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  • 1
    ISSN: 1432-0533
    Keywords: Ultrastructural cytochemistry ; Concanavalin A receptors ; 5′-Nucleotidase ; Blood-brain barrier ; Vesiculo-canalicular transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Plant lectin concanavalin A conjugated with ferritin (Con A-F) injected i.v. was used for the detection of the specific monosaccharide residues (α-d-mannosyl and α-d-glucosyl) on the luminal surface of endothelial cells (ECs) in brain micro-blood vessels (MBVs). Both normal mice and animals with mechanically damaged blood-brain barrier (BBB) were used in this study. In addition, the activity of 5′-nucleotidase (5′N), the putative receptor for Con A, was studied cytochemically. Various methodologic experiments indicated that the reaction product formed on the luminal plasmalemma of ECs after incubation of samples in the cytochemical medium for the detection of 5′N activity results from the action of unspecific phosphatase hydrolyzing both specific and nonspecific substrates. The abluminal side of the wall of MBVs seems to be a major location of 5′N activity. Thus, no correlation between cytochemically demonstrable 5′N activity and Con A receptor sites on the luminal surface of ECs was noted. After damage of the BBB, extensive internalization of the luminal plasmalemma forming the limiting membranes of pinocytotic vesicles, vacuoles, and endothelial channel-like structures was observed. This process was represented by a relatively rapid translocation of Con A receptors from luminal surface into the interior of the ECs and to the abluminal side of the vessel wall.
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  • 2
    ISSN: 1432-0533
    Keywords: Cold-lesion injury ; Brain edema ; Blood-brain barrier ; Alkaline phosphatase ; Anionic sites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Micro-blood vessels (MBVs), located in the area of edema, were studied in cat brain at various time intervals (1 h, 24 h, 7 days) after cold-lesion injury. Both cold-injured and adjacent gyri were examined for blood-brain barrier (BBB) permeability to i. v. injected horseradish peroxidase (HRP) with circulation times of 40 min and 24 h. Evans blue (EB) was used as a tracer for gross evaluation of the extension of brain edema. Localization of alkaline phosphatase (AP) and binding of cationized ferritin (CF), considered as a marker of anionic sites, were also studied ultrastructurally. Twenty-four hours after cold injury, the extravasated edema fluid, outlined by EB tracer, was observed to be spreading through the white matter (WM) into the adjacent gyrus. At this time, numerous, larger than capillary MBVs, presumably arterioles and venules located in the edematous WM, showed accumulations of HRP injected at the time of the operation, in the basement membrane, in abluminal pits, and in numerous pinocytotic vesicles and vacuoles of endothelial cells (ECs). The animals killed after 24 h with 40 min HRP circulation showed extravasation of HRP tracer in a zone underlying the necrotic cold injury lesion. On the other hand, there was no evidence of an abnormal HRP leakage in the further removed areas of edema in the WM, particularly in the adjacent gyrus. These observations suggest that a reverse, vesicular transport of HRP across the ECs of some MBVs represents one of several possible mechanisms responsible for the removal of extravasated proteins and of edematous fluid from brain extracellular space. This reverse transport is accompanied by a disruption of the surface anionic layer and changed polarity of ECs manifested by the relocation of AP activity from luminal to abluminal plasmalemma.
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  • 3
    ISSN: 1432-0533
    Keywords: Blood-brain barrier ; Endothelial cells ; Horseradish peroxidase ; Native ferritin ; Alkaline phosphatase ; Pinocytic transport system ; Canalicular transport system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary An ultracytochemical investigation was performed to study the origin of pinocytic vesicles and canalicular structures within endothelial cells (EC) of the injured mammalian blood-brain barrier (BBB). To accomplish this goal, two electron-dense tracers, native ferritin (NF) and horseradish peroxidase (HRP), were used in conjunction with the detection of alkaline phosphatase (AP) activity, a known marker of EC plasmalemma of brain micro-blood vessels. Brain ECs from (1) mice subjected to crude leptomeningeal damage for 1, 2, or 3 days and (2) cats subjected to cold lesion injury for 1, 4, or 24h were evaluated for tracer transport and AP activity. Fine structural analysis of leaking segments of micro-blood vessels from damaged cerebral cortex or basal ganglia demonstrated pinocytic vesicles, deep invaginations of the luminal plasmalemma and elongated, tubular profiles, all containing tracer. Because we observed in ECs from both experimental models of brain injury a positive reaction for AP activity in the luminal plasmalemma, in its deep invaginations, in deliminating membranes of pinocytic vesicles, and in tubulo-canalicular structures, we conclude that all types of transport structures derive from the same 100Å thick exoplasmic plasmalemmal membranes. Further, besides the pinocytic vesicular transport system (PTS), the canalicular transport system (CTS) appears to serve as an additional important mechanism for macromolecular transport across the damaged mammalian BBB.
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  • 4
    ISSN: 1432-0533
    Keywords: Ultrastructural cytochemistry ; Plasma membrane phosphatases ; Blood-brain barrier ; Scrapie ; Endothelial cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Alkaline phosphatase, 5′-nucleotidase nucleoside diphosphatase and thiamine pyrophosphatase activities were studied by cytochemical method applied to electron microscopy of brain microvasculature in normal and scrapie infected mice. In control mice, the major location of all phosphatases studied was the luminal plasma membrane of the endothelial cells. In scrapie infected mice, changes in activity and distribution of the above mentioned phosphatases manifested themselves in the appearance of the reaction product on the abluminal side of the vessel wall. Our data presents evidence that following scrapie infection, these enzymes change their specific localization along the endothelial cell membranes. These enzymatic changes may serve as useful indicators of some alterations in the mammalian blood-brain barrier following infection by scrapie agent in the mouse.
