On the components of Bruch's membrane.
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Animals↗
Biomedical subjects
E Essner
Publications and source records attributed to E Essner.
Plasma membrane-associated vesicles in retinal capillaries of the rat.
The structure and function of abluminal vesicles in endothelial cells of rat retinal capillaries was examined using glutaraldehyde-tannic acid fixation and the hemeproteins--horseradish peroxidase, microperoxidase, and lactoperoxidase--as tracers. Numerous vesicles, delimited by a tannic acid-positive membrane, were distributed along the abluminal front. Other vesicles were arranged in clusters and chains or tubule-like structures. Such vesicles were not found in the vicinity of the capillary lumen. When the retina was exposed to hemeproteins, either in vitro or after intravitreal injection, the abluminal vesicles became labeled with tracer reaction product. Apparently "free" vesicles and tubules seen in tangential sections through the basal lamina were also labeled, suggesting that they were in continuity with the plasma membrane in another plane of section. No enzyme reaction product was present in the capillary lumen. Peroxidase-positive multivesicular bodies were observed, suggesting that some protein was endocytosed and directed to lysosomes where it was presumably degraded. The results suggest that abluminal endothelial vesicles represent pits or invaginations of the plasma membrane and, as such, are not involved in the transendothelial transport of protein from the perivascular space to the capillary lumen. Tannic acid treatment revealed a population of similar vesicles associated with the plasma membrane of pericytes. After exposure to hemeproteins, enzyme reaction product was localized in these vesicles and in a few multivesicular bodies. The results suggest that the majority of these vesicles are in continuity with the plasma membrane and are not involved in endocytosis.
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Demonstration of microfibrils in Bruch's membrane of the eye.
The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.
Alcian Blue↗
Proteoglycans in Bruch's membrane.
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Animals↗
Observations on the permeability of the choriocapillaris of the eye.
The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW - approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.
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Permeability of the neonatal rat choriocapillaris to hemeproteins and ferritin.
The permeability of the endothelium of the capillaries of the rat choriocapillaris to circulating macromolecules was examined during the first post-natal week of development using hemeproteins of different molecular dimensions and ferritin. At this stage of development capillaries and photoreceptor cells in the neural retina are not fully formed, but the choriocapillaris has an adult-like morphology. Thus, through these differences in neural and vascular architectures the possible functional relationships between the retina and choriocapillaris can be explored. High levels of horseradish peroxidase [Einstein-Stokes radius (ESR) 3 nm] activity were localized in Bruch's membrane within 2 minutes after intravenous tracer injection. In contrast, scant levels of hemoglobin (ESR, 3.2 nm) and no catalase (ESR, 5.2 nm) activity were observed here at 75 and 90 minutes, respectively. Only a few ferritin particles (ESR, 6.1 nm) crossed the choriocapillary endothelium after a 90-minute circulation. The results demonstrate that in the neonatal rat the choriocapillaris has the same restrictive properties as have been described for the adult eye (Pino and Essner, 1980, 1981), even in the absence of retinal capillaries and functional photoreceptor cells.
Animals↗
In situ demonstration of actin in normal and injured ocular tissues using 7-nitrobenz-2-oxa-1,3-diazole phallacidin.
The fluorescent derivative of the actin-binding toxin phallacidin, 7-nitrobenz-2-oxa-1,3 diazole phallacidin, has been used to cytologically demonstrate the presence of actin in lens epithelium, corneal endothelium, and retinal pigment epithelium. In these noninjured tissues, no stress fibers are observed and fluorescence is confined mainly to an area at or near the cell membrane, although some diffuse cytoplasmic staining can also be seen. However, following injury to either the lens epithelium or corneal endothelium of rats and frogs, stress fibers are detected, but only in those cells that migrate into the wound area. Cells on the periphery of each tissue do not partake in would repair and thus maintain their normal appearance. After the tissue has regenerated, stress fibers disappear, and those cells involved in the injury response return to their normal morphology. When rabbit corneal endothelium is placed in tissue culture, stress fibers are observed as the cells migrate away from the initial explant. Upon reaching confluency, these cells spread out and each is surrounded by thick actin-containing bands. Furthermore, they exhibit some stress cables within their cytoplasm. This is in contrast to their appearance in vivo where stress fibers are absent and fluorescence is limited to a region near the cell membrane.
Actins↗
Location and chemical composition of anionic sites in Bruch's membrane of the rat.
The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.
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Distribution of anionic sites in Bruch's membrane of the rabbit eye.
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.
Animals↗
Microbodies: peroxisomes and glyoxysomes.
