Effects of external electrical stimulation on laser-beam-induced experimental thrombosis.

External electrical stimulation (EES) has demonstrated venous antithrombotic properties. The aim of this investigation was to study its antithrombotic properties using unfractionated heparin (UFH) as a reference in arterial and venous mesenteric microvessels. For this purpose a rat model of laser-beam-induced thrombosis was used. Four groups of rats were investigated (n = 7): control, UFH (5 mg /kg), UFH (2.5 mg/kg) and electrical stimulation [e-vascular, E = 10, alternate polarity, in two protocols: (i) 30 min before and during thrombosis induction and (ii) only 30 min before thrombosis induction as preventive stimulation]. This effect was tested in arterial and venous microvessels in the four groups using the continuous protocol and only in arterial microvessels using the preventive protocol. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli and the duration of embolization. Induced hemorrhagic time (IHT), platelet aggregation induced by ADP and coagulation tests (activated partial thromboplastin time, aPTT, prothrombin time, pTT, and fibrinogen level) were also determined. Continuous electrical stimulation decreased the number of emboli and the duration of embolization both in arterioles and in venules. It also significantly reduced the amplitude and velocity of the ex vivo platelet aggregation induced by ADP. Contrary to UFH, neither aPTT nor pTT were affected in platelet-poor plasma collected after the induction of thrombosis by laser beam, nor did it modify IHT. Preventive electrical stimulation produced similar results with fewer effects on induced venous thrombosis. These data suggest that EES has a potent antithrombotic effect on arterial and venous thrombosis without any hemorrhagic adverse effects, making it a very promising tool in the prevention and treatment of venous and arterial thromboembolic complications.

Increase of arterial thrombosis parameters in chronic Helicobacter pylori infection in mice.

An animal model was developed to study arterial thrombosis and determine if animals infected with Helicobacter pylori behave differently after induction of direct damage to blood vessels. Twenty-one C56/BL6 mice inoculated with the "Sydney strain" of H. pylori and 19 uninfected animals were kept for 1 year before testing. Vascular lesions were induced to mesenteric arterioles (15-25 microm diameter) by Argon laser. The dynamic course of thrombus formation was continuously monitored by a video camera for 10 min. Three parameters were assessed: (1) the number of laser pulses required to induce thrombus formation, (2) the number of platelet emboli removed by the blood flow and, (3) the duration of embolization. Additionally, blood was tested for platelet aggregation, fibrinogen, and cell count. Of the parameters measured, statistical differences between infected and uninfected mice concerned the number of emboli formed (6.00+/-2.18 infected vs. 3.89+/-1.37 non-infected, p=.0006) and the duration of embolization (2.41+/-0.73 min infected vs. 1.47+/-0.61 min non-infected p>.0001). A significant difference was also found in the fibrinogen levels between infected and uninfected mice. Chronic infection of mice with H. pylori leads to increased platelet embolization after damage to arterioles. These results are in favor of the possible involvement of H. pylori infection in the acute phase of coronary heart disease (CHD).

Thrombogenic potential of contrast media in an experimental model of laser-induced thrombosis.

Controversy still exists about the pro- or antithrombotic side effects of contrast media used in daily medical practice. Recent reports have shown that various contrast media, including ionic compounds, have deleterious prothrombotic actions. A new evaluation of these adverse side effects is reported here, with the study of the dose-effect relationship. Two ionic (ioxaglate and diatrizoate) and two non-ionic contrast media (iopamidol and iohexol) were studied. Experiments were done on 22 groups of 5 Wistar male rats each, using a Laser Argon-induced thrombosis model in mesenteric microvessels. Three parameters were studied: the number of laser beams needed to induce platelet thrombus formation, the number of emboli, and the duration of embolization. Platelet count and platelet aggregation also were determined. Iopamidol and iohexol induced a significant rise in both the number of emboli and the duration of embolization in mesenteric microvessels at doses up to 1 mL/kg. Ioxaglate and diatrizoate also significantly increased these parameters at doses up to 2 mL/kg. All the products tested decreased platelet count, inducing a -17 to -30% variation from control values. Diatrizoate and ioxaglate inhibited platelet aggregation, while iopamidol and Iohexol behaved as activators. All non-ionic, and to a lesser extent, all ionic contrast media demonstrated prothrombotic properties.

