Recruitment of Labelled Monocytes by Experimental Venous Thrombi

2001 ◽  
Vol 85 (06) ◽  
pp. 1018-1024 ◽  
Author(s):  
C. L. McGuinness ◽  
J. Humphries ◽  
M. Waltham ◽  
K. G. Burnand ◽  
M. Collins ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Vessel Wall ◽  
Time Course ◽  
Thrombus Formation ◽  
Vena Cava ◽  
Steady Increase ◽  
Time Interval ◽  
Venous Thrombus ◽  
Scanning Electron

Summary Objective. Infusing monocytes that have been stimulated to produce fibrinolytic activators and factors that regulate cell proliferation, migration and maturation, might enhance venous thrombus resolution. The aim of this study was to determine the time course of infused monocyte recruitment into venous thrombus in an appropriate model of this disease. Design and Methods. Thrombus was induced in the inferior vena cava of male Wistar rats using reduced flow (80-90% stenosis). The vessel wall was examined at 1hr by scanning electron microscopy. Resolving thrombi with surrounding vena cava were obtained at 1, 7, 14 and 21 days after induction (n = 8). Sections, taken at 0.5 mm intervals (10-15 sections per thrombus), were stained using haematoxylin, Martius Scarlet Blue and antibodies against monocytes, platelets and fibrin. Sections from human venous thrombi (n = 4) were similarly stained. The area occupied by monocytes (in relative pixel units, RPU) was determined using computer aided image analysis. Peripheral rat blood monocytes were extracted, fluorescently labelled and injected intravenously into 7 rats prior to thrombus induction. Vena cava with thrombus was harvested 1 h, 2, 3, 4, 7, 14 and 25 days after induction and their fluorescence measured. The fluorescent content of the caval wall and thrombus was analysed in greater detail at 2 and 25 days after thrombus induction (n = 4 at each time interval). Results. Experimental thrombi were structurally similar to human thrombus and resolved within 14-21 days. Scanning electron microscopy showed minimal endothelial damage at 1 h with signs of early thrombus formation (platelet, red cell leukocyte and fibrin deposition). Neutrophils were the predominant leukocyte in the thrombus at 1 day, with monocytes making up only 0.3% (0.04% sem) of the area of the thrombus. There was a steady increase in thrombus monocyte content and by 21 days the percentage area of thrombus covered by monocytes had increased by over 35 fold to 11.5% (2.3% sem) (p <0.001). Initially, monocytes appeared around the edge of the thrombus and became more evenly distributed through the thrombus as resolution progressed. Labelled monocytes could be found in the circulation up to 1 week after infusion. The fluorescent content (RPU) of the thrombus increased over 25 days (mean RPU At 2 days 0.012, sem 0.005; mean RPU at 25 days 1.062, sem 0.252, p = 0.008). The number of labelled monocytes in the vessel wall peaked at 2 days and decreased thereafter.

The time course of calcium deposition in shells of the barnacle (Balanus amphititre amphititre) during cyprid-juvenile metamorphosis

1994 ◽  
Vol 52 ◽  
pp. 178-179
Author(s):  
Nancy R. Wallace ◽  
Craig C. Freudenrich ◽  
Karl Wilbur ◽  
Peter Ingram ◽  
Ann LeFurgey
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Time Course ◽  
Vertical Plane ◽  
Mantle Cavity ◽  
Qualitative Assessment ◽  
Calcium Deposition ◽  
Free Swimming ◽  
Freeze Dried ◽  
Scanning Electron

The morphology of balanomorph barnacles during metamorphosis from the cyprid larval stage to the juvenile has been examined by light microscopy and scanning electron microscopy (SEM). The free-swimming cyprid attaches to a substrate, rotates 90° in the vertical plane, molts, and assumes the adult shape. The resulting metamorph is clad in soft cuticle and has an adult-like appearance with a mantle cavity, thorax with cirri, and incipient shell plates. At some time during the development from cyprid to juvenile, the barnacle begins to mineralize its shell, but it is not known whether calcification occurs before, during, or after ecdysis. To examine this issue, electron probe x-ray microanalysis (EPXMA) was used to detect calcium in cyprids and juveniles at various times during metamorphosis.Laboratory-raised, free-swimming cyprid larvae were allowed to settle on plastic coverslips in culture dishes of seawater. The cyprids were observed with a dissecting microscope, cryopreserved in liquid nitrogen-cooled liquid propane at various times (0-24 h) during metamorphosis, freeze dried, rotary carbon-coated, and examined with scanning electron microscopy (SEM). EPXMA dot maps were obtained in parallel for qualitative assessment of calcium and other elements in the carapace, wall, and opercular plates.

