in the connective tissue that surrounded the lingual muscle during myogenesis of the rat tongue.

Expression of keratin 14 in the basal cells of the lingual epithelium of mice during the morphogenesis of filiform papillae: visualization by fluorescent immunostaining and confocal laser-scanning microscopy in the transmission mode.

We examined the expression of keratin 14 (K14) on the lingual epithelium by immunofluorescent staining while monitoring morphological changes in the filiform papillae of mice by confocal laser-scanning microscopy in the transmission mode of the same sections to define both the histology and the morphology of cells. It is difficult to visualize histological details of the fetal lingual epithelium of the mouse on semi-ultrathin sections by light microscopy after immunohistochemical staining because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize the immunoreactivity specific for K14, we analyzed the results of immunofluorescent staining of semi-ultrathin sections in combination with an examination of the corresponding images by laser-scanning microscopy in the transmission mode after staining of specimens with toluidine blue. No immunoreactivity specific for K14 was detected on the lingual epithelium of fetuses on embryonic day 15 (E15), but immunoreactivity was distinct at all postnatal stages from postnatal day 0 (P0) to P21.

Expression of keratin 18 in the periderm cells of the lingual epithelium of fetal rats: visualization by fluorescence immunohistochemistry and differential interference contrast microscopy.

We examined the expression of keratin 18 (K18), by immunofluorescence staining, while monitoring morphological changes in the periderm on the lingual epithelium of rats by laser-scanning microscopy of epoxy resin-embedded, semi-ultrathin sections. We also examined differential interference contrast (DIC) images of the same sections to define the histology and morphology of the cells. It is difficult to visualize histological details of the fetal lingual epithelium of the rat on semi-ultrathin sections by light microscopy after immunohistochemical staining, because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize keratin 18 (K18), we used a combination of immunofluorescence staining of semi-ultrathin sections and corresponding differential contrast (DIC) images, obtained by laser-scanning microscopy.