Aspirin and fraxiparine in the prevention of laser induced thrombosis in the presence of iodinated contrast media.

The purpose of this study was to investigate the thromboembolic properties of ionic and nonionic contrast media in rats pretreated with aspirin and/or fraxiparine using an experimental model of laser induced thrombosis in the mesenteric microvessels of 17 groups of five male Wistar rats each. Two ionic (ioxaglate and diatrizoate) and two nonionic contrast media (iopamidol and iohexol), alone or associated with antithrombotic drugs (aspirin and/or fraxiparine) were studied. To evaluate the effects of these substances in this model, the number of laser beams needed to induce platelet thrombus formation, the number of emboli detached from the thrombus and the duration of embolization were quantified. Platelet aggregation induced by ADP, induced hemorrhagic time (IHT) and haemoglobin loss level were also determined. Both contrast media injected at 3 ml/kg caused a significant increase in the number of emboli and the duration of embolization (p<0.05). Pretreatment with aspirin and/or fraxiparine in the presence of ionic contrast media showed antithrombotic activities equal to those obtained when they were tested alone (p<0.05), while in the presence of nonionic contrast media, these drugs only neutralised the prothrombotic effects. There were no differences with the NaCl treated group (p>0.05). The ionic contrast media, and to a lesser extent the nonionic contrast medium: iohexol, inhibited platelet aggregation, while iopamidol behaved as an activator. The antithrombotic drugs tested in this study prevent the prothrombotic activities of contrast media therefore suggesting their use before radiographic procedures.

Combination of two doses of acetyl salicylic acid: experimental study of arterial thrombosis.

The antithrombotic effect of high dose acetylsalicylic acid is well known, and recently, in vitro studies hinted the potent thrombotic effect of ultra-low dose of acetylsalicylic acid (<1mg/day) showing a significant decrease in bleeding time. In this study, we investigated the effect of a combination between a high and an ultra-low dosage (100 mg/kg+ 10(-30) mg/kg) on an arterial thrombosis induced by a laser beam. We used an intravital microscopic technique, allowing to evaluate (anti)-thromboembolic events at previously determined locations of microvasculature. Thrombus formation was induced by argon-laser shot. The instrumental test setup was completed with a video system, to select mesenteric arterioles with the same diameter (between 15 and 25 microm). The changes in platelet aggregability were determined by Cardinal and Flower method, and the concentration of acetylsalicylic acid in the plasma was measured by high pressure liquid chromatography. Antithrombotic effect of high dose (100 mg/kg) acetylsalicylic acid was confirmed in all results obtained. Asa injected at ultra-low dose (10(-30) mg/kg) had a potent thrombotic properties and decreased significantly the bleeding time. The subcutaneous administration of the combination of the two doses permitted to come back to the control values, and the bleeding time was shortened compared to control group.

Fibrinogen as a factor of thrombosis: experimental study.

Epidemiological, clinical, and experimental studies have clearly demonstrated the strong association between baseline fibrinogen level and risk of thromboembolic complications. The pathogenesis of postoperative or post-traumatic thrombosis in man is associated with fibrinogen level in plasma. The purpose of this study was to evaluate the effects of fibrinogen administration on thrombus formation at different dosages. To investigate these effects, we used an experimental model of induced thrombosis in rat microcirculation. This model allows single endothelial cell destruction by laser injuries, thus leading to thrombus formation. Fibrinogen was injected intravenously via penis vein and tested at various dosages (50, 100, and 200 mg/kg), 60 minutes after injection on arterial thrombosis induction and 120 minutes after injection on venous thrombosis induction. Results showed that the administration of fibrinogen increases the number of emboli, the duration of embolization, the amplitude, and the velocity of the ex-vivo platelet aggregation induced by ADP (p < 0.05). A positive correlation between the percent of fibrinogen increase in plasma and the enhancement of thromboembolic risk in the experimented animals was observed.

Thromboembolic complications several days after a single-dose administration of aspirin.

