Ultrastructural Study Of Experimental Venous Thrombosis

1981 ◽  
Author(s):  
D P Thomas ◽  
R E Merton ◽  
K F Hiller ◽  
D Hockley
Keyword(s):  
Vessel Wall ◽  
Direct Action ◽  
Venous Stasis ◽  
Endothelial Lining ◽  
Jugular Veins ◽  
Ultrastructural Studies ◽  
Platelet Deposition ◽  
Scanning Electronmicroscopy ◽  
Venous Segment

Venous stasis is known to be a necessary but usually not a sufficient cause of venous thrombogenesis. The nature of the additional factor(s) required is uncertain, but the two most likely candidates are local generation of thrombin and vessel wall damage. Autopsy studies show that most venous thrombi develop in apparently normal vessels, although this evidence is based primarily on light microscopy. If thrombin generation is the additional factor, it is uncertain whether such thrombin acts solely on the blood or also damages the endothelium, leading to platelet deposition on exposed subendothelium. We studied the effect of locally injected thrombin on the jugular veins of rabbits, using transmission and scanning electronmicroscopy. One unit of thrombin was sufficient to clot blood contained in an isolated venous segment within a few minutes. The thrombus so formed was then embolised and autologous 11 indium-labelled platelets were injected. Blood flow was re-established for 30 minutes, following which the venous segment was fixed in situ and removed for study.We found no evidence of significant vessel wall damage, as judged by ultrastructural studies or the deposition of labelled platelets. At least 10 u. of injected thrombin were required before the radioactivity in the thrombosed segment exceeded that found in a control vein. Even then, the endothelial lining appeared intact by electronmicroscopy. Following administration of aspirin (10 mg/kg) there was a marked increase in radioactivity present in the thrombosed venous segment, suggesting that inhibition of vessel wall cyclo-oxygenase had led to increased platelet deposition. We conclude that a fresh stasis thrombus resulting from the direct action of thrombin on platelets and fibrinogen does not damage the endothelium, which appears to be relatively resistant in vivo to even high local concentrations of thrombin.

The Effects of Heparin and Annexin V on Fibrin Accretion after Injury in the Jugular Veins of Rabbits

1993 ◽  
Vol 69 (03) ◽  
pp. 227-230 ◽  
Author(s):  
J Van Ryn-McKenna ◽  
H Merk ◽  
T H Müller ◽  
M R Buchanan ◽  
W G Eisert
Keyword(s):  
Vessel Wall ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Specific Effect ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Jugular Veins ◽  
Prothrombinase Complex ◽  
Wall Injury

SummaryWe compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely deendothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p <0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins. We conclude that, i) mild vessel wall injury (selective de-endothelialization) in veins results in a thrombogenic vessel wall; ii) the thrombogenecity of which is not inhibited by prophylactic doses of heparin; but iii) is inhibited by annexin V, which binds to injured vessel wall surface, and inhibits thrombin generation independently of antithrombin III.

The Role of Prostaglandins in Platelet-Vessel wall Interactions

1979 ◽  
Author(s):  
R.H. Boutgain ◽  
F. Six ◽  
P. Potvliege
Keyword(s):  
Vessel Wall ◽  
In Vivo Model ◽  
Cell Contact ◽  
Synthetase Activity ◽  
Endothelial Lining ◽  
Electron Microscopic ◽  
Cell Hypertrophy ◽  
Microscopic Studies ◽  
Wall Interaction

In an in vivo model developed for the study of arterial thrombosis in the white Wistarrat, it was demonstrated that superfusion of the investigated arterial segment with cyclo oxygenase inhibitors (ASA, indomethacin, flurbiprofen) resulted in a marked decrease of ADP-induced thrombosis while tranylcypromine enhanced the thrombotic phenomenon. As other MAO inhibitors were without effect, it was concluded that tranylcypromine possibly decrsibsed the PGE2-synthetase activity of the vessel wall. In the light of these findings we then suggested that the platel et-vessel wall interaction was dependent at least to some extent on the cyclic endoperoxides-PGI2 ratio of the endothelium and eventually of the deeper vascular structures. Electron microscopic studies on arterial segments used in our model revealed that suloctidil markedly decreases smooth muscle cell hypertrophy following local deendothelialization. Furthermore reconstitution of the endothelial lining consisting in endothelial cell hypertrophy and sliding over the dcendothelialized are until cell contact is made was also delayed. Following the intravenous administration of suloctidil (0.5 mg/kg) in our model the thrombus enhancing effect of locally superfused tranylcypromine was Offset. These experiments suggest that a decreased PGI2-synthetase activity is inhibited by a platelet function active drug such as suloctidil.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Osseointegration interface of calcified bone and endosseous implants

