The ultrastructural basis of transcapillary exchanges

A brief survey is given of current views correlating the ultrastructural and permeability characteristics of capillaries. Observations based on the use of peroxidase (mol wt 40,000), as an in vivo, and colloidal lanthanum, as an in vitro, ultrastructural tracer, are presented. In capillaries with "continuous" endothelium, the endothelial intercellular junctions are thought to be permeable to the tracers, and are regarded as maculae occludentes rather than zonulae occludentes, with a gap of about 40 A in width between the maculae. Some evidence for vesicular transport is also presented. It is inferred that the cell junctions are the morphological equivalent of the small-pore system, and the vesicles the equivalent of the large-pore system. Peroxidase does not apparently cross brain capillaries: the endothelial cell junctions are regarded as zonulae occludentes, and vesicles do not appear to transport across the endothelium. This is regarded as the morphological equivalent of the blood-brain barrier for relatively large molecules. The tracers appear to permeate the fenestrae of fenestrated capillaries, and the high permeability of these capillaries to large molecules is attributed to the fenestrae. Capillaries with discontinuous endothelium readily allow passage of the tracers through the intercellular gaps. A continuous basement membrane may act as a relatively coarse filter for large molecules. In general, the morphology of capillaries correlates well with physiological observations.     

The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer

The permeability of the alveolar-capillary membrane to a small molecular weight protein, horseradish peroxidase (HRP), was investigated by means of ultrastructural cytochemistry. Mice were injected intravenously with HRP and sacrificed at varying intervals. Experiments with intranasally instilled HRP were also carried out. The tissue was fixed in formaldehyde-glutaraldehyde fixative. Frozen sections were cut, incubated in Graham and Karnovsky's medium for demonstrating HRP activity, postfixed in OsO4, and processed for electron microscopy. 90 sec after injection, HRP had passed through endothelial junctions into underlying basement membranes, but was stopped from entering the alveolar space by zonulae occludentes between epithelial cells. HRP was demonstrated in pinocytotic vesicles of both endothelial and epithelial cells, but the role of these vesicles in net protein transport appeared to be minimal. Intranasally instilled HRP was similarly prevented from permeating the underlying basement membrane by epithelial zonulae occludentes. Pulmonary endothelial intercellular clefts stained with uranyl acetate appeared to contain maculae occludentes rather than zonulae occludentes. HRP did not alter the ultrastructure of these junctions.     

Junctions in the central nervous system of the cat

Interendothelial membrane contacts in different segments of brain blood vessels were investigated by the freeze-etching technique. The study demonstrated that the endothelial cells of the pre- and postcapillary segments were coupled by elaborate zonulae occludentes. These tight junction formations encompassed gap junctions of different sizes and distribution. The globules of the pre- and postcapillary tight junctions revealed a great fragility which led to an atypical distribution of the sealing elements. In the "typical" brain capillaries the endothelial cells were connected by continuous tight junctions. In contrast to the structural continuity of these formations the fenestrated segments of capillaries in the choroid plexus and area postrema demonstrated discontinuous fasciae occludentes. They were composed of rows of individual particles which should be regarded primarily as focal in nature.     

Permeability of muscle capillaries to microperoxidase

In this study we attempted to identify a morphologic counterpart of the small pore of muscle capillaries. The existence of such a pore has been postulated by physiologists to explain the permeability of muscle capillaries to small macromolecules. We injected mice intravenously with microperoxidase (MP) and fixed specimens of diaphragm at intervals of 0-250 s after the injection to localize the tracer by electron microscopy. The small size of MP (1,900 mol wt and 20 A molecular diameter [MD]) ensures its ready passage through the small pore since the latter is thought to be either a cylindrical channel 90 A in diameter or a slit 55 A wide. MP appears in the pericapillary interstitium within 30 s of initiation of its intravenous injection. The patterns of localization of MP observed within clefts between adjacent capillary endothelial cells indicate that some endothelial junctions are permeable to this tracer. Although small vesicles transfer MP across the endothelium, we do not believe that the vesicles transfer substantial amounts of MP into the pericapillary interstitium. We did not obtain evidence that MP crosses the endothelium of capillaries through channels formed either by a single vesicle or by a series of linked vesicles opening simultaneously at both surfaces of the endothelial cell. From our observations we conclude that some endothelial junctions of capillaries are permeable to MP, and that these permeable junctions are a plausible morphologic counterpart of the small pore.     

A new occludens-like junction linking endothelial cells of small capillaries (probably venules) of rat jejunum.

