Biomedical subjects

A S Lossinsky

Publications and source records attributed to A S Lossinsky.

At least 37 records · Page 2Linked to original sources

PubMed1994

A quantitative immunocytochemical study of the osmotic opening of the blood-brain barrier to endogenous albumin.

The time sequence of the blood-brain barrier opening to endogenous albumin in rat brain after intracarotid infusion of hyperosmolar L(+)arabinose was studied using quantitative immunocytochemistry. Brain samples obtained 1, 5, and 30 min after insult were immersion-fixed in formaldehyde-glutaraldehyde mixture and embedded at low temperature in Lowicryl K4M. Untreated rats or rats exposed only to Ringer's solution were used as a control. Ultrathin sections were exposed to anti-rat albumin antiserum followed by protein A-gold. The density of immunosignals (gold particles per square micrometre) was recorded over four compartments: vascular lumen, endothelium, subendothelial (perivascular) space including basement membrane, and brain parenchyma (neuropil). The labelling density of the vessel lumen, containing blood plasma, was considered to represent 100% of the circulating albumin. Morphometric and statistical analysis indicated that in control animals only 0.4-0.6% of circulating albumin appears in the subendothelial space and in the basement membrane. As soon as one minute after L(+)arabinose infusion, this value increased to 3%, followed by a further increase to 25% and 56% after 5 and 30 min, respectively. A slow increase of the labelling density in the adjacent neuropil suggests that the basement membrane represents an obstacle for escaping albumin, which apparently sticks to or is trapped by this membrane. The results indicate that the applied procedure, although based on morphometric analysis of static electron micrographs can also be used for studying dynamic processes such as transvascular passage of albumin after disruption of the brain-blood barrier.

Animals↗

PubMed1994

Ultracytochemical studies of the effects of aluminum on the blood-brain barrier of mice.

We studied the effect of chronic exposure (6 weeks and 6 months) of mice to drinking (tap) water containing 1.76% (0.06 M) aluminum lactate on some cytochemical properties of the blood-brain barrier (BBB). The plasmalemma-bound enzymatic activities of alkaline phosphatase (AP) and Ca(2+)-activated adenosine triphosphatase (Ca(2+)-ATPase) were studied at the ultrastructural level. Anionic sites were localized with cationized ferritin in a pre-embedding procedure and with cationic colloidal gold in a post-embedding procedure applied to brain samples embedded in Lowicryl K4M. Intravenously injected Evans blue and horseradish peroxidase (HRP) were used for evaluation of the functional state of the BBB. The results indicate that chronic exposure to aluminum does not noticeably affect barrier function of the endothelium of cerebral cortex blood microvessels. Focal leakage of larger than capillary microvessels (presumably arterioles and venules) was observed only in a few areas, such as the basal ganglia and amygdaloid nuclei. The localization of both enzymatic activities (AP and Ca(2+)-ATPase) in microvessels remained essentially unchanged. The localization of anionic sites was also unchanged except on the luminal surface of the endothelium of a few blood microvessels located in areas of the brain where leakage of the injected HRP was noted. In these vessels the injected HRP was often attached to the luminal surface of the endothelial cells, suggesting its increased stickiness. These data, compared with our previous observations on brain microvascular endothelial cells growing in vitro, indicate that cytotoxicity of aluminum is evidently less pronounced in the living organism, presumably due to action of detoxicating and regulatory mechanisms.

Administration, Oral↗

PubMed1994

Platelet occlusion phenomenon after short- and long-term survival following complete cerebral ischemia in rats produced by cardiac arrest.

