Yoshifumi Hirabayashi

Histopathologic evaluation of the internal limiting membrane surgically excised from eyes with diabetic maculopathy.

Purpose: To histopathologically evaluate the internal limiting membrane (ILM) in diabetic eyes with macular edema as compared to nondiabetic controls. Methods: The authors ultrastructurally and immunohistochemically studied ILM specimens that were intentionally peeled from five eyes with diabetic maculopathy, comprising four with diffuse diabetic macular edema and one with macular hole accompanying diabetic retinopathy (DM group), and five with nondiabetic idiopathic macular hole (MH group). They compared ultrastructural and immunohistochemical findings between the two groups. Results: A larger amount of cellular elements was observed on the vitreous side of the ILM in the DM group. The thickness of the ILM in the DM group was significantly increased (mean 4.8 ± 1.6 &mgr;m) compared with that in the MH group (1.8 ± 0.6 &mgr;m) (P < 0.0001). Immunoreactions for heparan sulfate proteoglycan in the ILM were more abundant in the DM group than in the MH group. Conclusion: The ILM thickening and cell abundance on the vitreous surface might contribute to the course and the pathogenesis of diabetic maculopathy.

Cloning and functional expression of ASIC-β2, a splice variant of ASIC-β

We have isolated a cDNA encoding a splice variant of ASIC (acid-sensing ion channel)-β from the rat trigeminal ganglion. This clone, designated ASIC-β2, showed a 342 base deletion just after the first transmembrane domain in ASIC-β. RT–PCR experiments revealed that ASIC-β2 was expressed exclusively in the trigeminal ganglion and dorsal root ganglion. In situ hybridization showed that ASIC-β2 mRNA was concentrated in both small diameter and large diameter neurons and co-localized with ASIC-β mRNA within single sensory neurons in the trigeminal ganglion. When expressed in Xenopus oocytes, ASIC-β2 was inactive by itself. However, it associated with ASIC-β to form heteromers, which display lower affinity for protons than ASIC-β alone.

Strongyloides venezuelensis: longitudinal distribution of adult worms in the host intestine is influenced by mucosal sulfated carbohydrates

Mechanisms for the longitudinal distribution of parasitic females of Strongyloides venezuelensis in the host intestine were investigated in mice. Adult worms were mostly recovered from the anterior-most one-third of the small intestine throughout the infection after infective larvae inoculation. Surgically implanted adult worms established well in the small intestinal mucosa, either in the duodenum or in the ileum, whereas a few worms could establish in the large intestine. Implanted worms in the small intestine remained where they were implanted until expelled. Mucosal mast cells were induced in the whole small intestine after the worm implantation. In the large intestine, a considerable number of adult worms settled in the mucosa of mutant mice, whose goblet cell mucins were undersulfated because of a mutation in sulfate-activating enzymes. In these mice, the degree of sulfation of goblet cell mucins in the large intestine was significantly reduced to the level of normal small intestine goblet cell mucins. Our results suggest that sulfated glycoconjugates, either from mucosal mast cells or goblet cells, have important effects on the longitudinal distribution of parasitic females of S. venezuelensis.

Apoptosis is associated with formation and persistence of the embryonic fissure

PURPOSE The role of apoptosis in the transitory ocular embryonic structures has not been clarified yet, therefore, in the present study we focused on one of the transitory ocular structures, the embryonic fissure. To elucidate the developmental mechanism of the embryonic fissure, we observed cell death by apoptosis in the optic cup during early development in mice. METHODS Pairs of C57BL6N/Jcl mice, each comprising an estrous female and a potent male, were caged together overnight. Females that had vaginal plugs the next morning were considered at day 0 of pregnancy. The embryos or fetuses were removed by laparotomy on days 9, 10, 11, 12, 13, 14, 16, and 18 of gestation. Tissue blocks of the eyes were fixed and embedded in paraffin wax. Serial frontal sections of the eye were cut and stained with the TUNEL method and then counterstained by hematoxylin or methyl green solution. We examined TUNEL-positive cells in the optic cup by light microscopy. RESULTS TUNEL-positive cells were seen at the lower nasal side of the optic cup, corresponding to the presumed embryonic fissure area, on day 9 of gestation before the formation of the embryonic fissure. Many TUNEL-positive cells were present at the lips of the embryonic fissure on days 10, 11, and 12. In contrast, TUNEL-positive cells were not detectable in the corresponding area on day 13 after the complete closure of the embryonic fissure. CONCLUSIONS Apoptosis is anatomically closely associated, and appears to be essential for the formation and persistence of the embryonic fissure.

