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The permeability properties of fenestrated capillaries in the colon and exocrine and endocrine pancreas to exogenous and endogenous molecules were examined. The exogenous tracers horseradish peroxidase (Einstein-Stokes radius [ESR], 3.0 nm), hemoglobin (ESR, 3.2 nm), and ferritin (ESR, 6.1 nm) were injected intravenously and allowed to circulate for 5-90 min. Tissues were removed and processed for cytochemical or standard electron microscopic examination. The endogenous plasma proteins albumin (ESR, 3.5 nm) and IgG (ESR, 5.5 nm) were localized by immunocytochemistry using the protein A-gold technique. All vessels examined were permeable to HRP in less than 5 min. In contrast, these vessels were restrictive to the slightly larger hemoglobin molecule (60-min circulation) and to ferritin (90-min circulation). Capillaries in the exocrine and endocrine pancreas were restrictive to albumin and IgG. These results demonstrate the presence of fenestrated capillary beds, in addition to the choriocapillaris, that are restrictive to molecules with ESR greater than or equal to 3.2 nm. Capillaries in the mucosa of the colon were restrictive to hemoglobin and ferritin but did not restrict albumin or IgG. This indicates that these vessels are of the permeable type. However, the rate of transendothelial movement of molecules is slower than that of other permeable vessels, such as in the ileo-jejunum. This study has provided further evidence for the existence of fenestrated endothelia that are restrictive to exogenous and/or endogenous molecules.

The permeability of retinal capillaries in RCS rats with inherited retinal degeneration was investigated with horseradish peroxidase used as a tracer. Five-week-old rats showed typical degeneration of photoreceptor cells and accumulation of outer segment debris, but retinal capillaries were not permeable to peroxidase. At 10 weeks of age, capillaries in the inner retina appeared normal, but many in the outer retina were leaky. Peroxidase activity in these latter vessels was demonstrable in the basal laminae of endothelial cells and pericytes and in vesicles on the luminal and abluminal sides of the endothelium. Tracer also permeated the intercellular spaces in the surrounding area. The number of leaky capillaries in the outer retina increased during the course of the dystrophy. The site of the leak in permeable capillaries has not yet been established; it may be due to an alteration of the endothelial cell junctions or of transcellular vesicular transport. In the choriocapillaris, peroxidase permeated Bruch's membrane and the basal infoldings between adjacent pigment epithelial cells; tracer progression along the intercellular spaces was blocked at the zonulae occludens at the apicolateral border. The RCS rat may be a useful model for studying the morphological basis of changes in capillary permeability associated with retinal degeneration.

The choriocapillaris is the fenestrated capillary network that supplies a large portion of the nutrients required by the retinal pigment epithelium, photoreceptor cells, and other cells of the outer neural retina. The permeability of these capillaries was investigated in the rat by the use of ferritin (mol. wt. approx. 480,000; mol. diam. 110A) as a tracer. Ninety minutes after intravascular ferritin administration, a high concentration of tracer particles was distributed uniformly in the capillary lumina but few particles were present in Bruch's membrane, the multilayered basement membrane that separates the choriocapillary endothelium from the retinal pigment epithelium. The bulk of the tracer remained in the capillary lumina with a definate blockage seen at fenestral, channel, and vesicle diaphragms. These results indicate that the ratchoriocapillary endothelium, unlike the fenestrated endothelia lining other capillary beds, constitutes an important barrier to the passage of ferritin and presumably of circulating native molecules of similar size.