Platelet interaction with cerebral microvessels was studied in rats after global brain ischemia. Studies were performed with a model of global central nervous system ischemia produced by cardiocirculatory arrest in normothermic rats. The ischemic period of 5-min was followed by times of recirculation lasting 5 and 15 mins, 1, 3, 6, 24, and 48 hrs, and 6, 10, and 12 months. After different periods of survival, blood vessels from brains were examined by transmission electron microscopy. Our investigation revealed numerous platelet aggregates of varying sizes within both arterial and venous intraparenchymal vessels. At the same time, we also noted platelets in various stages of disintegration. Platelets close to the endothelial cells were often degranulated, with shape changes including pseudopodia. Aggregates of platelets were focal, random, and more widespread occurring in the cerebral cortex, thalamus, basal ganglia, hippocampus, and cerebellum. We also observed recent aggregates of platelets and thrombi, which presented varying degrees of degranulation. In one time period, some platelets were found outside the brain vessels (in the perivascular space after 24 hrs of survival). We present evidence that platelet aggregation was repeated a long time after the ischemic incident, i.e., after one year. The main result was a prominent and local accumulation of platelets in microvessel branches or regions of vessel bifurcations, which correlated well with blood-brain barrier alterations observed previously in this model. The platelet aggregations increased in frequency with longer periods of recirculation. We noted that local platelet adhesion/aggregation was present after brain ischemia requiring no denudation or exposure of the basal lamina.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Cellular mechanisms of the blood-brain barrier (BBB) opening to albumin-gold complex.

Cold lesion injury applied to mouse brain and infusion of hyperosmolar L(+) arabinose solution into rat carotid artery were used as extravascular and intravascular insults, respectively, leading to blood-brain barrier (BBB) disruption. To study the cellular mechanisms of the BBB opening, heterologous (bovine) and homologous (mouse and rat) albumin-gold complexes were used as a macromolecular tracer. Both insults rapidly induce the leakage of the blood-borne tracer, although the mechanisms of their action appear to be different. Cold lesion injury (cryoinjury) leads to the opening of interendothelial junctions and concomitantly to an endothelial-platelet reaction. This insult is followed by irreversible changes such as desquamation, degeneration and necrosis of the endothelial lining, formation of thromboses, and disruption of the basement membrane. Osmotic opening occurs through at least the four mechanisms (presumably temporal and reversible) that follow: 1) opening of a part of the junctional complexes; 2) the formation of transendothelial openings (interendothelial gaps or penetrating, crater-like excavations); 3) the uncontrolled passage of tracer particles through the cytoplasm of the injured endothelial cells; and 4) segmental denudation of the endothelial lining. The basement membrane appears to represent one of the main obstacles in the passage of blood-borne albumin-gold complexes to the extracellular space in the brain parenchyma.

Albumins↗

PubMed1991

Reassessment of a new model of complete cerebral ischemia in rats. Method of induction of clinical death, pathophysiology and cerebrovascular pathology.

The present study was undertaken to ascertain the role of the microcirculation in the phenomenon of hypoperfusion following complete cerebral ischemia. The experiments were performed on rats under superficial ether anesthesia. Cerebral ischemia was induced by cardiac arrest for 3.5 or 10 min, with survival periods that lasted from 3 min to 7 days. A special metal hook-like device was inserted into the chest cavity at the third intercostal spaces for occluding the cardiac vessel bundle. The effect of this procedure was total cessation of systemic circulation, i.e., clinical death. In 52% of animals with 10-min clinical death, resuscitation (external heart massage and artificial ventilation) restored heart activity. When brain circulation was restored, respiratory activity, pain reaction, corneal reflex, bioelectric activity of the cortex, and normal activities of the rats returned. Scanning electron microscopy was applied to study the effect of ischemia on the vessel wall and endothelial cells (EC). Ischemia produced a remarkable increase in the numbers of microvilli and pit-like invaginations on the luminal EC surface. The luminal wall surface of many of the microvessels (MV) formed ridges. Frequently, microthrombi of varying sizes were observed. The most prominent changes were noted from 3 min to 6 h of recirculation, and they correlated with hypoperfusion after ischemia. Seven days later, these changes completely disappeared. The data presented here indicate that progressive hypoperfusion after ischemia occurs with significant alterations in the MV walls. These studies collectively suggest that the focal responses in select MVs may be associated with receptor molecule up-regulation of some, but not all, affected ECs. Our data provide further characterization of a new and unique chronic model of brain ischemia that can be applied to relevant clinical studies.

Animals↗

PubMed1991

Mechanisms of inflammatory cell attachment in chronic relapsing experimental allergic encephalomyelitis: a scanning and high-voltage electron microscopic study of the injured mouse blood-brain barrier.