Freeze-cleave replicas of small capillaries of rat jejunum have revealed the presence of a new type of junction linking endothelial cells. This new junction reveals tight junctions (zonulae occludentes) in that the adjacent plasma membranes are held closely together along lines of attachment organized in the form of a loose, but frequently discontinuous network. In contrast to tight junctions, the A-face ridges possess a very low profile, and only at low shadowing angles can a repeating, particulate substructure occasionally be resolved. The shallow B-face furrows lack any particulate components. Images of cross-fractured focal points of attachment suggest that the external leaflets of adjacent membranes are closely apposed but not actually fused, as is the case with zonulae occludentes. It appears that this new type of endothelial junction is characteristic of small venules. Thus we propose that it be termed small venule endothelial junction.     

STUDIES ON BLOOD CAPILLARIES : I. General Organization of Blood Capillaries in Muscle

The wall of the blood capillaries of skeletal muscles (diaphragm, tongue, hind legs) and myocardium of the rat, guinea pig, and hamster consists of three consecutive layers or tunics: the endothelium (inner layer), the basement membrane with its associated pericytes (middle layer), and the adventitia (outer layer). The flattened cells of the endothelium have a characteristic, large population of cytoplasmic vesicles which, within the attenuated periphery of the cells, may attain a maximum frequency of 120/µ² of cell front and occupy ~18% of the cytoplasmic volume; these values decrease as the cells thicken toward the perikaryon. The vesicles are 650–750 A in over-all diameter and are bounded by typical unit membranes. They occur as single units or are fused to form short chains of two to three vesicles. Each configuration may lie entirely within the cytoplasm or open onto the cell surface. In the latter case, the unit membrane of the vesicle is continuous, layer by layer, with the plasmalemma. Chains of vesicles opening simultaneously on both the blood and tissue fronts of the endothelial tunic have not been observed either in sections or in a tridimensional reconstruction of a sector of endothelial cell cytoplasm. Adjacent endothelial cells are closely apposed to one another and appear to be joined over a large part of their margins, possibly over their entire perimeter, by narrow belts of membrane fusion (zonulae occludentes). Except for tongue capillaries, patent intercellular gaps are rare or absent. The middle layer is formed by a continuous basement membrane (~500 A thick) and by pericytes which lie in between leaflets of this membrane. The tips of the pericyte pseudopodia penetrate through the inner leaflet of the basement membrane and join the endothelium in maculae occludentes. The adventitia is a discontinuous layer comprising cellular (macrophages, fibroblasts, mast cells) and extracellular (fibrils, amorphous matrix) elements. The same general type of construction appears to be used along the entire length of the capillary.     

Fine structure and permeability of capillaries in the stria vascularis and spiral ligament of the inner ear of the guinea pig

Summary The blood capillaries in the stria vascularis and the spiral ligament of guinea pigs were studied by electron microscopy with freeze-fracture and thin section methods, including tracer experiments with horseradish peroxidase (HRP) and microperoxidase (MP). The endothelial cells of the capillaries of both tissues are connected by tight junctions, and contain about the same number of micropinocytotic vesicles. In cases of intravascular administration before fixation, both of the tracers stained the perivascular space and almost all endothelial vesicles in the stria vascularis. On the other hand, the perivascular space and many vesicles in the spiral ligament were unstained. The endothelial tight junctions in the stria vascularis prevented the penetration of HRP, but sometimes allowed the penetration of MP. Those of the spiral ligament were impermeable to both tracers. In cases of tracer administration after fixation, leakage spots of HRP from capillaries were sparsely located all over the stria vascularis. Transendothelial channels and isolated fenestrae formed by micropinocytotic vesicles were detected. It is concluded that the capillaries of the stria vascularis are similar to the muscle capillaries and to the capillaries of the elasmobranch brain, whereas those in the spiral ligament are similar to the brain capillaries of higher vertebrates.     

Retinal capillary junctions: Ultrastructural tight junction artefacts induced by sodium ions and membrane reduction in streptozotocin diabetes

