Primary culture of capillary endothelial cells from the spiral ligament and stria vascularis of bovine inner ear

Summary Methods for isolation and culture of microvascular endothelial cells of the inner ear were devised to provide an in-vitro system for studying endothelial functions in this tissue. Capillaries from the stria vascularis and spiral ligament were treated enzymatically to free them from surrounding tissue. Contamination by extraneous tissue was minimized by banding capillary segments in Percoll gradients and culture in plasma-derived serum on a fibronectin-coated substrate. Although only small amounts of inner ear tissue were available, tritiated thymidine autoradiography demonstrated that considerable growth in culture was possible. Addition of heparin and endothelial cell growth supplement to the medium enhanced proliferation. The endothelial origin of the cultured cells was confirmed by immunofluorescent demonstration of the presence of Factor VIII-related antigen and angiotensin-converting enzyme. In addition, tight junctions between cells were observed in both thin sections and platinum replicas obtained by freezefracture techniques. Endothelial cells from neither the stria vascularis nor the spiral ligament allowed passage of horseradish peroxidase across the monolayer during a 5-min period. However, endothelial cells from the stria vascularis exhibited a greater amount of pinocytotic activity than those of the spiral ligament, a difference that is also observed in vivo. Methods for expanding a small population of endothelial cells with retention of specialized properties into one of sufficient size for morphologic and biochemical studies have been demonstrated for the inner ear.     

Vascular permeability to proteins and peptides in the mouse pineal gland

Summary The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.Part of this study was presented at the EMCELL-76 meeting, Copenhagen, 1976     

THE INFLUENCE OF INTRAVASCULAR FLUID VOLUME ON THE PERMEABILITY OF NEWBORN AND ADULT MOUSE LUNGS TO ULTRASTRUCTURAL PROTEIN TRACERS

The permeability of the alveolar-capillary membrane of newborn and adult mice to horseradish peroxidase (HRP) and catalase was studied by means of ultrastructural cytochemistry, and the permeability to ferritin was studied by electron microscopy. The influence of varying volumes of intravenously injected fluid on the rate of leakage of the tracers from pulmonary capillaries was examined. The tracers were injected intravenously and the mice were sacrificed at timed intervals. Experiments on newborn mice with intranasally instilled HRP were also done. The tissues were fixed in formaldehyde-glutaraldehyde fixative. Chopped sections were incubated in Graham and Karnovsky's medium for peroxidase and in a modification of this medium for catalase. Tissues were postfixed in OsO₄ and processed for electron microscopy. In both newborn and adult mice, the ready passage of peroxidase through endothelial clefts was dependent on the injection of the tracer in large volumes of saline. When the tracer was injected in small volumes of saline, its passage through endothelial clefts was greatly reduced. Endothelial junctions of newborn mice were somewhat more permeable to HRP than those of adult mice. In all animals, alveolar epithelial junctions were impermeable to HRP. Catalase and ferritin did not pass through endothelial junctions. Intranasally instilled HRP in newborn mice was taken up by pinocytotic vesicles and tubules of flat alveolar cells.     

Elongated pericyte-like cells connect discrete capillaries in the cochlear stria vascularis of gerbils and rats

 Bridging structures between discrete capillaries in the stria vascularis of the cochlea were studied morphologically in gerbils and rats. Serial thin sections for transmission electron microscopy revealed (1) that elongated cells surrounded by the basal lamina provided the structural basis for the bridging structure, (2) that the basal lamina surrounding the elongated cell extended to the basal lamina around the capillary endothelial cell, (3) that the electron density of the cytoplasm was similar to that of the pericytes around the capillaries, and (4) that the cell was attached to the capillaries at both ends only. Visualization of the basal lamina by immunofluorescent methods revealed (1) that capillaries were often bent at the site of attachment of the bridging cell, (2) that the bridging cell bifurcated occasionally, and (3) that the density of the bridging cell was much higher in the stria vascularis than in the underlying spiral ligament. Filamentous actin visualized by fluorescent phalloidin was not apparent in the bridging cell. We propose that the bridging cell provides mechanical strength to the tortuous capillary network in the stria vascularis and participates in the specific function of the stria vascularis in cooperation with other types of cells. Received: 26 October 1998 / Accepted: 8 January 1999     

Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current “two-cell model” of the stria vascularis.     