The distribution of anionic (negatively charged) groups on the surface of retinal pigment epithelial cells (RPE), rod photoreceptor cells (RP) and discarded rod outer segments (R0S) was studied by labeling these groups with cationic ferritin (CF) at different pH values. In normal neonatal rats, characteristic CF binding patterns were observed on the RPE cell surface at pH values ranging from 1.8-7.2. Similar distributions were seen on the surface of RPE cells from RCS-p+ rats with inherited retinal degeneration. Treatment of RPE cells from normal neonates with several different degradative enzyme preparations prior to CF labeling, did not alter the density or distribution of CF molecules at any of the pH values tested. In normal adult rats, CF binding to RPE cells was essentially similar to that observed in neonates. In RP cells, heavy CF labeling at pH 1.8 was seen on the surface of the inner and outer segments and connecting cilium. CF labeling patterns on discarded R0S undergoing phagocytosis were similar to those on RPE cells at all pH values except pH 5.5. The findings indicate that anionic sites are present on the surface of RPE and RP cells and on discarded R0S undergoing phagocytosis. There is no evidence that such sites are involved in the phagocytic defect characteristic of RCS-p+ rats. Adult-like patterns of anionic sites on RPE cells appear early in postnatal development; their resistance to a wide range of degradative enzymes suggests that the carbohydrates or proteins containing these anionic moieties are organized in a complex fashion in the RPE cell surface.

The bone-marrow sinusoidal endothelium is a cellular barrier that separates developing blood cells in the extravascular space from the peripheral circulation. Mature blood elements enter the circulation via transendothelial migration pores. In the present study, monosaccharide constituents on the bone marrow endothelium were examined using lectin-affinity cytochemistry. With lectin-horseradish and lectin-ferritin conjugates, mannosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl, and sialic acid were localized to the luminal plasmalemma, bristle-coated pits and diaphragmed fenestrae. These were conspicuously reduced on the abluminal plasmalemma. When the tissue was treated with biotinylated lectins followed by avidin-ferritin, only a localization with wheat-germ agglutinin (sialic acid; N-acetylglucosaminyl) was observed. Pretreatment of the bone marrow with neuraminidase enabled the localization of the other monosaccharide components by the biotin-avidin method. Accumulations of carbohydrate residues were identified near the endothelium subjacent to migrating cells. Fucosyl moieties marked by Ulex europaeus agglutinin ( UEA ) reagents on the endothelium were not present. All binding was abolished by incubation of tissue and lectin conjugates with specific hapten sugars. labeling was also not present after Pronase E treatment, indicating that the identified monosaccharides are components of glycoproteins rather than glycolipids. The possible function of endothelial-surface glycoproteins as receptors for the surfaces of mature blood cells and their role in transmural migration are discussed.

Cytochemical methods have been used to examine the vascular endothelium. With hemeproteins and immunocytochemistry, investigators have demonstrated the pathways that blood-borne molecules can take to gain access to the extravascular space (Ghitescu et al. 1986; Milici et al. 1987; Schneeberger and Karnovsky 1971; Simionescu et al. 1975). These same cytochemical methods have also provided evidence that morphologically similar endothelia may have different permeability properties (Hart and Pino 1985b, 1986; Pino 1985; Pino and Essner 1980, 1981). Differences in the location and chemical composition of cell surface moieties have been ascertained with enzyme digestion methods, lectins, and cationic ferritin (De Bruyn and Michelson 1978; Pino 1984c, 1986a, b; Simionescu et al. 1981a). The author hopes that he has provided the reader with representative examples of how investigators have used these cytochemical methods for their studies. As new methods are developed and applications are found for existing techniques such as ultracryomicrotomy (Milici et al. 1987) and colloidal gold markers (Pino 1987b), cytochemistry will remain a fundamental tool for the study of the structure and function of the vascular endothelium.

A procedure using a 2.5% acrylamide-0.5% agarose gel for slab or tube isoelectric focusing is described. This composite gel is durable and enables a rapid focusing of high-molecular-weight compounds.