Neutrophil Derived Cathepsin G Induces Potentially Thrombogenic Changes in Human Endothelial Cells: a Scanning Electron Microscopy Study in Static and Dynamic Conditions

1994 ◽  
Vol 72 (01) ◽  
pp. 140-145 ◽  
Author(s):  
Valeri Kolpakov ◽  
Maria Cristina D'Adamo ◽  
Lorena Salvatore ◽  
Concetta Amore ◽  
Alexander Mironov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Thrombus Formation ◽  
Cathepsin G ◽  
Arterial Segment ◽  
Dynamic Conditions ◽  
Activated Neutrophils ◽  
Scanning Electron

SummaryActivated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer.We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions.Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment.Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an “in vivo” model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential.This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.

scholarly journals Bioengineering of Improved Biomaterials Coatings for Extracorporeal Circulation Requires Extended Observation of Blood-Biomaterial Interaction under Flow

2007 ◽  
Vol 2007 ◽  
pp. 1-10 ◽  
Author(s):  
Kris N. J. Stevens ◽  
Yvette B. J. Aldenhoff ◽  
Frederik H. van der Veen ◽  
Jos G. Maessen ◽  
Leo H. Koole
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Thrombus Formation ◽  
Blood Parameters ◽  
Systematic Evaluation ◽  
Loop Model ◽  
Biomaterial Surfaces ◽  
Chandler Loop ◽  
Scanning Electron

Extended use of cardiopulmonary bypass (CPB) systems is often hampered by thrombus formation and infection. Part of these problems relates to imperfect hemocompatibility of the CPB circuitry. The engineering of biomaterial surfaces with genuine long-term hemocompatibility is essentially virgin territory in biomaterials science. For example, most experiments with the well-known Chandler loop model, for evaluation of blood-biomaterial interactions under flow, have been described for a maximum duration of 2 hours only. This study reports a systematic evaluation of two commercial CPB tubings, each with a hemocompatible coating, and one uncoated control. The experiments comprised (i) testing over 5 hours under flow, with human whole blood from 4 different donors; (ii) measurement of essential blood parameters of hemocompatibility; (iii) analysis of the luminal surfaces by scanning electron microscopy and thrombin generation time measurements. The dataset indicated differences in hemocompatibility of the tubings. Furthermore, it appeared that discrimination between biomaterial coatings can be made only after several hours of blood-biomaterial contact. Platelet counting, myeloperoxidase quantification, and scanning electron microscopy proved to be the most useful methods. These findings are believed to be relevant with respect to the bioengineering of extracorporeal devices that should function in contact with blood for extended time.

Scanning Electron Microscopy Study of Endothelial Injury and Thrombus Formation in WT and VWF Deficient Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3062-3062
Author(s):  
Justin Barr ◽  
Jennifer Barr ◽  
Marielle Meurice ◽  
David Motto
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Thrombus Formation ◽  
Endothelial Damage ◽  
Wild Type ◽  
Subsequent Formation ◽  
Time Points ◽  
Endothelial Surface ◽  
Deficient Mice ◽  
Scanning Electron

Abstract Abstract 3062 Poster Board II-1038 VWF is a large plasma glycoprotein required for normal hemostasis, and performs its function through binding to coagulation Factor VIII, and via interactions with both platelet surface glycoproteins and the activated and/or damaged vascular surface. We have developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation in wild-type and VWF-deficient mice. Thrombus formation is initiated by ferric chloride, and subsequently at defined time points, the circulation is rapidly flushed and aldehyde fixed. The carotid artery is removed, externally fixed, sectioned (both longitudinally and in cross-section), processed for SEM, and visualized. With this protocol we have obtained high-quality images (exceeding 100,000x) of FeCl3-induced endothelial damage and thrombus formation in C57BL/6 and VWF-deficient mice at baseline, and at 30, 60, 90, 120, 240, and 300 seconds post-injury (please access http://sites.google.com/site/mottolab/ to view images). Interestingly, we find that FeCl3 induces little, if any, endothelial denudation and collagen exposure at these time points, with the endothelium clearly appearing changed from baseline, but not damaged. Thus, initial platelet adhesion seems to be occurring in the absence of collagen exposure in this model. In wild-type mice, platelets adhere rapidly to the endothelial surface and assume a cross-linked appearance by 90 seconds, with continual inward growth of the thrombus through the 300 second time point. In VWF-deficient mice, platelets also adhere rapidly to the endothelial surface, but in contrast, remain recognizable longer without assuming a highly-activated phenotype. Compared with wild-type, at all time points examined the VWF-deficient thrombus appears smaller with considerably less cross-linking and platelet activation. Interestingly, during the course of these experiments we also have identified what appears to be red blood cells (RBCs) participating in thrombus formation. Similar to platelets, RBCs interact directly with the endothelial surface, and subsequently become elongated in the direction of blood flow. These elongated RBCs are often observed to cluster and bind platelets, with the subsequent formation of large platelet-erythrocyte complexes. Further characterization of these complexes and the role they may play in thrombus formation is currently in progress. Additionally, similar SEM studies are underway with both ADAMTS13-deficient and GPIb alpha-deficient mice, and with mice transiently expressing in vivo biotinylated VWF for visualization of this molecule at high magnification and resolution. These studies should help better define the mechanisms of endothelial activation and thrombus formation as they occur in situ. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelet Aggregation and Fibrin Deposition: The Yin and Yang of Thrombin Activity Regulation By Platelet Glycoprotein Ibα