Impermeability of hagfish cerebral capillaries to horseradish peroxidase

Summary Brain capillaries and their permeability to intravenously injected horseradish peroxidase, HRP, (MW: 40,000) were examined electron-microscopically in an attempt to find a structural explanation for the poorly developed blood-brain barrier in the hagfish, Myxine glutinosa. In particular, it was the aim of this study to examine the role of the numerous endothelial vesicles and tubules in the transport of this tracer between blood and brain. Many of the vesicles and tubules were found to be in continuity with the luminal or abluminal surfaces, but tubules generating channels through the endothelial cells were never observed. The cleft between adjacent endothelial cells was obliterated by punctate junctions. HRP, which was allowed to circulate for up to 35 min, was not found in the basal lamina or in the surrounding brain parenchyma. Few of the luminal vesicles and tubules were marked by the tracer. In the intercellular cleft HRP was stopped by the junctions. It is concluded that the hagfish like other vertebrates has a blood-brain barrier to HRP, and the numerous vesicles and tubules occurring in hagfish brain endothelium are not involved in the transendothelial transport of this macromolecule.     

Ultrastructure and permeability of lymph node microvasculature in the mouse

Summary The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used.All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.     

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.     

Horseradish peroxidase as a tracer for capillary permeability studies

The permeability of capillaries was investigated utilizing an in vivo injection of horseradish peroxidase (HRP) and an in situ perfusion of a balanced salt solution containing HRP and lanthanum chloride. In the continuous capillaries of heart and muscle, HRP diffuses mainly through intercellular junctions, while in testicular capillaries, the transport is via micropinocytotic vesicles. The diffusion and micropinocytotic transport of HRP was demonstrated in both directions, i.e. from the capillary lumen to the interstitium and vice versa. Lanthanum can be used as a bidirectional inhibitor of micropinocytosis. The transport of HRP is then almost completely hindered in testicular capillaries. In heart muscle, the effect on HRP transport is not significant, due to second transport pathway, i.e. intercellular cleft passage.     

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.     

The penetration of exogenous tracers through the enameloid organ of developing teleost fish teeth

In order to determine whether exogenous materials permeate to the forming tooth enameloid matrix, teleost species were injected intramuscularly with horseradish peroxidase (HRP) or myoglobin, or; intracardially with lanthanum nitrate or HRP, then killed a predetermined intervals post-injection. Tooth bearing bones were processed for transmission electron microscopy. At the enameloid matrix formation stage, capillaries associated with the enameloid organ were few in number and rarely fenestrated. Both organic tracers reached the matrix at cervical but not coronal, regions of the teeth in all species examined. Lanthanum was rarely observed extravascularly and never extended to the enameloid matrix at the secretion stage. At the enameloid mineralization stage, fenestrated capillaries were closely associated with the outer dental epithelial cells (ODE). All tracers were observed in the plasma membrane invaginations of the ODE. Only intracardially injected HRP compromised the apical intercellular junctions of the inner dental epithelial cells (IDE) to reach the mineralizing enameloid Lanthanum did not extend past the ODE-IDE cell junctions. It is concluded that the close association of mineralization stage fenestrated capillaries with the highly invaginated ODE cells result in increased tracer penetration compared to the secretory stage. The deeper penetration of the organic tracers, compared with lanthanum, between mineralization stage IDE cells may be due to longer in vivo circulation of the former material. The apical junctions of mineralization stage IDE cells, however, remained impermeable to the organic tracers. The absence of mineral in secretory stage enameloid mineral could not be due to specialized cell junctions preventing access of molecules to the matrix. It is suggested that controlling factors other than cellular permeability initiate enameloid mineralization.     

