The intercellular junctions of guinea-pig placental capillaries: a possible structural basis for endothelial solute permeability

The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars.     

Evidence for tight junctions between odontoblasts in the rat incisor

Odontoblasts are known to be involved in the process of dentinogenesis but it is not clear whether substances may also be deposited in predentine and dentine by passing between these cells. Although tight junctions have been described, it is not clear if they are macular or "leaky" as opposed to continuous or "tight". In this study use has been made of the permeability of fenestrated capillaries amongst the odontoblasts to deposit the penetrative tracer lanthanum in the interodontoblastic space. This was done by perfusion of anaesthetized rats with physiological solutions containing lanthanum nitrate at 37 degrees C. Immersion fixation of transverse segments of mandibular incisors and examination with an electron microscope showed that lanthanum could permeate 40-50 microns between the odontoblasts to reach the peripheral pulp. Towards the predentine, often less than 10 microns from the capillaries, its progress was abruptly and completely halted by the junctions at the apical ends of the odontoblast cell bodies. Lanthanum was not found in the predentine. The mature secretory odontoblasts in the rat incisor have therefore been shown to be joined by continuous tight junctions. In the process of dentinogenesis this means that all substances deposited in predentine and dentine must arrive by passing through the odontoblasts.     

The Use of Lanthanum Chloride as a Marker for Intercellular Junctions in Rat Exocrine Pancreas

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly by electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.     

Freeze-fracture study of the epidermal cells of a teleost with particular reference to intercellular junctions and permeability to tracer

The plasmatic membranes, the intercellular junctions and the intercellular spaces of the epidermis of the fish Pimelodus maculatus were studied by freeze-fracture and by lanthanum methods. The observations has confirmed the presence of desmosomes. Gap junctions were not found and the tight junctions can be seen very rarely, arranged to form small discrete maculae. The finger-print pattern due to the microridges of the apical plasma membrane of the superficial cells was studied by direct replicas. The tracer penetrates all the intercellular epidermal spaces but failed to penetrate the dermis, suggesting the presence of a barrier at the dermo-epidermal level.     

The permeability of capillaries among the small granule-containing cells in rat superior cervical ganglia: an ultrastructural lanthanum tracer study.

The permeability of blood capillaries associated with small granule-containing (SGC) cells in rat superior cervical ganglia was investigated at ultrastructural level by employing ionic lanthanum as an electron dense tracer. In rat superior cervical ganglia, the majority of blood capillaries were nonfenestrated. Both fenestrated and nonfenestrated capillaries were observed in the area associated with SGC cells. Lanthanum tracer was observed in the luminal surface, the interendothelial cleft and the subendothelial perivascular spaces of both fenestrated and nonfenestrated capillaries associated with SGC cells. The external lamina of the Schwann cell which surrounded the neurons, nerve fibres and SGC cells were clearly delineated by the lanthanum tracer. Furthermore, the perineuronal space, the periaxonal space, and the pericellular space of the SGC cells were readily accessible to the lanthanum ion. The results demonstrated an absence of blood-nerve barrier, blood-ganglionic and blood-SGC cell barrier to the lanthanum ion in the parenchymal area of the SGC cells in rat superior cervical ganglia. It is proposed that lanthanum may pass through the endothelial cells via 1) the fenestrae of fenestrated capillaries, 2) the intercellular junctions of both fenestrated and nonfenestrated capillaries, i.e., a paracellular pathway; and 3) the process of endocytosis/exocytosis, i.e., a transcellular pathway, to reach the subendothelial space and be distributed in the parenchyma of SGC cells in rat superior cervical ganglia.     

The use of lanthanum chloride as a marker for intercellular junctions in rat exocrine pancreas

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly be electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

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.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES

The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous.     