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 114-114
Author(s):  
Alessandro Zarpellon ◽  
Patrizia Marchese ◽  
Antonella Zampolli ◽  
Grazia Loredana Mendolicchio ◽  
Zaverio M. Ruggeri
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Shear Stress ◽  
Carotid Artery ◽  
Platelet Activation ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Type I ◽  
Scanning Electron

Abstract Generation of α-thrombin (FIIa) in response to vascular injury is a key mechanism influencing thrombus formation. Platelet activation by FIIa is mediated by different protease activated receptors (PARs), although the most abundant FIIa binding site on platelets, but not a substrate for proteolysis, is glycoprotein (GP) Ibα in the GPIb-IX-V complex. The functional role of GPIbα in mediating/regulation thrombin functions relative to that of different PARs remains unclear. The goal of these studies was to define how binding to GPIbα can modulate FIIa functions. In mouse platelets we replaced endogenous GPIbα with either its human wild type counterpart (huGPIbα-WT) or with huGPIbα mutated at key residues involved in thrombin binding (D277N, Y276-8-9/F). Because these two mutations resulted in an undistinguishable phenotype, they are designated collectively as huGPIbα-Mut hereon. Mice expressing huGPIbα, WT or Mut, were evaluated in intravital models of arterial thrombosis induced by a ferric chloride-induced carotid artery lesion and venous thromboembolism induced by intravenous α-thrombin injection. Moreover, the blood of huGPIbα WT or Mut mice was also tested in an ex vivo model of thrombus formation upon perfusion over a thrombogenic surface under controlled flow conditions and platelets were evaluated for their responses to FIIa-induced activation. Mice expressing huGPIbα - WT or Mut - have comparable platelet counts and GPIbα surface density. Moreover, huGPIbα-WT platelets bind FIIa similarly than their normal human control counterpart, while huGPIbα-Mut platelets have essentially no detectable FIIa binding. Upon FIIa stimulation, which on mouse platelets is mediated by PAR4, aggregation and Ca2+ transients were significantly enhanced in huGPIbα-Mut as compared to huGPIbα-WT. In contrast, blocking FIIa binding to GPIbα on human platelets essentially abolished FIIa mediated activation, which in human occurs predominantly through PAR1. These results are compatible with the conclusion that, in mice, GPIbα is a competitive inhibitor of FIIa for PAR4-mediated functions. In the presence of metabolically inactive (PGE1 treated) huGPIbα-Mut washed platelets, the clotting time of a purified fibrinogen solution was significantly shorter when triggered by relative high concentration of FIIa (4 nM), but pronouncedly prolonged at a lower FIIa concentration (0.5 nM). Clot visualization showed a much more structured fibrin mesh in the presence of huGPIbα-WT platelets, which was lost with in the presence of huGPIbα-Mut platelets. Mutant mice tested in a model of carotid artery injury exhibited a pronounced prothrombotic phenotype, with a shorter time to occlusion. However they were protected from death induced by I.V. injection of α-thrombin. In ex vivo perfusion studies, the total volume of platelet aggregates formed in huGPIbα-Mut mouse blood exposed to acid-insoluble fibrillar collagen type I was slightly bigger than in huGPIbα-WT mice, but the number of thrombi was increased and their individual size smaller. These huGPIbα-Mut platelets exhibited clear signs of increased activation, as visualized by scanning electron microscopy (SEM). Strikingly, fibrin was almost totally absent in the huGPIbα-Mut thrombi. This was in striking contrast with what observed in huGPIbα-WT mice, in which the surface of platelet thrombi with directly and tightly connected with thick fibrin fibers as visualized by scanning electron microscopy. Possibly because of the reduced platelet membrane-fibrin fibril connection in huGPIbα-Mut platelets, these mice were significantly less susceptible to death when injected with an α-thrombin dose that caused 80% mortality plus in huGPIbα-WT mice. Thus, mice whose platelets have defective α-thrombin binding to GPIbα have a prothrombotic phenotype in high shear stress flow arteries and are protected from thromboembolic death in the low shear stress venous circulation. Our findings identify GPIbα as a relevant FIIa activity modulator in hemostasis and thrombosis through distinct and opposite mechanisms affecting platelet activation (The Yin) and fibrin formation (The Yang). Disclosures No relevant conflicts of interest to declare.