Brain and spinal cord blood vessels from mice subjected to chronic relapsing experimental allergic encephalomyelitis were examined by scanning (SEM) and high-voltage electron microscopy (HVEM). SEM analysis of veins and venules from affected tissue regions demonstrated inflammatory cells (ICs), primarily lymphocytes or monocytes, attached to the luminal endothelial cell (EC) surface adjacent to the junctional complexes. In transverse section these cells were shown by HVEM to extend and to insert filopodia (lymphocytes) or flap-like lamellapodia (monocytes) into the luminal EC surfaces. Affected ECs often expressed increased microvillar projection as well as parajunctional crater-like structures on their luminal surfaces. Based on scanning and high-voltage electron microscopy, we present morphological evidence that some populations of sensitized ICs do not penetrate the EC junctions initially during EC attachment but instead insert pseudopodial projections into specialized openings in the ECs that are formed in response to chronic inflammation.

Animals↗

PubMed1991

Structural and functional aspects of the interaction of inflammatory cells with the blood-brain barrier in experimental brain inflammation.

Interaction between various subclasses of inflammatory cells (ICs) and endothelial cells (ECs) lining selective blood vessels of the mammalian blood-brain barrier (BBB) is an initial, important event during inflammatory conditions of the central nervous system (CNS). In this review, we will present a brief ultracytochemical and immunocytochemical assessment of our perspective on this intimate cellular interaction which has been described recently in conditions that involve immunological alterations of the BBB. We will discuss some morphological aspects of what is currently known about acute and chronic inflammatory BBB disorders that are involved in inflammatory processes. We will focus, in particular, on experimental allergic encephalomyelitis (EAE), an animal model for human multiple sclerosis (MS). Many of the past and more recent concepts found in the literature concerning IC attachment and diapedesis, as well as our own experimental efforts over more than two decades will be presented.

Animals↗

PubMed1990

Sequential appearance of anionic domains in the developing blood-brain barrier.

The distribution of anionic sites in the walls of mouse brain micro-blood vessels (MBVs) during development and maturation of the blood-brain barrier (BBB) was studied by electron microscopy. Cationic colloidal gold (CCG) and Lowicryl K4M-embedded brain samples obtained from mouse fetuses (13th and 19th days) and from 1-, 5-, 12- and 24-day-old and adult mice were used. The labeling of anionic sites with CCG was more intense on the abluminal than on the luminal front of the endothelial cells (ECs) in fetuses and in newborn mice. Only a few anionic sites appear on the luminal front of the ECs of proliferating blood vessels invading the neural tissue in 13-day-old fetuses. They become slightly, although steadily, more abundant during further stages of development, and their number rapidly increases between the 12th and 24th day of life at which time they attain the density typical for mature animals. The maturation of the basement membrane (BM), which occurs during the myelinization period (12th-24th day of life), also coincides with an increasing concentration of anionic sites. These observations suggest that the gradual appearance of anionic sites on both fronts of the endothelium, as well as in the developing and maturing BM, represents one of the mechanisms responsible for differentiation of cerebral microvasculature into BBB-type MBVs.

Aging↗

PubMed1990

Ultrastructure of the cells forming amyloid fibers in Alzheimer disease and scrapie.

Ultrastructural, three-dimensional reconstruction of cells surrounding the amyloid star in classical plaques in Alzheimer disease (AD) and histochemical studies of the cells associated with the deposits of amyloid fibers in scrapie were carried out. These studies showed that in both diseases, the fibers appear within the smooth endoplasmic reticulum (ER) and infoldings of cytoplasmic membranes of microglia/macrophages. Additional information about the site of formation of the amyloid fibers derives from histochemical studies of the localization of nucleoside diphosphatase (NDPase) activity. In normal microglia, this enzyme is associated with smooth ER and cell membranes. In the cells that form amyloid fibers, the NDPase activity is associated with the newly formed amyloid fibers within the distended cisternae of ER and the finger-like cytoplasmic projections. In the center of the amyloid star, the NDPase activity disappears. The presence of NDPase-positive amyloid fibers in the same location, where the enzyme is found in non-amyloid-forming cells, further supports our conclusion that the microglia/macrophages are the source of amyloid deposits. These studies also show that in spite of the differences in the proteins that produce the amyloid fibers in AD and scrapie, in both diseases, the microglia/macrophages play a key role in amyloid formation.

