Gentaro Usuku

Electron microscopical studies on the genesis of white adipocytes: Differentiation of immature pericytes into adipocytes in transplanted preadipose tissue

SummaryIn order to clarify the relationships between perivascular cells, capillaries and fat cells, with a special reference to the origin of fat cells, we have made a light and electron microscopical study on the developing epididymal adipose tissue of newborn to 5-week-old rats, and also on the differentiating, transplanted epididymal preadipose tissue from 6-day-old rats. Development of epididymal preadipose tissue progressed rapidly 6 or 7 days after birth. The preadipose tissue on the 5th day after transplantation consisted of differentiated areas with many mature fat cells, and of undifferentiated areas in which these cells were scanty. In the differentiated areas of developing epididymal preadipose tissue, both in situ and transplanted, many fat cells seemed to develop in the area immediately adjacent to growing capillaries, but cells intermediate between perivascular cells and preadipocytes were seldom observed. However, in undifferentiated areas of transplanted tissue, we found ultrastructural evidence that immature pericytes of capillaries can differentiate into preadipocytes.

Possible systemic smooth muscle layer dysfunction due to a deficiency of dystrophin in Duchenne muscular dystrophy

The localization of dystrophin was examined immunohistochemically in various tissues from mice and rats as well as from biopsied human muscle specimens, using polyclonal antibodies against dystrophin. Although dystrophin was completely absent in biopsied muscle specimens from 3 male DMD patients, it was faintly observed in the surface membrane of almost all muscle fibers examined in a female DMD patient. In all controls, human and animal, a strong dystrophin reaction was observed in the surface membrane of intrafusal muscle fibers and at the neuromuscular junctions, rather than in the surface membrane of skeletal and cardiac muscle fibers. In addition, dystrophin was clearly localized in the surface membrane of smooth muscle fibers in the viscera, bronchial system, ureter, and tunica media of blood vessels, including arteries and veins, in all examined animal tissues. In mdx mice, dystrophin was absent in almost all muscle and smooth muscle fibers in various tissues and blood vessels. These results suggested a possible systemic dysfunction of smooth muscle layers, especially those of blood vessels, as well as skeletal muscle fibers, due to a deficiency of dystrophin in Duchenne muscular dystrophy.

An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.

Type VI Collagen in Healing Rabbit Corneal Wounds

A type-specific monoclonal antibody was used to examine the localization of type VI collagen in healing full-thickness corneal wounds (3 mm in diameter) in rabbits. By immunofluorescence, in 1-week-old wounds, type VI collagen was found only at the wound periphery. Subsequently, type VI collagen showed a rapid increase throughout the wounded area. In 3-week-old wounds, type VI collagen immunofluorescence had a laminar pattern as seen in normal corneal stroma, although it was irregular in arrangement. By electron-microscopic immunohistochemistry, type VI collagen was found in the pericellular region of fibroblastic cells possibly derived from keratocytes. These results suggest that type VI collagen might originate from the fibroblastic cells and play an important role in the regeneration of the corneal lamellar structure.

Distribution of type VI collagen in the bovine cornea

Using a specific polyclonal antibody against type VI collagen, the distribution of type VI collagen in the bovine cornea was investigated by indirect immunofluorescence microscopy and immunoelectron microscopy. By immunofluorescence, type VI collagen was detectable throughout the stroma. In addition to the uniform but relatively weak signals, a laminar pattern similar to the arrangement of stromal lamellae was clearly seen. Consistent with the immunofluorescent pattern, immunoelectron micrographs showed strong linear reactions located in the interlamellar spaces. The reactions were also observed in the pericellular region of the keratocytes. Epithelium, Descemets membrane and endothelium were negative.

Endosteal New Bone Formation in the Long Bones of Adjuvant Treated Rats

Histopathological changes were examined mainly in the diaphyseal parts of long bones, especially femur in adjuvant-treated male Lewis-SPF rats, with reference to clinical symptoms of chronic osteoarthritis. The diaphyseal bone marrow of long bones in these rats sequentially showed three different processes of chronic pathological changes, which, however, partly overlapped each other. Initially, multiple epitheloid cell granulomas or granulomatous lesions containing fibrin deposits began to appear in the marrow space about 22 days after adjuvant injection, when the joint score of arthritis reached a peak in severity. Secondly, about a week after appearance of the granulomas, there occurred the intramembranous endosteal new bone formation proceeding from the endosteum towards the granulomatous lesions. The bone formation reached a maximum about 64 days after the treatment, when the redness of joints of feet and hands was already sedated. Finally, about 40 days after occurrence of the second event, the newly growing bone matrix began to be actively resorbed simultaneously. On the other hand, in the bone marrow of metaphyseal parts of long bones in these rats, severe acute osteomyelitis was observed from an early stage, with marked destruction of bone trabeculae and simultaneous new bone formation. In the diaphyseal bone marrow of affected long bones, the epitheloid cell granulomas appear to induce the endosteal new bone formation.

