Hiroshi Konomi

Distribution of Types I, II, III, IV and V Collagen in Normal and Keratoconus Corneas

By using type-specific antibodies to types I, II, III, IV and V collagens, distribution of distinct types of collagen in normal human cornea as well as keratoconus cornea were examined by indirect immunofluorescence microscopy. In normal human cornea, immunohistochemical evidence supported the previous biochemical finding that type I collagen was the major type of collagen in human corneal stroma. No reaction was observed to anti-type II collagen antibody in the whole cornea. Anti-type III collagen antibody reacted with the corneal stroma in a similar fashion as that of anti-type I collagen antibody. Type IV collagen was observed in the basement membrane of the corneal epithelium and in Descemets membrane. Anti-type V collagen antibody also reacted with the corneal stroma diffusely. Bowmans membrane was strongly stained only with he anti-type V collagen antibody. For further details of the distribution of type I, type III and V collagens in human corneal stroma, immunoelectron microscopic study was undertaken. The positive reaction products of anti-type I and anti-type III collagen antibodies were located on the collagen fibrils, while that of anti-type V collagen antibody was either on or close to collagen fibrils. In keratoconus cornea, no difference was observed in terms of the distribution of type I, III and V collagens, while the disruptive and excrescent distribution of type IV collagen was noted in the basement membrane of the corneal epithelium.

Developmental immunohistochemistry of catalase in the human brain

The immunohistochemical studies on a peroxisomal enzyme, catalase, were done on brains from human fetuses to adults. The catalase-positive neurons appeared in the basal ganglia, thalamus and cerebellum at 27-28 weeks of gestation, and in the frontal cortex at 35 weeks. They then increased in number with gestational age. The extent of immunopositive staining increased with enlargement of perikaryonal size. However, the extent gradually decreased with postnatal age. On the other hand, catalase-positive glia appeared in the deep white matter at 31-32 weeks of gestation, their appearance shifting from the deep to the superficial white matter with increasing age. These results suggest that peroxisomes are closely related to neuronal growth and myelinogenesis in the human brain during development.

Immunohistochemical study of the vasculature in the developing brain

In this study, the developmental proliferation of human brain vessels, from the fetal to the adult stage, was analyzed by immunohistochemical methods using antitype IV collagen, antilaminin, and antifibronectin antibodies. Examination of the frontal lobe indicates that these antibodies bind to the vessels, both arteries and veins. During cortical angiogenesis, the density and diameter of vessels increase rapidly from about 26 weeks gestation and peak at 35 weeks; after 35 weeks, the density and diameter of vessels are the same as those in adult brain. The white matter demonstrates no major changes in vessel density, although the pattern of the changes in vessel diameter resembles that of the cortex. Small immunopositive spots suggesting neovascularization reveal the same developmental tendency as the density of vessels in the cortex and white matter; therefore, it appears that neovascularization in the fetal brain during development is more rapid than cortical expansion and is equal to the growth of white matter. Neovascularization may be closely related to normal brain development and may play an undefined role in perinatal cerebrovascular insults.

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.

IMMUNOHISTOCHEMICAL LOCALIZATION OF TYPE I, III AND IV (BASEMENT MEMBRANE) COLLAGENS IN THE LIVER

The tissue distribution of type I, III and IV (basement membrane) collagens in normal human and bovine livers was examined by indirect immunofluorescence microscopy, by using type‐specific rabbit antibodies to individual types of bovine collagen. Type I and III collagens were found to distribute in the interstitium of portal tracts as thick fiber bundles and in perisinusoidal spaces as thin fibers like reticulin fibers, both in human and bovine livers. No significant distribution differences of type I and III collagens in the livers was observed under the experimental conditions employed, indicating that both collagens are Involved in in vivo collagen fibrillogenesis in the tissue, regardless of the sizes of collagen fibers, as in the skin (Conn. Tiss. Res. 7: 157–163, 1980). Type IV collagen, when examined with bovine liver, was located in hepatic arteries, portal veins and bile ducts of portal tracts, and was also distributed in the perisinusoidal spaces In a linear fasion.