Acid Anhydride Hydrolases↗

PubMed1990

Changes in the distribution of anionic sites in brain micro-blood vessels with and without amyloid deposits in scrapie-infected mice.

Cationic colloidal gold (CCG) and scrapie-infected mouse brain samples embedded in Lowicryl K4M were used for ultrastructural localization of negatively charged microdomains (anionic sites) in the cerebral microvasculature. The distribution of anionic sites on both fronts (luminal and abluminal) of endothelial cells and in the basement membrane (BM) in the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs (especially capillaries), however, located inside the plaque areas and surrounded directly by amyloid fibers contained attenuated endothelium, the luminal surface of which showed a segmental lack or diminution of anionic sites. In these vessels the BM was frequently infiltrated and replaced by the amyloid fibers. In some vessels located mainly in the areas of the neuropil vacuolization deposits of homogenous material causing the thickening of the BM were noted. These changes were accompanied by irregular labeling of the BM with gold particles. At the sites of bifurcation of some MBVs, predominantly in the area of the venular estuary at the mouth of capillary (at capillary-venular connections), a discontinuity in the distribution of anionic sites was noted. The observed disturbances in the distribution of anionic sites can be associated with a previously noted increased permeability of some MBVs in the brains of scrapie-infected mice.

Amyloid↗

PubMed1990

Combined conventional transmission, scanning, and high-voltage electron microscopy of the same blood vessel for the study of targeted inflammatory cells in blood-brain barrier inflammation.

The microvasculature of brains and spinal cords from mice subjected to chronic relapsing experimental autoimmune encephalomyelitis (CREAE) was studied using three different electron microscopic techniques. Blood vessels were initially examined by scanning electron microscopy. This allowed for the investigation of topographical changes of the luminal aspects of endothelial cells (ECs) and identification of targeted inflammatory cells (ICs) attached to the ECs. The same blood vessel areas with attached ICs examined by scanning electron microscopy were subsequently trimmed, processed for routine conventional transmission electron microscopy, and plastic embedded. Thin (80 nm) sections were cut and evaluated. Semithick (0.5-0.75 microns) serial sections of this material were examined by high-voltage electron microscopy. Data presented here described a useful technique for combining several ultrastructural techniques that permits simultaneous topographic and cross-sectional examination of selected regions of individual blood vessels or specifically targeted ICs.

Animals↗

PubMed1989

High voltage electron microscopic studies of endothelial cell tubular structures in the mouse blood-brain barrier following brain trauma.

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.

Animals↗

PubMed1989

Sites of egress of inflammatory cells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis.

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 evaluating 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.

Animals↗

PubMed1988

Influence of stereotaxically injected scrapie on neurotransmitter systems of mouse cerebellum.

The 22L strain of scrapie was injected stereotaxically into the cerebellum of C57BL/6J mice to determine its effect on several cerebellar neurotransmitter systems during the early clinical stages of the disease. In this model vacuolar lesions are restricted to the cerebellum with no evidence of vacuolization in other brain regions. Although vacuolar lesions develop throughout all cell layers of the cerebellum, they are most severe in the granule cell layer. Modest but significant (P less than 0.01) reductions in cerebellar weight, glutamate decarboxylase activity, and in the affinity of the N6-[adenine-2,8-3H]cyclohexyladenosine binding sites, were observed in scrapie affected mice. The densities of the high- and low-affinity adenosine receptors were unaffected. Adenosine receptors in the cerebellum are highly localized to the axon terminals of the glutamatergic, GABA receptive granule cells. GABA, benzodiazepine, glutamate, and muscarinic cholinergic receptors were not significantly altered. In addition, the high-affinity uptake of glutamate, and the activity of choline acetyltransferase were not significantly changed. GABA high-affinity uptake was slightly increased. Even though the granule cell layer of the cerebellum had undergone severe vacuolation, only modest neurotransmitter changes were apparent. Although these results suggest a tenuous relationship between scrapie pathology and the integrity of neurotransmitter systems, it is possible that compensatory neurochemical changes in uncompromised neuronal populations may have masked potentially specific neurotransmitter effects.

Animals↗

PubMed1988

Ultrastructural studies of glycoconjugates in brain micro-blood vessels and amyloid plaques of scrapie-infected mice.