The avascular islet tissue preparation, the pseudoislet, was transplanted into the liver of streptozotocin diabetic rats. The vascularization of the islet tissue was studied ultrastructurally over a 2-week period. At 2 days, proliferations of fibroblast-like cells and large amounts of collagen fibers were present at the periphery of grafts. By 7 days, vascular buds were present at the edge of the graft and invaginating into the islet tissue. At this time, diaphragmed fenestrae were present in the endothelial cells of these forming vessels and they were surrounded by a thin continuous basal lamina. By 12 to 14 days, vascularization was complete as determined by the presence of perfused vessels. At this time the mural architecture of the capillary endothelium was identical to that found in pancreatic islets in situ, i.e., diaphragmed fenestrae, transendothelial channels, and a continuous basal lamina. This contrasted sharply with normal hepatic sinusoidal endothelium which has larger open fenestrae, no channels, and a discontinuous or absent basal lamina. In animals that had been labeled with colloidal carbon before pseudoislet transplantation, the fenestrated endothelium in the grafts contained carbon-filled phagocytic vacuoles indicating the hepatic origin of these cells. Also present at this time was a change in the hepatic sinusoids near the graft sites to a near continuous endothelium. This study demonstrates that isolated avascular adult islet cells are capable of inducing a diaphragm-fenestrated endothelium.

Heparin-gold probes were used to localize regions of heparin binding on the luminal surface of the diaphragmed-fenestrated endothelium of the rat choriocapillaris. Structures of endothelial cells were unlabeled when rats were kept on ice and perfused with solutions at 4 degrees C. When the heparin-gold quantity was doubled, only a few heparin-gold particles marked some diaphragms spanning fenestrae, vesicles and channels, parajunctional regions of the plasmalemma, and coated pits. With solutions at 4 degrees C, but the animals kept at room temperature, all of these structures in the endothelial cells were labeled. This binding was not altered by the perfusion of low levels of unlabeled heparin, but was eliminated following high levels of unlabeled heparin, and by digestion with trypsin and pronase. At 37 degrees C, heparin was localized to the above structures and, in addition, was internalized into coated vesicles, endosomes, and multivesicular bodies, but not other types of lysosomes. Some particles were found in tubules adjacent to the Golgi stacks. Heparin-gold was also transported to the abluminal front of the endothelium by vesicles. A desulfated, non-anticoagulant, fraction of heparin bound to gold was localized to the endothelium in the same manner. These results demonstrate receptors for heparin on the surface of a fenestrated endothelium. Furthermore, they show the pathway of endocytosis and transport of heparin. The possible roles of heparin in the function of the endothelium is discussed.

Beef liver catalase has been used as a tracer in cytochemical studies of vascular permeability. The use of Sigma catalase C-40 and C-100 preparations in capillary permeability studies of the ileojejunum and choriocapillaris was examined. Catalase C-40 is restricted by the fenestrated capillaries of the ileojejunum in contrast to their permeability to C-100. The choriocapillaris restricts both catalase preparations. Sephadex G-200 chromatography of plasma samples incubated with catalase C-40, or from C-40-injected animals, demonstrated an increase in the molecular weight of the tracer. No increase in molecular weight was evident for catalase C-100. The isoelectric point of both preparations was 5.4-5.7. These findings indicate that Sigma catalase C-100 is the preferred preparation for use in vascular permeability studies.

Kupffer cells of fetal rat liver were examined by ultrastructural cytochemical methods to reveal acid phosphatase (AcPase) activity in lysosomes. Elongated cisternae, 940-1150 A in width containing AcPase reaction product, were identified in these cells. These cisternae were sometimes in continuity with phagosomes containing engulfed erythrocytes. Observations suggest that such cisternae may partly encircle these phagosomes. The relationships of these cisternae to GERL (Golgi Endoplasmic Reticulum Lysosomes) is discussed.