Developmental change in type VI collagen in human cerebral vessels

Vascular development in the human brain was studied by immunohistochemistry using an anti-type VI collagen antibody. Positive vessels were evident from an early gestational age in the meninges, from 21 weeks gestation in the basal ganglia and deep white matter, and from 38 weeks gestation in the cerebral cortex and superficial white matter; however, type VI collagen never appeared in the subependymal germinal layer. The absent or scarce type VI collagen in the subependymal germinal layer may be one of the important factors of subependymal/intraventricular/periventricular hemorrhage in premature neonates. The earlier appearance of positive vessels in the deep white matter than in the cortex and superficial white matter suggests that the medullary vein develops earlier than the cortical and subcortical veins and arteries. These characteristics of the developing vascular structure may be one cause of perinatal brain damage.

IMMUNOHISTOCHEMICAL LOCALIZATION OF TYPE I, II, III, AND IV COLLAGENS IN THE LUNG

Type specific rabbit antibodies to bovine type I, 11, 111, and IV (basement membrane) collagens showing no cross‐reaction with other types of collagen were prepared by cross‐adsorption and diethylamiuoethyl‐cellulose romatography. The antibodies to bovine type I and I11 collagens showed a high cross‐reaction with the corresponding human collagens, but those to type I1 and IV collagens did moderate and no cross‐reactions with human type I1 and IV collagens, respectively. By using these antibodies, tissue distribution of various types of collagen in normal bovine lung was examined by indirect immunofluorescence microscopy. Both type I and I11 collagens were found to distribute widely in the interstitium of bronchial tree, bronchial

Glycosaminoglycan and collagen distribution in the developing human vitreous

Abstract · Background: We determined the distribution of glycosaminoglycans and collagens in the developing human vitreous. · Methods: Eighty human eyes from 5 gestational weeks to 2 postnatal years of age were used. Glycosaminoglycan components were determined by enzyme digestion with hyaluronidase or chondroitinase AC and ABC and immunohistochemistry for chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate. Collagen distribution was determined by immunohistochemistry for types I, II, and III collagens. · Results: Enzyme digestion showed that throughout development hyaluronic acid is the main glycosaminoglycan in the vitreous and in the extraocular space at 5–7 gestational weeks. Both areas were filled with mesenchymal cells. Immunohistochemistry showed chondroitin-6-sulfate in the vitreous between 6 and 40 gestational weeks, and chondroitin-4-sulfate between 12 and 40 gestational weeks. Hyaluronic acid and chondroitin sulfate appeared in the retina and around the hyaloid vessels at 12–40 weeks. Immunohistochemistry showed type III collagen in the vitreous and around the mesenchymal cells at 5–7 weeks that was replaced by type II collagen after 8 weeks. · Conclusions: Hyaluronic acid is the major glycosaminoglycan in the vitreous throughout development, except for the transient appearance of chondroitin sulfate at 6–40 gestational weeks. Type III is the main collagen in the early developing vitreous that converts to type II collagen at 8 weeks. The primary and secondary vitreous has the same components as these macromolecules. These vitreous glycosaminoglycans and collagens seem to be produced by mesenchymal cells at an early stage and by the retina and hyaloid vessels during middle and late development.

Transitions in collagen types during endochondral ossification in human growth cartilage

Immunohistochemical staining for Types I and II collagen in human growth cartilage showed that cartilage matrix consists of Type II collagen, whereas bone matrix contains Type I collagen. Endochondral ossification began with the deposition of Type I collagen by cells derived from bone marrow on the surface of the eroded cartilage. However, territorial matrix of the last hypertrophic chondrocytes contained both Types I and II collagen, thereby indicating that the degenerating chondrocytes initiate the synthesis of Type I collagen. This matrix, consisting of Types I and II collagen, is then replaced by newly formed osteoid.

Immunohistochemistry of superoxide dismutase-1 in developing human brain

The developmental changes in superoxide dismutase (SOD)-1 were studied in brains ranging in age from human fetuses to adults by immunohistochemistry. SOD-positive neurons and glial cells appeared with maturation in each region, and increased progressively with gestational and postnatal age. This phenomenon implies a relationship between SOD-1 gene expression and the anti-oxidant defence mechanism in developing neurons and glia.