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Biomedical subjects

A S Lossinsky

Publications and source records attributed to A S Lossinsky.

At least 55 records · Page 3Linked to original sources
PubMed1986

Localization of alkaline phosphatase activity in endothelia of developing and mature mouse blood-brain barrier.

The relationship between alkaline phosphatase (AP) activity and maturation of blood-brain barrier (BBB) in mouse brain was studied ultracytochemically. The permeability of micro-blood vessels (MBVs) to intravenously injected horseradish peroxidase was considered to be a criterion of BBB maturation, which occurs between the 12th and 24th day of mouse life. This process coincides with the appearance of cytochemically detectable AP activity in luminal plasma membrane (PM) of the endothelial cell (EC) of capillaries or in both luminal and abluminal PM in arterioles. No reaction appears in ECs of venules. These observations indicate that the development of BBB function is accompanied by the formation of enzymatic barrier in MBVs endothelia. AP constitutes one of the components of this barrier. Comparative study indicates that in various MBVs of non-BBB type, except in skeletal muscle, no AP activity is detected in similar incubation conditions.

Aging↗
PubMed1986

Characterization of endothelial cell transport in the developing mouse blood-brain barrier.

The cerebromicrovasculature of immature mice ranging in age from 1-24 days after birth was studied by electron microscopy. A micromanipulator apparatus enabled us to facilitate direction of a needle cannula and injection into leg veins or hearts with horseradish peroxidase (HRP) or ferritin (anionic and cationic) tracers and subsequent perfusion fixatives. Numerous HRP-filled vesiculocanalicular transport structures appearing in endothelial cells (ECs) of brain microblood vessels (MBVs) were observed in time periods ranging from 1-14 days. In addition to HRP transport across the ECs by tubulovesicular profiles, some of these structures appeared to become connected to multivesicular bodies. Between 14 and 24 days after birth, limited HRP was transported across the ECs to the basement membrane in only a few short segments of subpial arterioles. The decoration pattern with cationized ferritin (CF) on the luminal surface of the ECs depends upon whether the surface was exposed to the ligand before or after fixation. Quenching of aldehyde groups in fixed brain tissue has critical importance for the decoration pattern of CF on the luminal plasmalemmal surface. The absence of CF labeling on the delimiting membranes of plasmalemmal vesicles and tubular structures suggests that these structures represent differentiated microdomains engaged in macromolecular transport in the ECs of the developing mouse brain.

Animals↗
PubMed1986

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels.

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.

Animals↗
PubMed1985

Ultrastructural observations on the transvascular route of protein removal in vasogenic brain edema.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1984

Ultrastructural studies of concanavalin A receptors and 5'-nucleotidase localization in normal and injured mouse cerebral microvasculature.

Plant lectin concanavalin A conjugated with ferritin (Con A-F) injected i.v. was used for the detection of the specific monosaccharide residues (alpha-D-mannosyl and alpha-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.

5'-Nucleotidase↗
PubMed1983

Enzyme cytochemistry of blood-brain barrier (BBB) disturbances.

Alkaline phosphatase (AP) is one of the enzymes which is highly active in the plasmalemma of endothelial cells (ECs) of BBB-type microvessels. In the ECs of non-BBB type vessels, the reaction for AP (and other phosphatases) is negative (e.g. choroid plexus, area postrema, hypophysis). After BBB damage, the leakage of the vessels can be demonstrated by the use of horseradish peroxidase (HRP). Concomitantly, changes in polar distribution of AP in the ECs occur, paralleled by the appearance of numerous pinocytic vesicles, deep invaginations of the plasmalemma and channel-like structures. The delimiting membranes of these structures possess AP, 5'-nucleotidase, nucleoside diphosphatase and Na+, K+-ATPase activities. These observations suggest that the redistribution of plasmalemma bound enzymes from luminal to abluminal surface results from membrane flow associated with formation of pinocytic vesicles and channel-like structures in affected ECs. In the area of brain where the process of resolution of brain edema occurs, the shift of the enzymatic activity from luminal to abluminal plasmalemma of the ECs is observed probably because of the need to remove various solutes present in the edematous fluid. The appearance of positive reaction for AP in the abluminal side of the EC can be a reflection of the changed functional polarity of these cells associated with reverse transport of solutes from brain, back into the blood stream.