Surface morphology characterization of industrial polymers by low-voltage scanning electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 1036-1037
Author(s):  
V. K. Berry
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Low Voltage ◽  
Collection Efficiency ◽  
Steady Increase ◽  
Brightness Field ◽  
Morphology Characterization ◽  
Voltage Scanning ◽  
Scanning Electron

There has been an increase in awareness in low voltage scanning electron microscopy (LVSEM) of polymers in recent years because not only is it possible to use uncoated or very lightly coated (1-5nm) polymer samples but also due to an inherent advantage of limited beam damage of polymer samples by low energy beams. A steady increase in the use of low accelerating voltages for characterizing polymers has been made possible due to some major developments, such as high brightness field emission source, better lens and column design to minimize lens aberrations, and newer and improved detector systems with higher collection efficiency, incorporated by instrument manufacturers in their newer commercial models in recent years. Although there is still room for further improvements, the described instrumental changes have made possible the appreciation of LVSEM in characterization of polymers and non-conducting, beam sensitive materials.

Living Plants Continue to Grow After Examination by Scanning Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 372-373
Author(s):  
Arthur E. Sowers
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Time Interval ◽  
Stinging Nettle ◽  
Living Plant ◽  
Hydroponic Solution ◽  
Scanning Electron ◽  
Ground Part

It has been found that, within the constraints and limitations described below, the same individual living plant, Urtica pilulifera (common name - Stinging Nettle), continues to grow after repeated brief (6 minute) examination by conventional scanning electron microscopy. Fig. 1-3 show the simultaneous initiation and synchronous elongation of many epidermal hairs in the same area on the first internode of the same plant. The time interval between examinations was 24 hours.Seeds of Urtica pilulifera were germinated in vermiculite, the above ground part separated from the roots by a perpendicular cut with a razor and transferred to an hydroponic solution. Germination and transfer of the above ground parts were timed such that the plants were at a stage of morphogenesis shown in Fig. 4 at the start of the daily SEM examinations.

Scanning Electron Microscopy of the Normal Rabbit Hepatocyte

1975 ◽  
Vol 33 ◽  
pp. 520-521
Author(s):  
Waykin Nopanitaya ◽  
Joe W. Grisham
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Inferior Vena ◽  
Healthy Adult ◽  
Three Dimensional ◽  
Vena Cava ◽  
Tissue Preparation ◽  
Renal Veins ◽  
Scanning Electron ◽  
Made In

Current interest in the ultrastructure of the liver arises partly from demonstrations that scanning electron microscopy is very useful for studying surfaces and interrelationships of intrahepatic structures in both normal and pathological situations. In recent years, the development of the scanning electron microscope and techniques of tissue preparation has provided a three-dimensional method for examination of the features of intrahepatic ultrastructure. Only reports on the rat liver were available. However, SEM observations on the liver from other species are currently in progress in our laboratories. In this report, the SEM observations on the rabbit hepatocyte and the relevant technique for preparing hepatic tissues are described.Healthy, adult New Zealand rabbits were anesthetized by either intravenous or intraperitoneal injection of pentobarbital. Both abdominal and thoracic walls of anesthetized animals were opened. A small incision was made in the inferior vena cava at the level just below the renal veins, immediately followed by intracardiac perfusion of chilled 0.9% saline for 5 minutes.

Wall Sculpturing in Vessel Elements of Juglans Species of the United States

IAWA Journal ◽  
2008 ◽  
Vol 29 (2) ◽  
pp. 153-160
Author(s):  
Young Geun Eom ◽  
Ohkyung Kwon ◽  
Robert B. Hanna ◽  
Robert W. Meyer
Keyword(s):  
Electron Microscopy ◽  
United States ◽  
Scanning Electron Microscopy ◽  
Vessel Wall ◽  
The United States ◽  
Diagnostic Value ◽  
Black Walnut ◽  
Large Vessel ◽  
Vessel Elements ◽  
Scanning Electron

Wall sculpturing in the vessel elements of some Juglans species of the United States, four black walnuts (J. major, J. nigra, J. microcarpa (= J. rupestris), and J. californica) and one butternut (J. cinerea), was investigated through light and scanning electron microscopy. This feature showed various forms such as helical and reticulate thickenings, and thickening-like structures formed by coalescent pit apertures and large vessel to ray pits. Light to prominent wall sculpturing occurred regularly in black walnuts but fine and indistinctive wall sculpturing was observed rarely in butternut. Thus, vessel wall sculpturing was of some diagnostic value for separating black walnut from butternut.

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