Formation of the blood-testis barrier in the rabbit

Summary The time of establishment of the blood-testis barrier in the rabbit was studied by electron microscopy using lanthanum nitrate. This electron-dense tracer was present in the intercellular spaces in all regions of the seminiferous cords in 7 to 9-week-old animals. In 10 and 11-week-old rabbits, the penetration of lanthanum nitrate was restricted to the basal region of the seminiferous cords. Closer examination revealed the presence of numerous tight junctions between adjacent Sertoli cells. The morphological appearance of these junctions was similar to those described previously in other mammals. Entry of the tracer substance was restricted at these junctions. Pachytene germ cells, which reside beyond the junctions, were never surrounded by the tracer. Based on our observations it was concluded that the blood-testis barrier in the rabbit is formed between the 9th and 10th postnatal week, and that it is functionally effective by the 10th week.     

Effects of hyperosmotic stress on cultured airway epithelial cells

Inhalation of hyperosmotic solutions (salt, mannitol) has been used in the treatment of patients with cystic fibrosis or asthma, but the mechanism behind the effect of hyperosmotic solutions is unclear. The relation between osmolarity and permeability changes was examined in an airway cell line by the addition of NaCl, NaBr, LiCl, mannitol, or xylitol (295–700 mOsm). Transepithelial resistance was measured as an indicator of the tightness of the cultures. Cell-cell contacts and morphology were investigated by immunofluorescence and by transmission electron microscopy, with lanthanum nitrate added to the luminal side of the epithelium to investigate tight junction permeability. The electrolyte solutions caused a significant decrease in transepithelial resistance from 450 mOsm upwards, when the hyperosmolar exposure was gradually increased from 295 to 700 mOsm; whereas the nonelectrolyte solutions caused a decrease in transepithelial resistance from 700 mOsm upwards. Old cultures reacted in a more rigid way compared to young cultures. Immuno-fluorescence pictures showed weaker staining for the proteins ZO-1, claudin-4, and plakoglobin in treated samples compared to the control. The ultrastructure revealed an increased number of open tight junctions as well as a disturbed morphology with increasing osmolarity, and electrolyte solutions opened a larger proportion of tight junctions than nonelectrolyte solutions. This study shows that hyperosmotic solutions cause the opening of tight junctions, which may increase the permeability of the paracellular pathway and result in increased transepithelial water transport. This study was supported by the Swedish Asthma and Allergy Association and the Swedish Heart Lung Foundation.     

Differential permeability of pineal capillaries to lanthanum ion in the rat (Rattus norvegicus), gerbil (Meriones unguiculatus) and golden hamster (Mesocricetus auratus)

Summary Capillaries in the pineal gland of the rat (Rattus norvegicus), gerbil (Meriones unguiculatus) and golden hamster (Mesocricetus auratus) were investigated by means of electron-microscopical tracer studies using lanthanum. The tracer was administered together with the fixative solution by perfusion via the left cardiac ventricle. In the rat, endothelial junctions of fenestrated capillaries are permeable to lanthanum. In the gerbil the tracer does not leave the capillaries, which are of the non-fenestrated type throughout the organ. In the golden hamster the two portions of the pineal system have different types of capillaries. While in the superficial pineal fenestrated capillaries permeable to lanthanum predominate, the deep pineal possesses capillaries that are, without exception, devoid of fenestrations and impermeable to the tracer. It is suggested that differences in the structure of the capillaries are related to differences in the extent of a specialized contact of the pineal gland to the third ventricle.The authors dedicate this paper to Professor H. Rollhäuser, Münster, on the occasion of his 65th birthday     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

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.     

THE ULTRASTRUCTURAL BASIS OF CAPILLARY PERMEABILITY STUDIED WITH PEROXIDASE AS A TRACER

The transendothelial passage of horseradish peroxidase, injected intravenously into mice, was studied at the ultrastructural level in capillaries of cardiac and skeletal muscle. Peroxidase appeared to permeate endothelial intercellular clefts and cell junctions. Abnormal peroxidase-induced vascular leakage was excluded. Neutral lanthanum tracer gave similar results. The endothelial cell junctions were considered to be maculae occludentes, with gaps of about 40 A in width between the maculae, rather than zonulae occludentes. Some observations in favor of concurrent vesicular transport of peroxidase were also made. It is concluded that the endothelial cell junctions are most likely to be the morphological equivalent of the small pore system proposed by physiologists for the passage of small, lipid-insoluble molecules across the endothelium.     

