Shigeki Yamabayashi

Ultrastructural Studies of Collagen Fibers of the Cornea and Sclera by a Quick-Freezing and Deep-Etching Method

The ultrastructure of collagen fibers of the cornea and sclera of rabbits was studied by a newly developed quick-freezing and deep-etching method. An isopentane-propane mixture (-193 degrees C) cooled in liquid nitrogen was used for quick-freezing of the tissues. The frozen tissues were fractured in nitrogen, deeply etched and shadowed with platinum and carbon. Corneal collagen fibers were observed to be longitudinally arranged and separated by moderately wide interfibrillar spaces. Interconnecting filaments were three-dimensionally radiating between the collagen fibers, which were 40.6 +/- 5.0 nm in diameter and 42.0 +/- 2.8 nm in periodicity. In contrast, scleral collagen fibers were compactly organized, with few interconnecting filaments. Five kinds of striations were clearly observed on the collagen fibers, which were 217.3 +/- 50 nm in diameter and 67.5 +/- 1.4 nm in major periodicity. These striations could hardly be observed in the fractured scleral collagen fibers. The present study reveals the ultrastructural differences between corneal and scleral collagen fibers through the use of a quick-freezing and deep-etching method.

Effect of Alpha-Human Atrial Natriuretic Peptides on Intraocular Pressure in Normal Albino Rabbits

The effect of alpha-human atrial natriuretic peptide (alpha-hANP) on the intraocular pressure (IOP) was studied in normal rabbits. Dose response studies to IOP were performed after intravenous administration of alpha-hANP at 6.25, 12.5, 25.0, 50 and 100 micrograms/kg to the rabbits. The maximum reduction in IOP was observed to occur with the alpha-hANP 50 micrograms/kg dose. The time course of the pressure response to alpha-hANP were measured prior to and following intravenous administration of 50 micrograms/kg of alpha-hANP. The change in IOP and blood pressure showed the same pattern in the simultaneous record. Silver grains of 125I-alpha-hANP (10 microCi/g body weight) were detected in the ciliary processes of mice by radioautography after intravenous injection. The pupillary response was not tested in this experiment. The mechanism of IOP reduction was discussed.

Histochemical Studies on the Conjunctival Goblet Cells

The difference in the histochemical results between AB-PAS and PAS-AB sequence was studied in the conjunctival goblet cells of monkeys and rats by light microscopy. These sequences demonstrated the pr

Incorporation of 3H-befunolol (beta-blocking agent) into melanin granules of ocular tissues in the pigmented rabbits

Summary3H-befunolol was administered intravenously to pigmented rabbits. Thirty minutes after the administration, the iris, ciliary body and retina were fixed, embedded and processed for light microscopic radioautography. Radioautographical silver grains were observed over the pigment granules of the iris, ciliary body, choroid and retina. From these results it is concluded that 3H-befunolol is incorporated into the pigment granules of these cells. The mechanism of incorporation is discussed.

Histochemical study on rat tear film and ocular surface epithelial cells

PURPOSE To investigate the histochemistry of the tear film, conjunctival goblet cells and corneal superficial epithelial cells by an in vivo cryofixation with the freeze-substitution method. METHODS Following rapid freezing of the eyeballs by an isopentane-propane mixture cooled by liquid nitrogen directly, the specimens were freeze-substituted and then embedded in Lowicryl K4M resin at low temperature, or in epoxy resin for histochemical study, using light and transmission electron microscopy. RESULTS The tear film was weakly positive with periodic acid-Schiff staining, but negative with alcian blue at pH 2.5 or pH 1.0 and high iron diamine staining. Conjunctival goblet cells were positively stained by all these techniques. There was a gradually weakening staining intensity of mucus from the goblet cells to the conjunctival surface. With the periodic acid-thiocarbohydrazide-silver protein method the tear film was weakly stained, while glycocalices, vesicles, glycogen in epithelial cytoplasm and the mucus secretory granules of goblet cells were all intensely stained. CONCLUSIONS The tear film consists mainly of dilute mucus and contains either neutral or acid glycoproteins.

Ultrastructural Studies on the Corneal Superficial Epithelium of Rats by in vivo Cryofixation with Freeze Substitution

The method of in vivo cryofixation with freeze substitution (VC-FS) was used to examine the natural ultrastructure of the corneal superficial epithelium under the transmission electron microscope, and the results have been compared with the conventional method. With the VC-FS method, the ultrastructure down to 8 microns in depth was well preserved in the specimen. There was a layer of homogeneous material thought to be tear film on the corneal surface. Except for the intercellular spaces presenting under the first and sometimes also the second layers of the epithelial cells, other adjacent cells were in close contact without any spaces between them or between the desmosomes. In contrast, when using the conventional method, intercellular spaces were found between adjacent cells in all the layers of epithelium, and no tear film remained on the corneal surface. VC-FS is considered to be a better way to preserve the structure of tissue in its natural state than the conventional method and to be a useful method for the morphological study of the corneal epithelium and the tear film. Intercellular spaces, a kind of artifact, can be reduced by this method.

Ultrastructural Study of Basal Lamina of Retinal Pigment Epithelium

The ultrastructure of basal lamina (BL) of retinal pigment epithelium (RPE) in guinea pigs was studied by the quick-freezing, deep-etching (QF-DE) method. The retinal tissue was quickly frozen, fractured and deeply etched. A replica was prepared by shadowing with platinum and carbon. Electron micrographs of the BL of RPE provide a three-dimensional (3-D) ultrastructure. The lamina lucida was filled with traversing filamentous structures, which connected the basal infolding of RPE with the lamina densa. The lamina densa consisted of a 3-D meshwork structure. It is suggested that the traversing filamentous structures in the lamina lucida might play a role in anchoring the basal infolding of RPE and in maintaining the normal architecture.

Newly Designed Continuous Corneal Irrigation System for Chemical Burns

A newly designed continuous irrigation system has been applied in our clinic. Six cases of corneal chemical burns have been treated with this system from September 1988 to February 1989. The present system has been available in any clinics by materials such as a disposable intravenous tube and a scalp vein tube for infants. Compared with the methods previously reported, this system showed good results, simplicity for setting up, good patient tolerance and low cost of the equipment.

Application of a Laser Scanning Microscope to the Morphological Study of Corneal Epithelium of Rabbits

A laser scanning optical microscope was used to study the structure of the rabbit corneal epithelium. From the results obtained, satisfactory images have been taken of unfixed, 0.05% glutaraldehyde-fixed and 4% paraformaldehyde-fixed materials. Continuous optical sections were taken between the superficial layer of the corneal epithelium and the basal layer. It was concluded that this device is useful for investigating the tissue structure of fresh biological materials.

Bupranolol, A β1- and β2-Blocker, Causes Constriction of the Pupil in Men

Two out of three normal eyes tested with frequent instillation of 30 µl of drops of 0.5% bupranolol, a β1 + β2 blocking drug with some intrinsic sympathomimetic and local anesthe