Experimental venous thrombosis induced by homologous serum in the rat

Thrombosis

Pergamon

Research, Vol. 81, No. 4, pp. 497-502, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in the USA. All rights reserved 0049-3848/96 $I 2.00 + .Oll

PII SOO49-3848(96)00023-O

BRIEF COMMUNICATION EXPERIMENTAL

VENOUS

THROMBOSIS INDUCED IN THE RAT

BY HOMOLOGOUS

SERUM

Jean Millet, Michel Vaillot, Jocelyne Theveniaux and Neil L. Brown Recherche et Developpement, Laboratoires Foumier SCA, 21121 Daix, France. (Received 6 June 1995by Editor B. 0sterud; revised/accepted 28 December 1996)

To investigate the effects of new drugs on the prevention of venous thrombosis, the Wessler stasis model (1) is extensively employed usually in the rabbit (2,3,4). In this model, a hypercoagulable state is achieved by the administration of either heterologous serum (often human serum) or an activated coagulation factor (see 3) followed by total stasis of the vein in which the thrombus is to be formed. Although classically the stasis model is performed in rabbits, it is possible to induce experimental venous thrombosis in a number of animal species, including rats (5,6,7,8). Howerever, the use of homologous serum as the activating or thrombogenic agent has been previously described only in the dog (9) and in the rabbit (3). The purpose of the present study was to compare the activities of heterologous and homologous serum as hypercoagulating agents in a rat stasis model of venous thrombosis using Wessler’s technique. Heparin was employed as a reference antithrombotic compound in order to compare the two thrombogenic challenges. MATERIALS

AND METHODS

Animals

The studies were performed on male Futth Wistar and Wistar rats (260-290 g) obtained from Iffa Credo (St Germain/L’Arbresle, France). Animals were used one week after quarantine, during which they were maintained at 20 f 2°C with free access to food and water. Serum preparation

Whole blood samples were obtained from either healthy human volunteers (aged 3040 years and free from medication for at least one week) by venepuncture of an antecubital vein or from the abdominal aorta of anaesthetized (12 % urethane, 10 ml/kg, intraperitoneal, i.p., injection) rats. Blood samples were collected directly into glass tubes and incubated at 37°C for one hour. The clots and their exudates were then poured into polystyrene hemolysis tubes and centrifuged at 3000 g for 10 min at 20°C. The serum of each species was pooled, aliquoted into 1 ml vdumes and immediately frozen at -70°C. The serum samples were thawed for 10 min at 37°C (water bath) before use. Corresponding author : Dr Jean Millet, Laboratoires Fournier, Developpement, BP 90, 50 rue de Dijon, 21121 Daix, France Key words: venous thrombosis; homologous serum; rat; stasis 497

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Experimental model The technique employed was a modification of the stasis model described by Wessler and adapted for the rat (see 10). Following anaesthesia (12 % urethane, 10 ml/kg, i.p.) and mid-abdominal laparotomy, a segment of the inferior vena cava was isolated between the left renal vein and the iliac bifurcation. All collateral vessels were carefully ligatured. Two cotton threads, 2 cm apart were placed around the vena cava and loosely tied. Various volumes of serum (human or rat) were injected via the penile vein and 30 set later the two ligatures around the vena cava were tightened. The stasis was maintained for 10 min after which, the venous segment was opened and the thrombus removed, blotted of excess blood and weighed. Experimental design In an initial study, thrombi wet weights were correlated against dry weights. A number (n=47) of formed thrombi were weighed fresh (wet weight) and then dried to constant weight (dry weight). In a first experimental group, one hundred rats were divided into two groups of 50 animals each, receiving either heterologous (human) or homologous (Wistar rat) serum as the thrombogenic agent. Within both groups and 5 minutes prior to their thrombogenic challenge, 10 animals in each groups were pretreated, by i.v. injection, with either 5, 10, 15 or 20 @kg heparin (150 IU/mg, reference TH/023 obtained from Terhormon, Italy). The heparin was dissolved in physiological saline (0.9 % NaCI). Control animals (n=lO) received an equal volume (1 ml/kg) of solvent. In a second experimental group using the Furth Wistar rat (genetically similar strain), 126 animals were divided into 3 groups of 38 to 48 animals. Each group received differents volumes (50 to 450 PI/kg) of either heterologous serum (n=48), homologous serum (n=38) or autologous serum (Furth Wistar rat : n=40). A further group of Furth Wistar rats received either heparin (20 pg/kg i.v., 8 animals) or solvent (0.9 % NaCI, 7 control animals) 5 min before thrombogenic challenge. Prior to assertaining the effects of heparin in Furth Wistar rats, and in order to compare the actions of heparin in both Wistar rats and Furth Wistar rats, 14 animals of each type received either heparin (20 yglkg) or 0.9 % NaCI, 5 min before a thrombogenic challenge with bovine factor Xa (see 4 for experimental details). Statistical analysis Thrombus weights (expressed as mean * sem) in control and treated groups were submitted to an analysis of variance followed by Student’s unpaired t test. The dose of heparin required to reduce control thrombus weight by 50 % (ED,) was calculated by regression analysis and was expressed as mean and corresponding 95 % confidence limits. RESULTS An excellent linear correlation (y = 0.313x - 0.114, r = 0.999) was observed (Fig. 1) between thrombus wet weights (x) and dry weights (y). This analysis took into account 47 values covering a range of thrombus weights from both treated (using both thrombogenic challenges) and control animals. Hence, in this Wessler model as adapted for the rat, thrombus wet weight could be employed for the evaluation of the thrombotic response. potential of heterologous (human) and In the first group, the thrombogenic homologous (rat) serum is shown in Fig. 2a. Heterologous serum produced a strong thrombogenic effect in which a volume as low as 125 @/kg induced the formation of