The antithrombotic properties of acetyl salicylic acid (ASA) used at current doses are largely demonstrated. However, our previous study showed unexpected thrombotic potencies associated with the use of this drug. In this study we investigate the effect of aspirin on an experimental thrombosis induced by laser beams, according to its in vivo plasma concentration. Experiments were done on nine groups of seven Wistar male rats. The groups are defined by the delay between aspirin administration time and the laser-induced thrombosis time. Results from this study showed an enhancement of thromboembolic complications when thrombosis was induced 8 or 10 days after aspirin administration; the number of emboli and the duration of embolization are increased, compared to the control group. The prothrombotic properties of ASA demonstrated in this study, might limit its therapeutic benefit and might explain thromboembolic complications observed in some ASA-treated patients. These results also suggest a biological monitoring several days after aspirin administration to patients.

Action of neurotransmitters: acetylcholine, serotonin, and adrenaline in an experimental arterial thrombosis induced by oxygen free radicals.

It is well known that high stress and particularly an enhancement of plasma catecholamines and myocardial infarction have a close relation. In addition, adrenaline is presented as a prothrombogenic agent in vivo. The role of the other agents such as serotonin or acetylcholine, in the development of arterial thrombosis is somewhat uncertain, although, the role of each of them is often considered at the level of vascular regulation only. Therefore, the present study was designed to investigate the effects of three neurotransmitters on experimental arterial thrombosis model induced by generation of free radicals. The results demonstrate that intravenously injection of adrenaline or serotonin (1 ng/kg) stimulated arterial thrombosis formation, whereas injection of high dose of acetylcholine (5 mg/kg) slackened the thrombosis formation.

Action of neurotransmitters: acetylcholine, adrenaline and serotonin on arterial thrombosis induced by a laser beam.

The releasing of catecholamines is increased in stress situation which promotes the formation of circulating platelet aggregates, and could participate in the arterial thrombosis formation in coronary diseases. The purpose of this study is to evaluate the thrombogenic action of some neurotransmitters, and their participation through the vessel's vasomotoricity, in the growth of an arteriolar thrombosis. Endothelial cells destruction, induced by a laser beam in mesenteric arteriole of the rat were observed to determine changes in thrombus growth, through the embolization and variation of vessel diameter. It is desirable to get insight into the interrelation of thrombus formation and local vasomotoricity in the presence of acetylcholine, adrenaline and serotonin. The administration of acetylcholine (5mg/kg) increases the number of emboli which detached from thrombus, and decreases the thrombus area. Therefore, acetylcholine induces a variation of the vessel's diameter, a vasodilation in the intact vessel and a vasoconstriction when the endothelium is removed. Two vasoconstrictor agents are used: adrenaline and serotonin which increase the number of laser injuries required to induce thrombus formation, decrease the number of emboli and the duration of embolization (p < 0.05). They cause a potent vasoconstriction. These neurotransmitters seem to be involved in the arterial thrombosis induced by laser beam, promoting or not the platelet aggregation, and modulating the vascular tone by the endothelium.

New class of heparin derivatives with a potent antithrombotic effect and a very limited hemorrhagic activity.

Eight heparin derivatives (HD1 to HD8) were prepared by mixing various doses of protamine with a fixed amount of heparin. After centrifugation and elimination of the formed complex, the supernatant was lyophilized and titrated. A dose of 5 mg (dry weight)/kg of these heparin derivatives was injected subcutaneously to rats in classical thrombosis model induced by stasis. Antithrombotic, hemorrhagic and anticoagulant activities are investigated and compared to those of Unfractionated Heparin (UFH) and LMWH (Enoxaparin). Seven rats in each group were studied. Significant antithrombic effect was exhibited by HD1, HD2, HD3, and HD4 which decreased progressively. Only HD1, HD2, and HD3 augmented hemorrhagic activity but to a lower degree than UFH and LMWH. No change was observed by coagulation assays; Activated Partial Thromboplastin Time (APTT) and Diluted Thrombin Time (dTT) and there was no effect on platelet aggregation except for UFH. These heparin derivatives might present advantages over UFH and LMWH in the treatment of thrombosis.