The permeability of fenestrated capillaries in an organ is believed to be homogeneous. However, the permeability of fenestrated capillaries in different organs and to various exogenous tracers varies from a complete restriction, as found in the eye (Pino and Essner 1980, 1981; Pino 1985a) to the freely permeable peritubular capillaries of the kidney (Venkatachalam and Karnovsky 1972). In the present report we demonstrate that within any single intestinal villus from the ileo-jejunum of the rat, the permeability of fenestrated capillaries is not uniform. Exogenous hemoglobin (Einstein-Stokes radius [ESR] = 3.2 nm) exists all capillaries at any villar level in less than 5 min. In contrast, all villar capillaries restrict catalase (ESR = 5.2 nm) at 5 min, but by 60 min the tracer is present extravascularly in crypt and lower villar regions. Apical capillaries are slightly permeable to catalase at 2 h, but the bulk of the tracer remains in the lumina. The particulate tracer ferritin (ESR = 6.1 nm) is restricted 3-10 times more by apical capillaries than basal ones and is found in increasing concentration extravascularly at lower villar and crypt levels after 20 min. Following an 18-h circulation, a second dosage of ferritin is restricted by the endothelium at all villar levels. Immunocytochemical localizations of the plasma proteins albumin (ESR = 3.5 nm) and IgG (ESR = 5.5 nm) revealed an apparent lack of restriction at all villar levels. These results demonstrate that apical villar capillaries in the ileo-jejunum are more restrictive to exogenous molecules with ESR greater than or equal to 5.2 nm. Also, the passage of tracer molecules out of an endothelium alters the subsequent permeability of that vessel.

This follow-up study assesses further suicidal risk and discusses the characteristics of patients who have repeatedly attempted suicide. Depth of coma alone due to overdose of medication does not seem to be a sufficient criterion for the assessment of further suicidal risk. Better predictors seem to be: age, family status, degree of loneliness and social disintegration, presence of existential anxiety, and lack of self-assertiveness. Patients who made repeated suicide attempts had a higher neuroticism score, were more often alcohol- and drug-dependent, and came more often from a broken home. Finally, the characteristics of various patient groups are delineated.

The organization of anionic (negatively charged) sites in Bruch's membrane of the rabbit eye at various stages of postnatal development was studied using the cationic polymer, polyethyleneimine (PEI). PEI-positive sites were demonstrable as rows of particles (diameter ca. 18 nm) located at intervals along either side of the basal laminae of the retinal pigment epithelium and choriocapillary endothelium. In tangential sections through Bruch's membrane, stained particles appeared to be arranged in a semi-regular, lattice-like pattern in which the sites were separated from each other by an interval of approximately 50 nm. PEI-positive particles were also observed on collagen fibers where they were distributed at regular intervals along the length of the fiber. In tangential sections, collagen fibers formed a loosely packed meshwork in the central zone of Bruch's membrane. In addition, individual fibers were frequently oriented so that one end was located close to or within the substance of the basal laminae, a result suggesting that the anionic sites on these fibers might contribute to the network present in the basal laminae. The findings lend further support to the suggestion that anionic sites in Bruch's membrane may serve as a charge barrier which retards the movement of anionic molecules that are in transit from the choriocapillaris to the retinal pigment epithelium and outer neural retina.

The choriocapillaris is a fenestrated capillary bed in the eye that restricts the egress of exogenous tracer molecules with Einstein-Stokes radii (ESR) greater than or equal to 3.2 nm. The present study examined the permeability of its endothelium to the endogenous plasma proteins albumin (ESR, 3.5 nm) and IgG (ESR, 5.5 nm) using ultrastructural immunocytochemistry. Reaction product indicative of the localization of albumin and IgG (using Fab-HRP conjugates) was high in the capillary lumen. In contrast, neither protein was localized extravascularly in Bruch's membrane, in endothelial vesicles, or in endothelial channels. The restriction was evident at the luminal side of the diaphragms spanning fenestrae, vesicles, and channels, and at the luminal front of cell junctions. In marked comparison, high levels of reaction product were localized in the extravascular space surrounding mucosal capillaries in the ileo-jejunum. Observations of tissue subjected to postembedment staining using a protein A-gold method were similar. These findings demonstrate for the first time the restriction of endogenous plasma proteins by a capillary endothelium identical in morphology to that of other vascular beds proven to be permeable.