5'-Nucleotidase↗
PubMed1983

Ultracytochemical studies of vesicular and canalicular transport structures in the injured mammalian blood-brain barrier.

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 24 h 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 delimiting membranes of pinocytic vesicles, and in tubulo-canalicular structures, we conclude that all types of transport structures derive from the same 100A 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.

Animals↗
PubMed1983

Increased blood-brain barrier permeability in scrapie-infected mice.

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of "slow" viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).

Animals↗
PubMed1982

Cytochemical localization of ouabain-sensitive, K+-dependent p-nitro-phenylphosphatase (transport ATPase) in the mouse central and peripheral nervous systems.

Enzyme activity, representing the sites of K+-dependent p-nitrophenylphosphatase (K+-pNPPase), a component of the transport adenosine triphosphatase (Na+,K+-ATPase) system, has been localized at the ultrastructural in both cerebral cortex and in sciatic nerve of the mouse. Normal mice and animals with mechanically injured blood-brain barrier (BBB) were used. In the cerebral cortex, positive reaction was found in synapses, plasmalemma of neurites (axons and dendrites), in endothelial cell of microblood vessels and in the plasmalemma of mesothelial cells of the pia mater. In the sciatic nerve, a strong reaction was present in the nodes of Ranvier, with weaker reaction in the internodal areas of the axolemma. In the endothelial cells of normal blood vessels, the reaction product was localized on the luminal and abluminal, or only on the abluminal plasmalemma. After damage of BBB, numerous invaginations, pits and pinocytic vesicles showing positive reaction in their limiting membrane appeared in the endothelial cells.

4-Nitrophenylphosphatase↗
PubMed1981

Evidence for induction of localized amyloid deposits and neuritic plaques by an infectious agent.

Mice inoculated intracerebrally with mouse brain homogenate infected with a particular strain of scrapie agent developed amyloid and neuritic plaques and amyloid tubercles along the injection track, solid wall-like infiltrations at the injection site, or both. Our observations implicate an infections agent in the localized formation of the amyloid and the neuritic plaque--the leading pathological change found in normal elderly people and animals and in patients with senile dementia of the Alzheimer type. Further, these studies indicate that microglia appear to play a central role in the pathogenesis of amyloid formation and could be involved in the processing, production, and assembly of the fibers.

Amyloidosis↗
PubMed1981

Ultracytochemical evidence for endothelial channel-lysosome connections in mouse brain following blood-brain barrier changes.

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.

Acid Phosphatase↗
PubMed1981

Ultrastructural cytochemical studies of cerebral microvasculature in scrapie infected mice.

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.

Alkaline Phosphatase↗
PubMed1981

Ultracytochemical studies of the blood-meningeal barrier (BMB) in rat spinal cord.

Alkaline phosphatase(AP),5'-nucleotidase(5'N) and nucleoside diphosphatase (NDPase) activities were studied by cytochemical methods applied to light and electron microscopy in the microvasculature of spinal cord leptomeningeal strips of normal and protamine sulfate (PS) treated rats. The increased permeability to intravenously injected horseradish peroxidase was observed in some segments of microvessels of PS treated rats. Enhanced formation of plasmalemmal pits and deep invaginations, formation of numerous pinocytic vesicles and the appearance of channel-like structures in the cytoplasm of endothelial cells were the most striking ultrastructural evidence of increased permeability of the affected microvessels. All of these structures also showed activity of AP, and to lesser extent, of NDPase; 5'N activity was mainly associated with the delimiting membranes of pinocytic vesicles. Our data present evidence that a shift of enzymatic activity from luminal to abluminal surface of affected endothelial cells results from membrane flow accompanying increased transport activity via formation of pinocytic vesicles and channel-like structures.