1992 ◽  
Vol 50 (2) ◽  
pp. 1096-1097
Author(s):  
K.E. Krizan ◽  
J.E. Laffoon ◽  
M.J. Buckley
Keyword(s):  
Structure And Function ◽  
Titanium Implants ◽  
En Bloc ◽  
Endosseous Implants ◽  
Ultrastructural Studies ◽  
The Past ◽  
Cacodylate Buffer ◽  
One Year ◽  
And Function

With increase use of tissue-integrated prostheses in recent years it is a goal to understand what is happening at the interface between haversion bone and bulk metal. This study uses electron microscopy (EM) techniques to establish parameters for osseointegration (structure and function between bone and nonload-carrying implants) in an animal model. In the past the interface has been evaluated extensively with light microscopy methods. Today researchers are using the EM for ultrastructural studies of the bone tissue and implant responses to an in vivo environment. Under general anesthesia nine adult mongrel dogs received three Brånemark (Nobelpharma) 3.75 × 7 mm titanium implants surgical placed in their left zygomatic arch. After a one year healing period the animals were injected with a routine bone marker (oxytetracycline), euthanized and perfused via aortic cannulation with 3% glutaraldehyde in 0.1M cacodylate buffer pH 7.2. Implants were retrieved en bloc, harvest radiographs made (Fig. 1), and routinely embedded in plastic. Tissue and implants were cut into 300 micron thick wafers, longitudinally to the implant with an Isomet saw and diamond wafering blade [Beuhler] until the center of the implant was reached.

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

1990 ◽  
Vol 63 (02) ◽  
pp. 220-223 ◽  
Author(s):  
J Hauptmann ◽  
B Kaiser ◽  
G Nowak ◽  
J Stürzebecher ◽  
F Markwardt
Keyword(s):  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Clotting Time ◽  
Thrombosis Model ◽  
Activity Factor ◽  
Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

An Electron Microscopic Study of Platelet Thrombus Formation in the Rabbit with Particular Regard to 5-Hydroxytryptamine Release

1967 ◽  
Vol 18 (03/04) ◽  
pp. 592-604 ◽  
Author(s):  
H. R Baumgartner ◽  
J. P Tranzer ◽  
A Studer
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Endothelial Damage ◽  
Electron Microscopic ◽  
Rabbit Ear ◽  
Basement Lamina ◽  
Platelet Thrombus

SummaryElectron microscopic and histologic examination of rabbit ear vein segments 4 and 30 min after slight endothelial damage have yielded the following findings :1. Platelets do not adhere to damaged endothelial cells.2. If the vessel wall is denuded of the whole endothelial cell, platelets adhere to the intimai basement lamina as do endothelial cells.3. The distance between adherent platelets as well as endothelial cells and intimai basement lamina measures 10 to 20 mµ, whereas the distance between aggregated platelets is 30 to 60 mµ.4. 5-hydroxytryptamine (5-HT) is released from platelets during viscous metamorphosis at least in part as 5-HT organelles.It should be noted that the presence of collagen fibers is not necessary for platelet thrombus formation in vivo.

A New Model for Quantitative In Vivo Microscopic Analysis of Thrombus Formation and Vascular Recanalisation: The Ear of the Hairless (hr/hr) Mouse

1997 ◽  
Vol 78 (05) ◽  
pp. 1408-1414 ◽  
Author(s):  
Frank Roesken ◽  
Martin Ruecker ◽  
Brigitte Vollmar ◽  
Nicole Boeckel ◽  
Eberhard Morgenstern ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Light Exposure ◽  
Thrombus Formation ◽  
Microscopic Analysis ◽  
Cell Interaction ◽  
Vascular Perfusion ◽  
Vessel Occlusion ◽  
Endothelial Cell Interaction ◽  
Platelet Deposition