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appreciable thrombi. Homologous serum induced thrombus formation in a dose (volume) related fashion, although significantly greater volumes were required to induce comparable thrombus weights to these acheived with heterologous serum (Fig. 2a). As such, volumes of serum required to produce equivalent thrombus formation (40 mg wet weight) were 204 (159-263) @/kg and 356 (312-407) @/kg respectively for heterologous and homologous serum (mean with 95 % confidence limits n = 5-8). In order to further validate the thrombogenic actions of the serums involved, various doses of heparin were administered to rats 5 min before the introduction of equithrombogenic volumes of heterologous or homologous serum. In control animals, 250 @/kg of human serum and 400 @/kg of rat serum produced thrombi of 41 .O t 5.2 mg and 42.4 f 4.8 mg respectively (mean + sem, n=lO per group), there being no significant difference between these weights. Heparin on the other hand produced comparable dose-related reductions in thrombus weight in both groups of rats (Fig. 2b), the ED,, for heparin being 9.4 (8.3-10.6) hg/kg and 10.0 (8.8-l 1.4) pg/kg (mean with 95 % confidence limits) against heterologous and homologous serum respectively. There was no significant difference between the two ED,, values. In a second group using Furth Wistar rats and Wistar rats (Fig. 3), the volumes of serum required to produce equivalent thrombus formation of about 40 mg were respectively : 225 (191-266), 341 (297-392), 410 (381-442) PI/kg, for heterologous, homologous and autologous serum (mean with 95 % confidence limits). In a previous study using Wessler’s technique in which factor Xa was the hypercoagulating agent, the mean thrombi weight appeared significantly different according to the rats species : Wistar or Furth Wistar rats. In these studies, these mean thrombi weights were respectively 40 or 23 mg. Nevertheless, the antithrombotic activity of heparin, injected (i.v.) at 20 @kg, 5 min before the thrombogenic challenge, was not different, giving 80 and 98.8 % reductions in thrombus weight for Wistar and Futth Wistar rat respectively. In comparison to the first experimentation, some Furth Wistar rat received heparin (20 p.g/kg/i.v.),

5 min before

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stimulus

(0.4 ml of autologous

this last study, the weight of thrombi were decreased

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WEIGHT

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FIG. 1. Correlation between wet and dry thrombus weights (n=47) from control treated rats receiving both thrombogenic challenges (human and rat serum).

and

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10

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HEPARIN

FIG. 2a.

20 @g/kg)

FIG. 2b.

Dose-response curves of the hypercoagulating effects of heterologous (0) and homologous serum in the rat. (0) Hypercoagulating effects are expressed as increases in wet thrombus weight (mean + s.e.m., 5-8 rats/volume) for various volumes of serum.

Dose-response curves of the antithrombotic effects of heparin against heterologous (0) and homologous (0) serum in the rat. Antithrombotic activity is expressed as % reduction (mean + s.e.m., 10 rats/volume) of wet thrombus weights compared to respective control values.