Alkaline Phosphatase↗
PubMed1981

A simple screening procedure for evaluating central nervous system tissue sections showing structural and cytochemical alterations of the blood-brain barrier.

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 micrometers and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.

Animals↗
PubMed1979

New ultrastructural evidence for a protein transport system in endothelial cells of gerbil brains.

Pathways for transfer of horseradish peroxidase (HRP) across the cerebral microvasculature were studied in Mongolian gerbils after inducing either unilateral carotid-artery ligation or intracarotid air embolism. Electron microscopy on samples from both ipsilateral and contralateral brain hemispheres showed the reaction product (HRP) in the endothelial cytoplasm of capillaries and arteriols in all animals; in the basal lamina, HRP was seen only after long (3--4 h) ischemic periods. HRP was seen within both spherical and tubular structures, with the latter appearing in obliquely or longitudinally sectioned blood vessels. These endothelial channel-like structures are part of a tubulovesicular network which seemingly exists in the normal endothelium of the gerbil brain. The tubules serve as pathways for proteins into the brain; protein transfer becomes enhanced following circulatory injuries such as ischemia.

Animals↗
PubMed1978

Neuronal ischemic injury: light microscopy, ultrastructure and biochemistry.

A uniform, predictable pattern of cellular abnormalities is seen after complete, irreversible ischemic injury to the central nervous system. This is in contrast to the heterogeneous, multifocal picture which characterizes incomplete ischemia. The range of abnormalities in neuronal soma after an arterial occlusion changes considerably as a function of time and site. There is no single pattern of neuronal alteration that can be ascribed exclusively to ischemia. Red neurons are a relatively late (about 18 h) indicator of ischemia and are seen only in areas where blood supply is marginal. In addition to depletion of high-energy-phosphate reserves, brain ischemia results in characteristic alterations of amino acid concentrations in the ischemic tissue. Glutamate, glutamine, and aspartate either decrease or remain constant while alanine increases. Proportional decreases in the former three amino acids may be explained by simple dilution due to edema. Increases in alanine relative to glutamate and aspartate may be utilized as a biochemical index of perfusion to various brain regions.

Alanine↗
PubMed1978

The effect of aldehyde fixation on selected substrates for energy metabolism and amino acids in mouse brain.

The effect of aldehyde fixation on concentrations of low molecular weight constituents was determined by comparing amounts of selected intermediates in brains of mice exposed to aldehyde fixative solutions with those perfused with phosphate buffer solution alone. Aldehyde perfusion resulted in excellent preservation of cerebral cortex ultrastructure in the presence of dramatic declines in adenosine triphosphate, phosphocreatine, glucose and glucose-6-phosphate that occureed before exposure of the tissue to aldehyde fixatives. Decreases in hexose were accompanied by approximately a 4-fold increase in lactate and a 2-fold increase in pyruvate. Glycogen levels decreased by about 60% during the initial operative procedure but remained constant after aldehyde fixation. Glycogen content declined approximately 90% in tissues that were not treated with aldehyde. Concentrations of aspartate and glutamate changed only slightly during the initial period (1-5 min) and remained constant for at least 90 min in cerebral cortices fixed with aldehydes. Alanine levels increased in both fixed and unfixed tissue; however, this increase was much smaller in tissues exposed promptly to aldehydes. Total ninhydrin-positive material in perchloric acid extracts of brain decreased in mice exposed to aldehyde solutions but increased in tissues that were not. These results indicated that several amino acids may be measured reliably in tissues preserved for light and electron microscopy. In addition, determination of glutamate: alanine ratios in tissues perfused with aldehydes may provide an indication of the timing of fixation.

Aldehydes↗