Summary Retinal capillary junctions were analysed in normal and diabetic rats and in a human retina with the electron microscope. Diabetes mellitus was induced with Streptozotocin. The retinae were fixed in Palade's osmium tetroxide containing sodium or calcium ions and block-stained in uranyl acetate.With Ca-fixation, no significant difference in interendothelial cleft width was detected between retinal layers or between normal and diabetic retinae. Diabetes caused a narrowing of the clefts in the Na-fixed tissue (X±SE, n=375; Normal: 78.6±3.00 Å; Diabetic: 57.7 ±2.42 Å; p0.001). A significant correlation was found between cleft width and the length of the tight junctions or zonulae occludentes (p<0.001). In the nerve fibre layer of the Nadiabetio retina, where cleft narrowing was greatest, there was an increase in length of the zonulae occludentes from 22.8±2.2% to 41.6±3.7% (p<0.001). Ca-fixation prevented these changes, indicating that at least some zonulae occludentes were interendothelial extraction artefacts.In the normal retina, endothelial cell membrane thickness was greater with Ca-than Na-fixation (p<0.001). Diabetes caused a decrease in membrane thickness of Ca-fixed tissue (p<0.001). The diabetic decrease in membrane thickness may explain the increased fragility and increased permeability of diabetic capillaries. Calcium binding by endothelial cell membranes is of primary importance in anticoagulation which is defective in diabetes.Work supported by a grant from the Taverna Estate and The Prince of Wales Hospital, Randwick, N.S.W., Australia. Presented in part at the 8th International Congress on Electron Microscopy held in Canberra, A.C.T., Australia, 1974.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Opening of blood-brain barrier in Triturus cristatus carnifex by hyperosmolar mannitol solutions

The permeability of the newt cerebral capillaries to lanthanum ion has been studied after perfusion with mannitol solutions of increasing molarity. In the control specimens lanthanum deposits were limited to the luminal side of the capillaries and tracer did not spread to the pericapillary spaces due to the tight junctions. Treatment with hypertonic solutions of mannitol (0.25M, 0.5M, 1M) caused opening of the blood brain barrier with a progressive increase in lanthanum between the endothelial cell edges, in the basal lamina and in the extracellular spaces of the nervous parenchyma in relation to the molarity of the mannitol solution. The spread of lanthanum is probably due to opening of the tight junctions between the endothelial cells, since pinocytotic vesicles labelled with tracer were not evident.     

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

The glomeruli of the human and the rat kidney studied by freeze-fracturing

Glomeruli isolated from rat and human kidneys were studied using the freeze-fracture technique. Discontinuous zonulae occludentes and gap junctions were found in the replicas of the split plasma membrane of the endothelial cells. A diaphragm across the endothelial pores was not demonstrated. The central layer of the basement membrane, corresponding to the lamina densa described in thin sections, revealed a coarse substructure. A slit membrane between the pedicles of the podocytes was not detectable; however, its position was indicated by the different texture of the replica, which abruptly changed at the transition of the basement membrane to the primary urinary space. Furthermore, at the level of the slit membrane arrays of particles were present within the cleaved membrane of the pedicles, probably representing the attachment points of the slit membrane. Isolated strands of a zonula occludens as well as gap junctions were seen on the split plasma membrane of the podocytes. The mesangial cells could be identified by their contiguity to the endothelial cells and by their numerous gap junctions.     

Electron microscopic research on the capillary permeability of the primary portal plexus in rats in the prenatal period

Permeability of portal capillaries to intravascularly injected ionic lanthanum, ferritin and horse-radish peroxidase has been examined in rats on the 18th fetal day, and on days 1 and 9 of postnatal life. For several minutes, tracer molecules pass through the capillary wall and reach the median eminence. In the case of immature capillaries, the materials pass freely through the endothelial cells, and to a lesser extent are transferred via occasional plasmalemmal vesicles and fenestrae. As the maturation of capillaries proceeds their permeability via plasmalemmal vesicles and fenestrae increases considerably due to a gradual rise in the number of these structures. The plasmalemma of differentiated endothelial cells becomes impermeable to all the tracers. Only ionic lanthanum appears to penetrate through transendothelial channels and intercellular junctions between adjacent endothelial cells.     

Ultrastructural observations at pineal gland capillaries in four rodent species.

The fine structure of the capillaries of the pineal glands of the rat, mouse, chinchilla, and ground squirrel were investigated. The pineal endothelial cells in the rat, mouse and ground squirrel were often composed of attenuated cytoplasmic portions which contained numerous fenestrations, in contrast to pineal capillaries in the chinchilla which were lined by thick non-fenestrated endothelial cells. Marked morphological differences were also apparent in terms of the types of vesicles within the cytoplasm and abutting on the cell surface of pineal endothelial cells from the various species investigated. The interendothelial junctions exhibited remarkable species differences with the chinchilla pineal possessing typical tight endothelial junctions while those in the rat, mouse and ground squirrel lacked such endothelial cell associations. Generally, capillary lining cells in the chinchilla pineal resembled similar cells within the brain, while endothelial cells in pineal glands of rat, mouse and ground squirrel were more typical of those found in other endocrine organs. Species differences in the structure of the pineal capillaries may represent physiological differences as well.     

