Ultrastructural Changes Associated with Reversible Stiffening in Catch Connective Tissue of Sea Cucumbers.

The dermis of sea cucumbers is a catch connective tissue or a mutable collagenous tissue that shows rapid, large and reversible stiffness changes in response to stimulation. The main component of the dermis is the extracellular material composed of collagen fibrils embedded in a hydrogel of proteoglycans. The stiffness of the extracellular material determines that of the dermis. The dermis has three mechanical states: soft (Sa), standard (Sb) and stiff (Sc). We studied the ultrastructural changes associated with the stiffness changes. Transverse sections of collagen fibrils in the dermis showed irregular perimeters with electron-dense protrusions or arms that cross-bridged between fibrils. The number of cross-bridges increased in stiffer dermis. The distance between the fibrils was shorter in Sc than that in other states, which was in accord with the previous report that water exuded from the tissue in the transition Sb→Sc. The ultrastructure of collagen fibrils that had been isolated from the dermis was also studied. Fibrils aggregated by tensilin, which causes the transition Sa→Sb possibly through an increase in cohesive forces between fibrils, had larger diameter than those dispersed by softenin, which antagonizes the effect of tensilin. No cross-bridges were found in isolated collagen fibrils. From the present ultrastructural study we propose that three different mechanisms work together to increase the dermal stiffness. 1.Tensilin makes collagen fibrils stronger and stiffer in Sa→Sb through an increase in cohesive forces between subfibrils that constituted fibrils; 2. Cross-bridging by arms caused the fibrils to be a continuous network of bundles both in Sa→Sb and in Sb→Sc; 3. The matrix embedding the fibril network became stiffer in Sb→Sc, which was produced by bonding associated with water exudation.

Rare case of the inferior mesenteric artery and the common hepatic artery arising from the superior mesenteric artery.

We found a case in which inferior mesenteric artery and the common hepatic artery arose from the superior mesenteric artery, forming the common hepatomesenteric trunk, during a routine dissection carried out at Iwate Medical University in 2002. This variation is rare, but can be embryonically explained. A change in the positions of the disappearance of the ventral splanchnic arteries and the longitudinal anastomotic channel results in variations in the system of arteries distributed to the digestive organs. In the present case, the longitudinal anastomotic channel between the superior and the inferior mesenteric arteries survived to form the common mesenteric artery, which was joined by the common hepatic artery, forming the common hepatomesenteric trunk.

Comparison of the thickness of basement membranes in various tissues of the rat.

The thicknesses of the lamina lucida and the lamina densa of basement membranes in various kinds of tissues, including the epidermis and mucosal epithelium, and basement membranes adjacent to a Schwann cell, perineurial sheath cell, endothelial cell, pericyte, muscle cell and fat cell, were compared on high-powered transmission electron micrographs. Each tissue and cell possessed a basement membrane with thicknesses of the lamina lucida and lamina densa that were specific to the particular tissue or cell. This indicates that the lamina lucida and lamina densa of the basement membrane of a tissue/cell have a particular constitution. New theories on the molecular constitution between the cell surface and basement membrane may need to be developed.

Abnormal anterior belly of the digastric muscle: a proposal for the classification of abnormalities.

We recognized an abnormal anterior belly of the digastric muscle in an 83-year-old male cadaver. Three muscle bundles were observed on the left anterior belly: (i) attached to the left digastric fossa; (ii) attached to the right digastric fossa; and (ii) attached to the raphe of the mylohyoid muscle. Four muscle bundles were recognized on the right anterior belly: (i) attached to raphe of the mylohyoid muscle; (ii, iii) attached to the exterior surface on the base of the mandible from the raphe of the mylohyoid muscle; and (iv) attached to the interior surface on the base of the mandible from the raphe of the mylohyoid muscle. The raphe of the mylohyoid muscle was curved significantly to right and the four abnormal bundles found on the right anterior belly (see above) were attached to its curved point.

Scanning electron microscopic observations of the basement membranes with dithiothreitol separation.