An ultrastructural study of the blood/air barrier in the guinea-pig

Horseradish peroxidase (HRP) was intravenously injected into guinea-pigs to ultrastructurally examine the permeability of the blood/air barrier. Adults were given 300 mg/kg of the tracer in a small volume of saline, anesthetized and sacrificed at intervals by either intratracheal filling or right ventricular perfusion with 3% glutaraldehyde. The reaction product had passed through endothelial clefts and accumulated in the interstitium as early as 1.5 min after injection. This same degree of penetration occurred with either fixation method used. Tight junctions between pneumocytes prevented passage of the reaction product into alveoli. Pinocytotic vesicles were numerous in both endothelial and epithelial cells, but did not significantly contribute to tracer transport. Ten minutes post-injection was selected as optimal for this model since the highest concentration of tracer was found in the tissues at this time.     

Uptake of horseradish peroxidase from CSF into the choroid plexus of the rat,with special reference to transepithelial transport

Summary Protein uptake from cerebral ventricles into the epithelium of the choroid plexus, and transport across the epithelium were studied ultrastructurally in rats. Horseradish peroxidase (HRP, MW 40,000) was used as protein tracer. Steady-state ventriculo-cisternal perfusion with subatmospheric pressure (-10cm of water) in the ventricular system was applied. HRP dissolved in artificial CSF was perfused from the lateral ventricles to cisterna magna for various times, and ventriculo-cisternal perfusion, vascular perfusion or immersion fixation with a formaldehyde-glutaraldehyde solution was performed.Coated micropinocytic vesicles containing HRP were seen both connected with the apical, lateral and basal epithelial surface and within the cells. Heavily HRP-labeled vesicles were often fused with the lining membrane of slightly labeled or unlabeled intercellular spaces. Since the apical tight junctions of the epithelium never appeared open or never contained HRP in the spaces between the fusion points, and since the intercellular spaces between adjacent epithelial cells below the junctions only infrequently contained tracer after 5 min, by increasing amounts after 15–60 min of HRP perfusion, a vesicular transport of HRP from the apical epithelial surface to the intercellular spaces, bypassing the tight junctions, is suggested.In addition to the transepithelial transport, micropinocytic vesicles also transported HRP to the lysosomal apparatus of the epithelial cells. With increasing length of exposure to HRP, a sequence of HRP-labeled structures could be evaluated, from slightly labeled apical vacuoles and multivesicular bodies to very heavily labeled dense bodies.     

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.     

Different regulation of connexin26 and ZO-1 in cochleas of developing rats and of guinea pigs with endolymphatic hydrops.

Using confocal microscopy and morphometry, we analyzed the expression of connexin26 (Cx26) and ZO-1 in rat cochlea during the postnatal period to elucidate spatiotemporal changes in gap junctions and tight junctions during auditory development. We also studied changes in these junctions in experimental endolymphatic hydrops in the guinea pig. In the adult rat cochlear lateral wall, Cx26 was detected in fibrocytes in the spiral ligament and in the basal cell layer of the stria vascularis, whereas ZO-1 was detected in the apical surfaces of marginal cells and in the basal cell layer. During postnatal development, Cx26 expression increased mainly in the spiral ligament, whereas ZO-1 expression increased in the basal cell layer. The morphometry of Cx26 showed a sigmoid time course with a rapid increase on postnatal day (PND) 14, whereas that of ZO-1 showed a marked increase on PND 7. In experimental endolymphatic hydrops, the expression of Cx26 significantly decreased, whereas there were no obvious changes in the expression of ZO-1. These results indicate that gap junctions and tight junctions in the cochlea increase in a different spatiotemporal manner during the development of auditory function and that gap junctions and tight junctions in the cochlea are differentially regulated in experimental endolymphatic hydrops. (J Histochem Cytochem 49:573-586, 2001)     