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  • 5
    ISSN: 1432-0533
    Keywords: Blood-brain barrier ; Chronic relapsing experimental allergic encephalomyelitis ; Inflammatory cell emigration ; Trans-endothelial cell transport ; Horseradish peroxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Results are reported of experiments designed to focus at attachment sites of inflammatory cells (ICs) on the luminal surface of brain endothelial cells (ECs) and on the mechanisms of horseradish peroxidase (HRP) transport across the altered blood-brain barrier (BBB) in a murine model of chronic relapsing experimental allergic encephalomyelitis. Cationized ferritin (CF) served as a marker for evaluting the electrostatic nature of brain microblood vessels (MBVs) on the plasma membranes of ICs or normal mouse peripheral white blood cells and erythrocytes. SJL/J mice demonstrating clinical illness were given HRP or CF, in vivo or in situ, respectively. Light microscopy and conventional transmission electron microscopy of cerebellum or thoracic and lumbar spinal cord regions demonstrated HRP leakage most pronounced in MBVs with perivascular infiltrates. HRP traversed across the ECs via numerous vesicles and tubular profiles located mostly in the parajunctional regions, while EC junctions appeared closed. Scanning electron microscopy demonstrated that IC attachment was primarily at parajunctional sites on the EC surface. We also observed increased microvillar projections extending from the EC surface into the lumen. CF demonstrated a patchy decoration on both the luminal EC surface and IC membranes but did not label uncoated invaginating membrane pits or tubular structures. Our data indicate that the points of attachment of the ICs on the EC surface may reflect specific receptor sites where the ICs eventually gain entrance into CNS across the BBB during brain inflammation.
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  • 6
    ISSN: 1432-0533
    Keywords: Blood-brain barrier ; Horseradish peroxidase ; Acid phosphatase ; Brain injury ; Vesiculo-channel canalicular system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary An investigation designed to define relationships between endothelial channels and lysosomes was conducted in the mammalian brain microvasculature. Microvessels from normal and mechanically injured mouse brains were studied ultracytochemically for: (1) transport of horseradish peroxidase (HRP) protein tracer through endothelial channels, and (2) for acid phosphatase (AcP) activity as an enzymatic marker of lysosomes. Following traumatic brain injury for 1 week with 2 h circulation of intravenously injected HRP, selected brain slices were processed for ultrastructural localization of either HRP, AcP, or for both reactions together within the same tissue slices. One week after blood-brain barrier (BBB) damage, the presence of HRP reaction product (RP) was observed within endothelial channels and vesicles of capillaries and arterioles with concomitant increase in lysosomal enzymatic activity of the endothelial cells bordering regions of brain damage. Lysosomes were observed to be directly connected to the endothelial channels. Our observations present cytochemical evidence for endothelial channel-lysosome connections which may suggest intralysosomal modification of blood-born materials before entering the neuropil. Such modification could have important immunological and/or metabolic significance.
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  • 7
    ISSN: 1432-0533
    Keywords: High-voltage electron microscopy ; Horseradish peroxidase ; Blood-brain barrier ; Endothelial cell ; Endothelial cell tubules and channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary High-voltage electron microscopy was applied to the study of endothelial cell (EC) transport of macromolecules in a murine model of blood-brain barrier injury to study the role of the EC canalicular system following brain insult. Semithick sections from mouse brains subjected to acute (2–3 h) mechanical trauma demonstrated permeation of intravenously injected horseradish peroxidase via tubular structures either (a) in the absence of lysosome-associated structures in close proximity, or (b) in association with lysosomes, dense bodies or multivesicular bodies. Our data suggest a dual-purposed system of tubules, one portion that supplies the metabolic requirements of the cell and another portion, suggested to be more limited, that opens up as a result of brain injury.
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  • 8
    ISSN: 1432-0533
    Keywords: Scrapie infection ; Blood-brain barrier ; Cerebrospinal fluid ; Ependymal cells ; Horseradish peroxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Spinal cord samples from IM or VM mice injected intracerebrally with the 87V scrapie agent were examined ultrastructurally at the clinical stage of disease for changes in blood vessel permeability and for pathological alterations. In several animals, (3 of 16), massive changes were noted in the cervical spinal cords in the subependymal area of the cortical gray matter immediately surrounding the central canal including ependymal cell changes, the presence of amyloid plaque in close association with microglial cells, extensive neuropil vacuolation, the appearance of reactive astrocytes, degenerating neurites and vacuolated neurons. In those regions showing structural damage, localized increased permeability to horseradish peroxidase across the blood-brain barrier was noticed along with the appearance of numerous vesiculo-canalicular profiles in micro-blood vessel endothelial cells with extravasation of the tracer to the neuropil. Some damaged neurons appeared flooded with this tracer. These changes were not observed in either the thoracic or lumbar spinal cord regions. The occurrence of pathological changes in the spinal cords of a small percentage of intracerebrally injected mice was probably due to a high concentration of the scrapie agent which localized in the cervical spinal cord, presumably after entering the spinal fluid via the lateral ventricle at the time of injection.
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