Bruch's membrane of the brachymorphic mouse.

Bruchs membrane exists between the retinal pigment epithelium and the choriocapillary endothelium. Its structure is very complicated, having five sublayers containing basement membranes of retinal pigment epithelium and choriocapillary endothelium, outer and inner collagenous layers, and a central elastic layer. In the development of Bruchs membrane in normal mice, both basement membranes are created first. Secondarily, collagen fibers are accumulated in the space between these basement membranes and then form a collagenous layer. Finally, the elastic layer elaborated in the collagenous layer separates this into outer and inner collagenous layers. Brachymorphic mice have a disorder in the sulfation pathway, resulting in undersulfation. Consequently, in Bruchs membrane of brachymorphic mice, the expression of decorin, a small proteoglycan containing chondroitin sulfate and an indispensable component in collagen assembly, is at a very low level. It is clear that hypoplasia of the collagenous layer in Bruchs membrane of brachymorphic mice induces a disorder in the following formation of the elastic layer. These findings suggest that the formation of the collagenous layer, regulated with acidic glycoconjugates such as decorin, is important in the development of Bruchs membrane.

Cytological and Lectin Histochemical Characterization of Secretion Production and Secretion Composition in the Tubular Glands of the Canine Anal Sacs

The study reports on secretion production and composition in the tubular glands of the canine anal sacs. For this purpose, light and electron microscopical (TEM, SEM) as well as several histochemical methods for the demonstration of lysosomal acidity, lipofuscin, and complex carbohydrates were used. The glandular tubules exhibited a pseudostratified epithelium with secretory cells of a different shape as related to secretion production activity, and regionally varying amounts of basal cells. Flat, cuboidal or columnar cells with or without apocrine-like protrusions were assembled in one glandular endpiece, although grouping of these cell types often occurred. Active secretory cells were columnar with many cytoplasmic vesicles and a typically merocrine and/or micro-apocrine exocytosis of vesicle contents. Additionally, many lysosomes of different sizes could be found, whereby in aged cells giant secondary lysosomes (autophagolysosomes, about 7 µm in diameter) occupied the major cell part. These giant lysosomes were shed by an apocrine-like process forming a final bottleneck stage of the upper cell part, and consisted of ceroid-type lipofuscin. The general carbohydrate histochemical and the lectin histochemical methods revealed that the secretion produced was composed of strongly concentrated neutral glycoproteins with the following saccharide residues: α-D-mannose, β-D-galactose, β-N-acetyl-D-glucosamine, α-L-fucose and N-acetyl-neuraminic acid (sialic acid); the luminal secretion contained only β-D-galactose and, especially, N-acetyl-neuraminic acid. This luminal secretion showed a spatially orientated maturation beginning in terminal tubular regions and finishing near the excretory duct, independent of the different secretory cell types. The results obtained demonstrated highly active secretion production, with a regional variation in the glandular tubule, and at least three different modes of secretion by the secretory cells, whereby the shedding of giant lipofuscin granules seems to be very specific. The high amounts of sialic acids in the glycoproteins found may influence the rheological properties of the secretion by their water-binding capacities.