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FIG. 3. Dose-response curves of the hypercoagulating effects of heterologous (0), homologous (a) and autologous (m) serum in the rat. Hypercoagulating effects are expressed as increases in wet thrombus weight (mean f s.e.m., 5-8 rats/volume) for various volumes of serum. DISCUSSION The purpose of this study was to compare the thrombogenic potential of heterologous and homologous serum as hypercoagulating agents in a simple, small animal experimental model of venous thrombosis. For this purpose, the Wessler technique was adapted for the rat. Using this model, we have clearly demonstrated that the

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thrombotic response could be evaluated by thrombus wet (fresh) weights. It was not the purpose of this study to evaluate the relevance of the Wessler model. It was shown that homologous serum could satisfactorily replace heterologous serum (usually human serum) as a thrombogenic or hypercoagulating agent in the rat. Previously, in the dog, it has been demonstrated that homologous serum is thrombogenic (9) whilst the sera of various animal species -including man- have been compared to homologous serum in the rabbit (3). In both these reports, the effects were assessed in a qualitative manner by scoring clot or thrombus formation, which is different from the present quantitative measurements of thrombus weight. However, the results obtained in rats are consistent with those described in the rabbit, whereby heterologous serum generally produced a stronger thrombogenic response than homologous or autologous serum. Heterologous serum was approximately 1.5 times more potent than homologous serum and 1.8 times more potent than autologous serum. While homologous serum was 1.2 times more potent than autologous serum. The difference in thrombogenic potency between heterologous, homologous and autologous serum is considered (3) to reflect the effect of other physiological stimuli formation of immune complexes, due to species incompatibility (hemolysis, hypotension, etc) becoming superimposed on the stimulation of the coagulation cascade. However, we have found for the rat that homologous serum, whilst being a less potent stimulus, demonstrated the same or greater coagulation efficacy as that of heterologous serum. This is in marked contrast to the findings in the rabbit in which Fareed et al. demonstrated that homologous serum was a less potent and a less efficacious hypercoagulating agent than heterologous (human) serum (3). In order to avoid activation of immune complexes, autologous serum provided to Furth Wistar rat (genetically similar strain) was employed. In this case, these immune complexes were totally excluded. Although this serum appeared less potent than heterologous and homologous, autologous serum did induce a thrombus in the Wessler model. This finding demonstrates that all serum injected into animals could induce a venous thrombosis, according to Wessler models, independantly of immune complex, hypotension, hemolysis etc. The volume required to induce comparable venous thrombosis only varied in fonction of the nature of this serum. In an attempt to further validate the activity of homologous or autologous serum compared to that of heterologous serum as the thrombogenic stimulus, the antithrombotic effects of heparin were compared against equi-effective doses (volumes) of these sera. Whatever the origin of the serum, heparin at the dose of 20 pg/kg (3 IU/kg) given 5 min before the induction of thrombosis, produced close to 100 % reduction in thrombus formation. These results are in complete agreement with those previously observed in rats, with human serum being employed as the thrombogenic agent (8). The effects of heparin thus appear identical against heterologous, homologous and autologous serum. This may not be surprising since the control thrombus weights were the same in the two experimental groups. Obviously, the potency of any antithrombotic agent, such as heparin, will vary as a function of the strength or potency of the thrombogenic challenge. It was for this reason that equieffective doses (volumes) of heterologous and homologous sera were employed. The results therefore suggest that the mechanism whereby heterologous, homologous and autologous serum induce a hypercoagulable state are the same, despite the observed differences in potency. An alternative and more plausible hypothesis is that different factor(s) responsible for the greater thrombogenic potency of heterologous serum is(are) not sensitive to heparin (see above). Using Wessler stasis model adapted to the rat, we have demonstrated that homologous serum can induce thrombosis, similar to that of heterologous (human)

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serum. Although the precise mechanism of action whereby injected serum induces a hypercoagulable state is not understood (see 3), the effects of homologous serum are preventable by prior treatment of the rats with heparin. The use of homologous serum as a thrombogenic challenge in the rat, in addition to conserving species specificity or compatibility, suggests that this model is a viable and safe alternative to employing human serum in testing for in vivo antithrombotic activity. Acknowledgements We thank Prof. M. M. Samama and Dr N. Martin for helpfull MS A. Prigent for preparing the manuscript.

criticism

of the work and

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8. HOBBELEN, P.M.J., VAN DINTHER, T.G., VOGEL, G.M.T., VAN BOECKEL, C.A.A., MOELKER, H.C.T. and MEULEMAN, D.G. Pharmacological profile of the chemically synthesized antithrombin Ill binding fragment of heparin (pentasaccharide) in rats. Thromb Haemost 63, 265-270, 1990. 9. WESSLER, S. and MORRIS, L.E. Studies on intravascular coagulation. IV. The effect of heparin and Dicumarol on serum-induced venous thrombosis. Circulation 72, 553-556, 1955. 10. MILLET, J. THEVENIAUX J. and BROWN N.L. The venous antithrombotic effect of LF 1351 in the rat following oral administration. Thromb Haemost 67(7), 176-179, 1992.