Antithrombotic effects of aspirin and LMWH in a laser-induced model of arterials and venous thrombosis.

Antiplatelet drug aspirin and anticoagulant low molecular weight heparin (LMWH) were compared as arterial and venous antithrombotic preparations in the rat experimental model of the laser induced thrombus formation. A method to induce microthrombi in small mesenteric vessel (15-25 microns) has been developed to investigate antithrombotic drugs and to study platelet reactions. Mesenteric injuries are induced in the vascular system of Wistar rats with an argon laser. The laser beam induced formation of the vessel wall injury with damage of endothelial cells. Thrombus was formed within seconds after laser injury and grew rapidly. The aggregate can be swept away by the flow and a new thrombus was formed again. This embolization began within the minute following the laser flash. Thrombus formation and embolization were repetitive phenoma. Aspirin (100 mg/kg) and LMWH (1 mg/kg) are approximately the same as to decrease the number of emboli detached from the thrombus and the duration of embolization; both in venules and in arterioles. This results suggest reflexion about the role of platelets in venous thrombosis induced by laser beam.

Effect of monoclonal and polyclonal antibodies against conjugated neurotransmitter on experimental venous thrombosis.

Vasomotoricity in veins is largely controlled by the sympathetic nervous system. Norepinephrine and acetylcholine modulate the vasoconstrictor effect mediated by alpha-adrenergic receptors. Our objective was to study the role of local vasomotoricity in venous thrombosis, and particularly to determine some of the factors regulating it. Although no attempt has been described until now, polyclonal and monoclonal antibodies against L-dihydroxyphenylalanine, acetylcholine, and dopamine were administered in an experimental model of venous thrombosis. In view of the rather high specific antibody recognition for each compound, the presence of endogenous epitopes of L-dopa-like molecules and acetylcholine-like molecules in the circulation can be postulated. The results of this study may open an important new approach in the treatment of vascular diseases.

The arterial antithrombotic activity of thioxylosides in a rat model of laser-induced thrombosis.

beta-D xylosides have been shown to have venous antithrombotic properties after simple oral administration. Therefore, the arterial antithrombotic effect of these compounds was investigated in vivo, using the experimental thrombosis model induced by laser injury. The products tested were administered orally, 4 h before the thrombosis induction. Two beta-D xylosides were tested (LF 09-0055 and LP 05-0030), either after a simple oral administration at 50, 100, 200, and 400 mg/kg, or after repetitive oral administration at 200 mg/kg twice daily during 5 days. These compounds increased significantly the number of laser shots required to induce arterial thrombosis and decreased the number of emboli and the duration of embolization. At single-dose or repeated administrations, these xylosides did not affect diluted thrombin time in platelet-poor plasma collected after thrombosis inductions. They induced a dermatan sulfate-like activity in the plasma of treated rats, as measured by heparin cofactor II-mediated thrombin inhibition assay. These data suggest that these xylosides are potent arterial antithrombotic agents after single or repetitive oral administrations. beta-D xylosides constitute a very promising therapeutic class of orally active antithrombotic drugs.

Experimental thrombosis model induced by free radicals. Application to aspirin and other different substances.

A large number of experimental studies suggests that oxygen free radicals play a major role in the pathogenesis of the myocardial lesions observed during the sequence ischemia-reperfusion. The purpose of this study was to determine whether oxygen free radicals can induce thrombosis. In so doing we have developed a new experimental thrombosis model. Reproducible focal thrombosis has been achieved by irradiating mesenteric arterioles of rat for variable time with green filtered light issuing from a mercury lamp after systemic injection of different rose bengal doses. The number of emboli that remove in the blood (N), the duration of total occlusion (T) and the number of emboli per minute were then measured. As control, no rose bengal administration was done and the vessels were exposed to the filtered light. In comparison with this control, results clearly showed that free radicals always induced thrombosis and the induced thrombus was mainly composed of platelets. In this new thrombosis model induced by free radicals antithrombotic drugs (aspirin, 200 mg/Kg, heparin, 2 mg/Kg) and antioxidants (vitamin C, 10 and 20 mg/Kg, allopurinol, 200 and 300 mg/Kg, vitamin E, 500 and 1000 mg/Kg) have been tested. Results have shown that only heparin and vitamin E had an antithrombotic effect on thrombus formation induced by free radicals. This model should be useful in studying the effects of different drugs and could lead to new treatment modalities for ischemic accident and other cardiovascular diseases.