Scanning electron microscopic (SEM) studies of the interstitial surface of the lamina densa can be performed with dithiothreitol separation, which is the only method of exposing this surface. SEM observation revealed the three-dimensional structures of the meshwork in the lamina densa and anchoring fibrils in dithiothreitol-separated specimens. Detection of the components of the basement membrane can be performed by immunoscanning electron microscopy on this exposed surface by comparing the backscattered and the secondary electron images. SEM observation also revealed the fine structure of the lamina fibroreticularis using separated dermis or the lamina propria mucosae.

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae.

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.

Regeneration of the epidermis and mucosal epithelium on the basement membranes.

The processes of degeneration and regeneration of the lip epidermis and mucosal epithelium after cryo treatment were observed by transmission electron microscopy. Epidermal and mucosal epithelial cells degenerated due to the formation of ice crystals and detached from the basement membranes, leaving a small amount of cell debris. Regenerating cells migrated over the cell debris, which was gradually phagocitized, and formed new hemidesmosomes with the preexisting lamina densa. Regenerating epidermal cells migrated from the undamaged areas and the hair follicles. Regenerating mucosal epithelial cells originated from surviving cells in the basal half of the epithelium, at the periphery of the blister cavity. They migrated in a multilayered fashion. Desmosomes were preserved even between dead cells and between dead and living cells.

Real electron microscopic images of collagen fibrils in the endoneurium.

Collagen fibrils run in parallel in the endoneurial space, forming fibre bundles. Spaces are evident between these bundles when examined by transmission electron microscopy (TEM). However, the procedures for TEM include chemical fixation, dehydration and embedding, which may cause morphological changes in the specimens. Ultracryo thin sectioning procedures may avoid the artefacts caused by these procedures. An examination of ultrathin frozen sections revealed that the endoneurial space was completely filled with collagen fibrils, with little space between the fibre bundles. These results suggest that the dehydration and/or embedding procedures cause shrinkage of the specimen, resulting in the appearance of a widened space in the endoneurium. Therefore, the widened space between the bundles of collagen fibrils may be a technical artefact.

Comparative observations on corneas, with special reference to Bowman's layer and Descemet's membrane in mammals and amphibians.

Corneas of tadpole, mouse, rat, guinea pig, rabbit, cat, cattle, and human were examined by TEM and SEM in a comparative study. The differences between species were noted mainly by using TEM. Bowman's layer showed a tendency to be well developed in higher mammals. Tadpoles lack a Bowman's layer, lower mammals have a thin Bowman's layer, and higher mammals have a thick Bowman's layer. The boundary between the substantia propria and Descemet's membrane was distinct in higher mammals. On the other hand, there are no differences in thickness of the collagen fibrils that constitute Bowman's layer and those of the substantia propria. NaOH digestion was utilized for SEM preparation. SEM imaging revealed a textured appearance of the epithelial side of Bowman's layer. In Descemet's membrane, fibrous long spacing (FLS) fiber-like structures, which are arranged in parallel to the endothelium, were observed by both TEM and SEM. To our knowledge, this is the first report of SEM observations of FLS fiber-like structures on the endothelial surface of Descemet's membrane. SEM at a plane normal to the plane of the cornea showed that Descemet's membrane has a piled laminar structure. Descemet's membrane is closely associated with the collagen layer of the substantia propria. Collagen fibrils invading from the substantia propria into Descemet's membrane were observed with both TEM and SEM.

Measurement of the repeat period of myelin sheath using ultrathin frozen sections.

The myelin sheath of peripheral nerves was observed by transmission electron microscopy (TEM) using plastic-embedded sections and ultrathin frozen sections. Repeat distances of myelin sheaths were measured in high-powered electron micrographs. The ultrathin frozen sections showed a longer repeat distance than the plastic-embedded sections. The ultrathin frozen sections were thought to contain fewer artefacts, as they had not been subject to dehydration and embedding. It is known that broken myelin sheaths are often observed under conventional TEM. It is thought that these procedures cause contraction and partial destruction of the myelin sheath.