Studies of the lung in diabetes mellitus

To evaluate the effects of chemically induced diabetes on lung tissue, we examined the ultrastructure of the lung of alloxan-induced diabetic rats. Fifty male Wistar rats were made diabetic by a single intraperitoneal injection of alloxan (200 mg/kg of body weight): they were sacrificed from one to four weeks later. The alloxan-induced diabetes produced significant morphological alterations in the lung. These include marked dilatation of the cisterna of the granular endoplasmic reticulum, dilation of the Golgi saccules and the appearance of glycogen granules as a cluster in the cytoplasm of the granular pneumocytes and the interstitium. These findings were well correlated with the severity of diabetes mellitus. The altered granular pneumocytes were observed in about 50% of animals and in most (87.5%) of the observed pneumocytes 2 weeks and 4 weeks after alloxan treatment respectively. The average number of lamellar inclusion bodies per granular pneumocyte decreased to about half of that of the control in diabetic rats 4 weeks after alloxan treatment, and minimum thickness of the capillary basement membrane was approximately 35% thicker than that of the control (diabetics; 879±189 Å, controls; 649±100 Å). The ultrastructural alterations of the lung in diabetic rats indicate disorders in the pulmonary capillaries and in the metabolism of pulmonary surfactant, which may cause pulmonary dysfunction in diabetic patients.

An ultrastructural study on the ear cartilage of rabbits after the administration of papain. Appearance of cross-striated collagen segments of an atypical FLS-type.

Crude papain was administered intravenously to young rabbits and the cartilage of the collapsed ear was examined electron-microscopically. Degeneration and recovery of chondrocytes, and decrease in and recovery of the electron-density of elastic fibers, were observed during the collapse and restoration of the ear. Some samples were stained with ruthenium red. In the collapsed ear, with a marked decrease of proteoglycan in the cartilage, loss of ruthenium red-positive granules was observed in the extracellular matrix. Collagen fibrils in the cartilage appeared to be somewhat increased in number, some of their diameters became slightly greater, and a part were assembled into bundles, occasionally accompanied by periodic crossstriation. Decrease of proteoglycan in the cartilage matrix probably brought about the unmasking and the assembly of collagen fibrils. In one of the experimental animals, collagen fibrous segments of an atypical fibrous long spacing (FLS-)type with symmetrical cross-striation were found around the chondrocytes in the ear cartilage, during the period of recovery. Some kind of the endogenous sulfated carbohydrate may have acted to affect the arrangement of type II collagen or procollagen molecules newly produced by the recovering chondrocytes.

STUDIES ON ANIMAL COLLAGENASE

During metamorphosis, the collagen layer (basement lamella) of the resorbing tail fin of bullfrog tadpole was markedly disintegrated, and was invaded by mesenchymal cells from below, which contained cytoplasmic vacuoles enclosing collagen fibrils. Participation of the locally produced collagenase in the process was discussed.

A freeze-fracture study of two types of collagen-phagocytosing cell in the post-partum rat endometrium

SummaryIn the post-partum rat endometrium, ultrastructural distinction could be made between stromal cells (fibroblast-like cells) and macrophages, especially by the freeze-fracture technic. The stromal cells were characterized by a well-developed rough-surfaced endoplasmic reticulum (RER) and intercellular junctions, while the macrophages had many vacuoles and vesicles, but no intercellular contact with each other. The freeze-fracture image showed that the stromal cells had many low linear elevations and gap junctions on the cleaved plane of the cell membranes, while the macrophages had no linear elevations or intercellular junctions. The cell membranes of the stromal cells had more intramembranous particles (IMP) (P-face 697 ± 63/μm2,E-face 303 ± 52/μm2) than those of the macrophages (P-face 467 +- 50/μm2,E-face 217 ± 35/μm2). It was confirmed that these two types of cell phagocytosed collagen fibrils.