An Improved Procedure for the Histochemical Demonstration of Cathepsin D by the Mercury-Labeled Pepstatin Method

The desirable fixation conditions for the histochemical demonstration of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor were examined biochemically and histochemically. Four well known fixatives, namely, glutaraldehyde (GA), paraformaldehyde (PFA), glutaraldehyde with paraformaldehyde (GA-PFA) and periodate-lysine-paraformaldehyde (PLP), were applied to the prefixation of tissues prior to the reaction of the labeled inhibitor to the enzyme-active site. The effects of fixatives on cathepsin D were biochemically examined using subcellular fractionated lysosomes. Cathepsin D from rat liver lysosomes was rapidly inactivated by the fixatives containing glutaraldehyde, i.e., GA and GA-PFA, whereas the activity of cathepsin D was sufficiently maintained after fixing the enzyme in the PFA or PLP preparations. Effects of the PLP fixative on lysosomal cathepsin D in liver tissues using the mercury-labeled pepstatin method were also studied histochemically. The best result for the visualization of lysosomal cathepsin D in liver tissues was obtained using the PLP fixative with the prefixation time of three hours or more.

Light-microscopic detection of acidic glycoconjugates with sensitized diamine procedures

SummaryTo enhance the efficiency and specificity of diamine methods in light microscopy, these methods were sensitized by sliver enhancement in combination with trichloro(ethylene) platinate (KTP). The sensitized diamine methods consisted of a diamine (high or low iron diamine: HID or LID), KTP, borohydride reduction (BH) and a physical development (PD) sequence. The new methods have been successfully applied to routinely prepared tissue sections obtained from rat organs, such as salivary glands, stomach, colon, kidney, lung and trachea. In the tissues subjected to the sensitized diamine methods, weakly diamine-stained histological structures exhibited vivid positive reactions. The combined sensitized diamine methods and selective procedures, such as enzyme digestion and chemical modification, have substantiated that these methods were of sufficient efficiency and specificity.

Enhanced expression of adhesion molecules of the retinal vascular endothelium in spontaneous diabetic rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a spontaneously diabetic strain with clinical features resembling those of human noninsulin-dependent diabetes mellitus. These rats show increased leukocyte entrapment in the retina. The present study was designed to investigate the immunolocalization of intercellular adhesion molecules-1 (ICAM-1) and P-selectin in the retinas of OLETF rats. Four 72-week-old male OLETF rats and 4 age- and sex-matched Long-Evans Tokushima Otsuka (LETO) control rats were used. Western blot analysis and immunohistochemical study were performed using an anti-P-selectin monoclonal antibody (mAb) and an anti-ICAM-1 mAb. Western blot analysis showed increased expression of both ICAM-1 and P-selectin in the retinas of OLETF rats. Immunohistochemically, OLETF rats expressed greater amounts of ICAM-1 and P-selectin in the retinal vascular endothelium than did LETO rats. These findings demonstrated the upregulation of ICAM-1 and P-selectin in the retina of OLETF rats. The enhanced expression of these adhesion molecules might participate in the pathogenesis of early diabetic microangiopathy in the retina.

HISTOCHEMICAL STUDIES OF GLYCOSAMINOGLYCANS IN DEVELOPING PERIODONTAL LIGAMENTS OF ICR MICE

Although the periodontal ligament (PL) contains an abundance of glycosaminoglycans (GAGs), there are only a few histochemical studies describing GAGs in the developing PL. In the present study, the relationship between the formation of principal fibers and the molecular species of GAGs in the developing PL was examined by light microscopic histochemistry. Jcl:ICR mice were killed on day 0 to day 28 after birth. Paraffin‐embedded tissue sections were routinely made and stained with hematoxylin‐eosin (H&E), Azan, or the sensitized high iron diamine (S‐HID) procedure combined with enzyme digestions. Before tooth eruption, thin threads of collagen fibers in the PL assembled and constructed principal fibers, which projected from both the side of the alveolar bone and the root of the tooth. After tooth eruption, the principal fibers from both sides were tightly entangled. In the developing PL, the molecular species of GAGs was mainly dermatan sulfate. Moreover, the relative amount of dermatan sulfate increased together with the maturation of the principal fibers, while the principal fibers adjacent to the alveolar bone and cementum contained chondroitin sulfate. These results suggest that dermatan sulfate contributes to collagen fiber assembly in the PL and that chondroitin sulfate relates to PL adhesion to the alveolar bone and to the cementum of the root. Anat Rec 254:465–473, 1999.