Distribution of endothelial vesicles in the microvasculature of skeletal muscle and brain cortex of the rat,as demonstrated by tannic acid tracer analysis

Summary Ultrathin serial sectioning and labeling with tannic acid have demonstrated that most plasmalemmal vesicles of rat vascular endothelial cells are not free, but rather are conjoined in three dimensions to form racemose invaginations from the cell surfaces. To elucidate the distribution of vesicles in these microvascular endothelial cells, we have examined terminal arterioles, capillaries and post-capillary venules of rat skeletal muscle and brain cortex, using tannic acid labeling and stereological methods, and have determined the proportions of free vesicles and the vesicles of luminal and abluminal invaginations, as well as the numerical density of vesicles. In the case of capillaries, regional differences in distribution have also been studied. The ratio of free vesicles is 6–7% and is constant throughout the muscle microvasculature. The distribution (proportions and numerical densities) of vesicles in the brain and muscle microvascular endothelial cells shows regionally distinctive patterns. In rapid-frozen, freeze-substituted endothelial cells, there are almost as many fused vesicles as seen in chemically fixed cells. Therefore, aldehydes do not seem to induce membrane fusion, and the distribution of vesicles seems to be preserved by chemical fixation. The structure and function of plasmalemmal vesicles are discussed.     

Distribution and origin of acetylcholinesterase activity in the capillaries of the brain

Summary Areas containing AChE-positive capillaries were mapped in the brain of the cat and the guinea pig. Regions with AChE-positive capillaries mostly also contain neuronal elements with AChE activity. Electron-microscopical cytochemistry revealed localization of AChE in basement membranes of endothelial cells and pericytes very often in continuity with activity of the extracellular space. Intraendothelial AChE activity was seen only in pinocytic vesicles. The vascular AChE is thought to be of neuronal origin since no cytochemical evidence has been obtained for a synthesis of this enzyme in endothelial or other non-neuronal cells in the CNS.Drs. H. Kaiya and L. Toth were recipients of research fellowships granted by the Max Planck Society     

Brain endothelial cells and the glio-vascular complex

We present and discuss the role of endothelial and astroglial cells in managing the blood-brain barrier (BBB) and aspects of pathological alterations in the BBB. The impact of astrocytes, pericytes, and perivascular cells on the induction and maintenance of the gliovascular unit is largely unidentified so far. An understanding of the signaling pathways that lie between these cell types and the endothelium and that possibly are mediated by components of the basal lamina is just beginning to emerge. The metabolism for the maintenance of the endothelial barrier is intimately linked to and dependent on the microenvironment of the brain parenchyma. We report the structure and function of the endothelial cells of brain capillaries by describing structures involved in the regulation of permeability, including transporter systems, caveolae, and tight junctions. There is increasing evidence that caveolae are not only vehicles for endo- and transcytosis, but also important regulators of tight-junction-based permeability. Tight junctions separate the luminal from the abluminal membrane domains of the endothelial cell (“fence function”) and control the paracellular pathway (“gate function”) thus representing the most significant structure of the BBB. In addition, the extracellular matrix between astrocytes/pericytes and endothelial cells contains numerous molecules with inherent signaling properties that have to be considered if we are to improve our knowledge of the complex and closely regulated BBB. Any work of our own cited in this review was supported by grants from the Deutsche Krebshilfe (to H.W.), the Deutsche Forschungsgemeinschaft (to H.W.), and the Hertie-Foundation (to H.W. and to Britta Engelhardt, Bern, Switzerland).     

Ultrastructure of the jugular body of Rana pipiens

Summary The jugular bodies in adult Rana pipiens, are surrounded by a capsule of mesothelium and connective tissue, and their parenchyma consists of cell cords arranged in a sinusoidal network. The cell cords are formed by irregular reticular cells, showing numerous filaments and joined together by zonulae adhaerents. The intercellular spaces are filled by reticular fibres and free cells. These latter are small and medium lymphocytes, lymphoblasts, and developing and mature plasma cells. Additionally, free macrophages, neutrophils and acidophils also occur. Sinusoidal blood vessels show thin walls with numerous filaments and pinocytotic vesicles. They exhibit a discontinuous basement membrane, and tight junctions frequently occur between endothelial cells. Occasionally, lymphatic vessels are found and the innervation is principally vasomotor, although nerve endings appear remarkably near reticular cells and lymphocytes. The jugular bodies of adult R. pipiens are plasma cell and antibody-forming organs, whose functional significance is discussed in relation to their ultrastructural organization.     

Cell junctions in amphioxus (Cephalochordata): a thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent 'poorly developed' septate junctions.