JUNCTIONS BETWEEN INTIMATELY APPOSED CELL MEMBRANES IN THE VERTEBRATE BRAIN

Certain junctions between ependymal cells, between astrocytes, and between some electrically coupled neurons have heretofore been regarded as tight, pentalaminar occlusions of the intercellular cleft. These junctions are now redefined in terms of their configuration after treatment of brain tissue in uranyl acetate before dehydration. Instead of a median dense lamina, they are bisected by a median gap 20–30 A wide which is continuous with the rest of the interspace. The patency of these "gap junctions" is further demonstrated by the penetration of horseradish peroxidase or lanthanum into the median gap, the latter tracer delineating there a polygonal substructure. However, either tracer can circumvent gap junctions because they are plaque-shaped rather than complete, circumferential belts. Tight junctions, which retain a pentalaminar appearance after uranyl acetate block treatment, are restricted primarily to the endothelium of parenchymal capillaries and the epithelium of the choroid plexus. They form rows of extensive, overlapping occlusions of the interspace and are neither circumvented nor penetrated by peroxidase and lanthanum. These junctions are morphologically distinguishable from the "labile" pentalaminar appositions which appear or disappear according to the preparative method and which do not interfere with the intercellular movement of tracers. Therefore, the interspaces of the brain are generally patent, allowing intercellular movement of colloidal materials. Endothelial and epithelial tight junctions occlude the interspaces between blood and parenchyma or cerebral ventricles, thereby constituting a structural basis for the blood-brain and blood-cerebrospinal fluid barriers.     

Vascular permeability to lanthanum in the rat incisor pulp

Summary Experiments were performed to compare the permeability of capillaries supplying the endoneurial environment, which is invested by perineurium, with vascular permeability in the pulp where perineurium is absent. Anaesthetised rats were perfused through the aorta with physiological solutions containing lanthanum nitrate at 37° C. Pieces of inferior alveolar nerve and segments of mandibular incisors were immersion-fixed and transverse sections were examined electron microscopically for the distribution of lanthanum. In the pulp the nerve fibres pass between lanthanum-impermeable arterioles and venules en route to the incisal end. In the peripheral pulp a few capillaries were permeable but the most permeable capillaries lay between the odontoblasts. Pulpal capillary permeability was attributed to the fenestrated endothelium and contrasted with the unfenestrated endoneurial capillaries which were impermeable to lanthanum. Whereas the tight junctions of endoneurial capillaries are known to prevent certain blood-borne substances from entering the endoneurium, it was not clear whether the permeability of the pulpal capillaries, which are distant from the nerve fibres, could affect the nerve fibre environment. No extravasated lanthanum reached the pulpal nerve fibres suggesting that they are not affected. Technically it was not possible to examine the incisal third of the tooth where the situation could be different because the volume of the pulp decreases and capillaries lie closer to the nerve fibres.     

Maturation of tight junctions in guinea-pig cecal epithelium

Summary By means of the freeze-fracture technique and in tracer studies it is demonstrated that the structure of tight junctions and the permeability to lanthanum of the guinea-pig cecal epithelium change during maturation of cells. Height and strand number of tight junctions in the apical-basal direction increase as crypt cells migrate to the surface of the epithelium. Likewise, the interlacing of continuous strands was greater in surface than in crypt junctions. The numerous free-ends, isolated individual freestrands and maculae occludentes found in crypt cells were absent in surface epithelial cells. Goblet cells, located at the bottom of crypts, displayed tight junctions similar in characteristics to those of cells located in the middle region of crypts. Cells at the surface and in middle regions of crypts possess tight junctions impermeable to lanthanum, whereas junctions between cells located at the bottom of crypts often were permeable to the tracer, indicating that permeability decreases as the epithelial cells mature. Genesis and maturation mechanisms related to structural configuration of tight junctions are discussed.     

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.