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Amino Acids↗
Permeability of rat choriocapillaris to hemeproteins. Restriction of tracers by a fenestrated endothelium.
The choriocapillaris is the fenestrated capillary bed in the choroid of the eye and is the major blood supply to the retinal pigment epithelium (RPE) and photoreceptor cells. Bruch's membrane (BM) is a multilaminated basement membrane that separates the choriocapillaris from the RPE. In a previous study (Pino RM, Essner E; Cell Tissue Res 208:21, 1980) we found that the choriocapillary endothelium restricted the egress of ferritin from the choriocapillaris. In the present study, hemeproteins were used to further establish the permeability characteristics of this capillary bed. Horseradish peroxidase (Einstein-Strokes radius (ESR), 30 A) rapidly crossed the capillary endothelium (less than 5 min) after intravenous administration and after 5 minutes filled BM and the basal infoldings of the RPE. In contrast, hemoglobin (Hg) (ESR, 32 A) and lactoperoxidase (LP) (ESR, approximately 40 A) are markedly restricted at the level of endothelial diaphragmed fenestrae, channels, and intercellular junctions. Little vesicular transport of these proteins was observed. The reaction product of the two hemeprotein activities was not demonstrable in BM for up to 30 min after injection; relatively low levels were detected after 75 min. HG and LP appear to be further restricted by BM, since their reaction products were not demonstrable between the RPE basal infoldings at this time. Catalase (ESR, 52 A) activity was not detected in BM for up to 4 hr after injection. These results indicate that the rat choriocapillary endothelium, unlike the fenestrated endothelia lining other vascular beds, substantially restricts the passage of large tracer molecules.
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Distribution of anionic sites on the surface of retinal pigment epithelial and rod photoreceptor cells.
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.
Animals↗
The metabolic basis for the lens growth inhibiting effect of hypophysectomy. II. The liver.
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Animals↗
Structure and permeability to ferritin of the choriocapillary endothelium of the rat eye.
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.
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Breakdown of blood. Retinal barrier in RCS rats with inherited retinal degeneration.
A breakdown in the blood-retinal barrier to certain proteins is described in mutant RCS rats with inherited retinal degeneration. Intravenously injected microperoxidase and horseradish peroxidase are extravasated from the outer (but not inner) retinal capillaries of these rats, at approximately 11 weeks of age and older. The number of affected capillaries increases with the age of the animals and progression of the retinal dystrophy until virtually all capillaries in the outer retina become permeable to these tracers. In such capillaries, enzyme reaction product is demonstrable in a greater proportion of luminal vesicles and in the majority of abluminal vesicles. Reaction product is also localized in cytoplasmic vacuoles, the basal laminae of endothelial cells and pericytes, and the perivascular spaces. The increased permeability of outer retinal capillaries in RCS rats appears to be due to an increase in transendothelial vesicular transport of the probe molecules. There was no evidence that either tracer permeated the interendothelial junctions or entered the basal laminae by reflux from the perivascular spaces. It is suggested that factors originating from the degenerated photoreceptor cells may play a role in stimulating the vesicular transport observed in permeable capillaries. In contrast to these findings, the outer retinal capillaries of RCS rats were not permeable to hemoglobin, catalase, or ferritin, regardless of the age of the animal or the degree of retinal degeneration. Since the vesicles that form at the luminal front are covered by a diaphragm, it is possible that this structure prevents entry of these larger proteins into the endothelial vesicle, even in capillaries that are demonstrably permeable to the smaller tracers.
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Permeability of retinal capillaries in rats with inherited retinal degeneration.
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.
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An electron microscopic study of macrophages in rats with inherited retinal dystrophy.
In retinas of rats (RCS) with inherited retinal dystrophy, cells interpreted as macrophages infiltrate the outer nuclear layer and subsequently appear in the interphotoreceptor space, where they accummulate during the course of the disease. The morphology and distribution of these cells and their relations to the pigment epithelial cells were investigated. Macrophages, regardless of their location, possessed morphological features that distinguished them from the pigment epithelial cells. Premelanosomes and melanosomes, typical of pigment epithelial cells, were never observed in macrophages. There was no evidence to indicate that, during the period studied, the pigment epithelial cells had become dedifferentiated or had migrated from Bruch's membrane. Macrophages, like pigment epithelial cells, phagocytized little or no outer segment material. The findings indicate that, at least during the interval studied, the cells that infiltrate the retina and interphotoreceptor space are macrophages rather than pigment epithelial cells.
Acid Phosphatase↗
Localization of carbohydrate components in rat colon with fluoresceinated lectins.
Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.
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