[Experimental models of venous thrombosis]. / Les modèles expérimentaux de thrombose veineuse.

Various experimental models have been developed in order to more clearly understand deep vein thrombosis, the mechanisms involved and its treatment. These models are based on venous stasis, either alone or combined with the injection of thrombogenic substances or endothelial lesions. Other models only use endothelial lesions. Thrombogenic substances are mostly composed of activated factor X or thrombin, which raises the problem of purity of the substances and determination of the antithrombotic activities of the substance tested, especially heparin and hirudin and their derivatives, and consequently their efficacy. Endothelial lesions can be induced by chemical, physical or electrical agents or by repeated application of clamps, or cellular crushing. These models result in the formation of various forms of venous thrombus. The development and improvement of experimental models is very important in every case. Experimental models of thrombosis constitute the best tool for the study of thrombosis, in which many points remain to be elucidated. They also allow the study and development of various antithrombotic substances the improvement of their efficacy. These models must be validated, standardized, reproductible and in agreement with local legislation in each country.

[Experimental models of arterial thrombosis]. / Modèles expérimentaux de thrombose artérielle.

Arterial thrombosis is clearly responsible for a very wide range of cardiovascular diseases, which is why many models of arterial thrombosis have been developed. These models are based on various techniques such as electrical, mechanical, biochemical, photochemical induction. They are an essential prerequisite to the understanding of molecular and cellular phenomena and are also essential to test the antithrombotic activity of new molecules before the first clinical trials in man. The large range of models means that the most appropriate model can be selected for the study of the test substance. However, the antithrombotic activity of a substance needs to be studied by several models. Most of these models induce total occlusion in which the number of parameters studied is limited. As a result of technological progress and the development of techniques such as laser and image analysers, new perspectives are now available for both basic and pharmacological research. Consequently, the already major value for these models should continue to grow over the years to come.

Thrombogenicity of ionic and nonionic contrast media tested in a laser induced rat thrombosis model.

Contrast media are used as substances for visualization of vascular system. But, their administration is often associated with thromboembolic complications. The purpose of this study is to evaluate the thrombogenic action of ionic and non-ionic contrast media on thrombus formation. The experimental destruction of endothelial cells by Laser injury leads to thrombus and emboli formation. Two ionic and two non-ionic contrast media were injected intravenously via penis vein and tested at various dosages (1.0 and 2.5 ml/kg) 5, 30, 45 and 65 minutes after injection. The administration of these contrast media decreases the number of Laser injuries required to induce thrombus formation, increases the number of emboli which detached from thrombus and prolongs duration of embolization (p < or = 0.05). These experimental results suggest that ionic and non-ionic contrast media induce thrombogenic effects. This thrombogenicity was the greatest for non-ionic contrast media. It was observed the decrease of the white cells, red cells and platelets.

Effect of the low molecular weight heparin/non steroidal anti-inflammatory drugs association on an experimental thrombosis induced by laser.

LMWH (Fraxiparine), and NSAIDs (Aspirin, Feldene, Indocid and Profenid) injected together in doses, 1 mg/kg (Aspirin was used at 100 mg/kg), subcutaneously into rats 30 minutes before the thrombosis induction by LASER beams, increased the number of LASER beams required to induce platelet thrombus formation, decreased the number of emboli and reduced the duration of embolization, compared with control (p < or = 0.05). Of all the studied NSAIDs being injected either with LMWH or separately 30 minutes before the thrombosis stimulation by LASER only Aspirin appeared to potentiate the antithrombotic effect of Fraxiparine. Neither LMWH nor NSAIDs (except for Aspirin) at the dosages used modified aggregatory parameters compared with control. But it was observed the inhibition of platelet aggregation by the associations of Fraxiparine with Aspirin, Feldene or Profenid, tested in the whole blood 90 minutes after the drug injections.