SummaryThe alteration of rheological blood properties as well as deterioration of vascular perfusion conditions and cell-cell interactions are major determinants of thrombus formation. Herein, we present an experimental model which allows for quantitative in vivo microscopic analysis of these determinants during both thrombus formation and vascular recanalisation. The model does not require surgical preparation procedures, and enables for repeated analysis of identical microvessels over time periods of days or months, respectively. After i.v. administration of FITC-dextran thrombus formation was induced photochemically by light exposure to individual arterioles and venules of the ear of ten anaesthetised hairless mice. In venules, epiillumination induced rapid thrombus formation with first platelet deposition after 0.59 ± 0.04 min and complete vessel occlusion within 7.48 ±1.31 min. After a 24-h time period, 75% of the thrombosed venules were found recanalised. Marked leukocyte-endothelial cell interaction in those venules indicated persistent endothelial cell activation and/or injury, even after an observation period of 7 days. In arterioles, epi-illumination provoked vasomotion, while thrombus formation was significantly (p <0.05) delayed with first platelet deposition after 2.32 ± 0.22 min and complete vessel occlusion within 20.07 ±3.84 min. Strikingly, only one of the investigated arterioles was found recanalised after 24 h, which, however, did not show leukocyte-endothelial cell interaction. Heparin (300 U/kg, i.v.) effectively counteracted the process of thrombus formation in this model, including both first platelet deposition and vessel occlusion. We conclude that the model of the ear of the hairless mouse allows for distinct in vivo analysis of arteriolar and venular thrombus formation/ recanalisation, and, thus, represents an interesting tool for the study of novel antithrombotic and thrombolytic strategies, respectively.

Thrombogenicity of the Injured Vessel Wall – Role of Antithrombin and Heparin

1994 ◽  
Vol 71 (01) ◽  
pp. 147-153 ◽  
Author(s):  
Siw Frebelius ◽  
Ulf Hedin ◽  
Jesper Swedenborg
Keyword(s):  
Vessel Wall ◽  
Antithrombin Iii ◽  
Rabbit Aorta ◽  
Continuous Treatment ◽  
Balloon Injury ◽  
Coagulant Activity ◽  
Risk For Thrombosis ◽  
Inhibition Of Thrombin

SummaryThe thrombogenicity of the vessel wall after endothelial denudation is partly explained by an impaired inhibition of thrombin on the subendothelium. We have previously reported that thrombin coagulant activity can be detected on the vessel wall after balloon injury in vivo. The glycosaminoglycans of the subendothelium differ from those of the endothelium and have a lower catalyzing effect on antithrombin III, but inhibition of thrombin can still be augmented by addition of antithrombin III to the injured vessel surface.In this study the effect of antithrombin III and heparin on thrombin coagulant activity on the vessel wall was studied after in vivo balloon injury of the rabbit aorta using biochemical and immunohistochemical methods and thrombin was analysed after excision of the vessel. Continuous treatment with heparin, lasting until sacrifice of the animal, or treatment with antithrombin III resulted in significant reduction of thrombin coagulant activity on the injured aorta. Heparin given only in conjunction with the injury did not prevent thrombin coagulant activity or deposition of fibrin on the surface.The capacity of the injured vessel wall to inhibit thrombin in vitro was improved on aortic segments obtained from animals receiving antithrombin III but not from those given heparin. It is concluded that treatment with antithrombin III interferes with thrombin appearance on the vessel wall after injury and thereby reduces the risk for thrombosis.

scholarly journals Signaling via β2 Integrins Triggers Neutrophil-Dependent Alteration in Endothelial Barrier Function

2000 ◽  
Vol 191 (11) ◽  
pp. 1829-1840 ◽  
Author(s):  
Narinder Gautam ◽  
Heiko Herwald ◽  
Per Hedqvist ◽  
Lennart Lindbom
Keyword(s):  
Protein S ◽  
Endothelial Barrier ◽  
Cheek Pouch ◽  
Cross Linking ◽  
Endothelial Lining ◽  
Hamster Cheek Pouch ◽  
Cationic Protein ◽  
Edema Formation ◽  
Β2 Integrins

Activation of polymorphonuclear leukocytes (PMNs) and adhesion to the endothelial lining is a major cause of edema formation. Although known to be dependent on the function of β2 integrins (CD11/CD18), the precise mechanisms by which adherent PMNs may impair endothelial barrier capacity remain unclear. Here, the role of transmembrane signaling by β2 integrins in PMN-induced alterations in tight junctional permeability of cultured endothelial cell (EC) monolayers was investigated. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD11b/CD18, mimicking adhesion-dependent receptor engagement. CD18 cross-linking in PMNs added to the EC monolayer provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca2+ and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cell-free supernatant obtained after CD18 cross-linking in suspended PMNs triggered an EC response indistinguishable from that induced by direct PMN activation, and caused clear-cut venular plasma leakage when added to the hamster cheek pouch in vivo preparation. The PMN-evoked EC response was specific to β2 integrin engagement inasmuch as antibody cross-linking of l-selectin or CD44 was without effect on EC function. Our data demonstrate a causal link between outside-in signaling by β2 integrins and the capacity of PMNs to induce alterations in vascular permeability, and suggest a paracrine mechanism that involves PMN-derived cationic protein(s) in the cellular crosstalk between PMNs and ECs.

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