Lectin or glycoprotein-gold complexes and samples of scrapie-infected mouse brain embedded in Lowicryl K4M were used for ultrastructural localization of glycoconjugates. The lectins tested recognize the following residues: beta-D-galactosyl [RCA, Ricinus communis agglutinin (aggl.) 120], N-acetyl and N-glycolyl neuraminic acid (LFA, Limax flavus aggl.), N-acetyl-D-glucosaminyl and sialyl (WGA, Wheat germ aggl.), N-acetyl-D-galactosaminyl (HPA, Helix pomatia aggl., and DBA, Dolichos biflorus aggl.), alpha-D-mannosyl/alpha-D-glucosyl (Con A, Concanavalin A), alpha-D-galactosyl and alpha-D-galactopyranoside (BSA, Bandeirea simplicifolia aggl., izolectin B4). Labeling of the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs, however, located inside the plaque area and surrounded directly by amyloid fibers showed attenuation of the endothelium, the surface of which was scarcely and irregularly decorated with RCA, LFA, WGA and Con A. These abnormalities in the composition of glycoconjugates can be associated with previously noted increased permeability of some MBVs in the brains of scrapie-infected mice. Some vessels in the plaque area were encapsulated by perivascular deposits of homogeneous or flocculogranular material containing several glycoconjugates. A very intimate structural relation between reactive (microglial-like) cells and amyloid fibers suggests the participation of these cells in elaboration of plaque material. Labeling of the cell surface and adjacent amyloid fibers with the same lectins (RCA, WGA, DBA, Con A) suggests the possibility that the glycosylation of these fibers occurs extracellularly. Only WGA and DBA were occasionally labeling some Golgi elements of the reactive cells.

Amyloid↗

PubMed1987

Ultrastructural observations of spinal cord lesions and blood-brain barrier changes in scrapie-infected mice.

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.

Amyloid↗

PubMed1986

A comparative ultrastructural study of endothelial cell tubular structures from injured mouse blood-brain barrier and normal hepatic sinusoids demonstrated after perfusion fixation with osmium tetroxide.

Mice subjected to surgical leptomeningeal traumatic injury were fixed by perfusion with solutions containing either: (1) osmium tetroxide, (2) a mixture (cocktail) of osmium tetroxide and glutaraldehyde, or (3) a standard aldehyde fixative following the circulation of intravenously injected solutions of native ferritin (NF) or horseradish peroxidase (HRP) tracers. Endothelial cells (ECs) from injured cerebral cortex from all the above groups were examined ultrastructurally for the presence of tubular transport structures. These ECs were compared to endothelia of hepatic sinusoids which normally express numerous EC tubular profiles. Because we observed EC tubular structures in ECs of both injured brain and from liver sinusoids irrespective of fixation regime employed, we present evidence that the tubular profiles are real structures that form in vivo and which do not represent postmortem fixation artifacts.

Animals↗

PubMed1986

Distribution of anionic sites and glycoconjugates on the endothelial surfaces of the developing blood-brain barrier.

The distribution of anionic sites detected in vitro with cationized ferritin and lectin-binding sites on the endothelial cell (EC) surface of brain micro-blood vessels was studied by electron microscopy. Gold-labeled lectins and glycoproteins and Lowicryl K4M-embedded brain samples obtained from mouse embryos (19th day), and from 1-, 5-, 12-, 24- and 48-day-old and adult mice were used. It was shown that the functional maturation of the blood-brain barrier (BBB) occurring in the mouse after birth between the 12th and 24th day of life is accompanied by a disappearance of vesicular transport in capillaries and by the formation of a uniform, thin, negatively charged layer on the surface of the EC. Concomitantly the binding of lectins specific for beta-D-galactosyl (RCA) and sialyl (LFA and WGA) residues become progressively more intense and uniform on both luminal and abluminal fronts of the EC. The concentration of HPA-binding sites on the abluminal side of the EC and in the basement membrane increases. Similarly the binding of Con A becomes more intense on abluminal than on luminal front of the EC. These observations suggest that extensive remodeling of anionic sites and surface glycoprotein layer and also the elaboration of ECs polarity occur during BBB maturation.

Aging↗