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 permeability of the primary decidual zone in the rat uterus: an ultrastructural tracer and freeze-fracture study

The rat primary decidual zone (PDZ) is a transitory avascular region of transformed fibroblasts surrounding the implanting embryo. Studies using fluorescein-labeled tracers have shown that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight. In the present study we investigated the morphologic basis of the permeability barrier. Horseradish peroxidase (HRP) or HRP-labeled immunoglobulin G (IgG-HRP) was administered i.v. to rats on Day 7 of pregnancy, and the animals were killed 30 min to 2 h later. The reaction product of HRP was the same density in uterine blood vessels as in the intercellular spaces of the endometrium and PDZ at 30 min and 1 h after administration. Two hours after administration, the reaction product of IgG-HRP was dense in uterine blood vessels, much less dense in the interstitial spaces of the endometrium, and was not detected in the PDZ. There was an abrupt change in the density of the IgG-HRP reaction product at the intercellular clefts between endothelial cells, where cellular junctions were observed in control tissue. This suggests that the passage of large macromolecules from blood to the implantation chamber is limited initially by cellular junctions between capillary endothelial cells. The exclusion of IgG-HRP from the PDZ indicates that an additional barrier(s) to macromolecules in this region. Lanthanum nitrate tracer was uniformly present throughout the intercellular spaces of the PDZ except at tight junctions between decidual cells. Freeze-fracture replicas of the PDZ showed tight junctions that varied from single strands to interconnected networks of strands oriented mainly parallel to the long axis of the PDZ. Some strands were discontinuous. The tight junctions between decidual cells appear to be functionally discontinuous because HRP readily penetrated the PDZ, but such junctions may retard larger macromolecules such as IgG-HRP. The biological significance of the permeability barrier of the PDZ is discussed.     

Roles of gap junctions in glucose transport from glucose transporter 1-positive to -negative cells in the lateral wall of the rat cochlea

Despite the importance of glucose metabolism for auditory function, the mechanisms of glucose transport in the cochlea are not completely understood. We hypothesized that gap junctions mediate intercellular glucose transport in the cochlea in cooperation with facilitative glucose transporter 1 (GLUT1). Immunohistochemistry showed that GLUT1 and the tight junction protein occludin were expressed in blood vessels, and GLUT1, the gap junction proteins connexin26 and connexin30, and occludin were also present in strial basal cells in the lateral wall of the rat cochlea. Gap junctions were found among not only these GLUT1-positive strial basal cells but also GLUT1-negative fibrocytes in the spiral ligaments and strial intermediate cells. Glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the stria vascularis and spiral ligament, whereas EBA was localized only in the vessels. The gap junctional uncouplers heptanol and carbenoxolone inhibited the distribution of 6-NBDG in the spiral ligament without decreasing the fluorescence of EBA in the blood vessels. These findings suggest that gap junctions mediate glucose transport from GLUT1-positive cells (strial basal cells) to GLUT1-negative cells (fibrocytes in the spiral ligament and strial intermediate cells) in the cochlea.     

FINE STRUCTURAL LOCALIZATION OF A BLOOD-BRAIN BARRIER TO EXOGENOUS PEROXIDASE

Horseradish peroxidase was administered to mice by intravenous injection, and its distribution in cerebral cortex studied with a recently available technique for localizing peroxidase with the electron microscope. Brains were fixed by either immersion or vascular perfusion 10–60 min after administration of various doses of peroxidase. Exogenous peroxidase was localized in the lumina of blood vessels and in some micropinocytotic vesicles within endothelial cells; none was found beyond the vascular endothelium. Micropinocytotic vesicles were few in number and did not appear to transport peroxidase while tight junctions between endothelial cells were probably responsible for preventing its intercellular passage. Our findings therefore localize, at a fine structural level, a "barrier" to the passage of peroxidase at the endothelium of vessels in the cerebral cortex. The significance of these findings is discussed, particularly with reference to a recent study in which similar techniques were applied to capillaries in heart and skeletal muscle.