Induction of experimental arterial occlusive thrombi in rats

Atherosclerosis, 17 (1973) 369-382 0 Elsevier Scientific Publishing Company, Amsterdam

INDUCTION IN RATS

OF EXPERIMENTAL

G. HORNSTRA

AND

ANNA

369 -

Printed in The Netherlands

ARTERIAL

OCCLUSIVE

THROMBI

VENDELMANS-STARRENBURG

Unilever Research, Vlaardingen (The Netherlands) (Received, June 5th, 1972)

SUMMARY

A method is described for the induction of arterial occlusive thrombi in rats. A loop-shaped cannula is inserted in the abdominal aorta. Histological investigation revealed that the cannula tip damages the endothelial wall as a result of which platelets adhere to the subendothelial tissue. Passing platelets aggregate to the adhered ones, thus forming a platelet thrombus which is stabilized by fibrin formation. The technique is simple and inexpensive. Thrombosis incidence, which can be checked easily, is 100x, while mortality is negligible. The usefulness of this technique is shown by measurement of the antithrombotic effect of intravascularly administered prostaglandin Er and of dietary sunflowerseed oil.

Key words: Aorta - Arterial thrombosis - Histology - Prostaglandin EI - Rat Sunjlowerseed oil - Technique

INTRODUCTION

Occlusive arterial thrombosis is the principal complication of atherosclerosisi. Moreover, mural platelet thrombi are thought to play an initiating role in the atherosclerotic process2>s. In the search for antithrombotic drugs or diets, an in viva technique for the production of arterial thrombi is required, which has to meet the following criteria : - simple and inexpensive; - high thrombosis incidence, low mortality rate; - degree of thrombus formation simple to be established; - structure and composition of thrombus comparable to that of arterial thrombi in man.

G.

370

HORNSTRA,

A. VENDELMANS-STARRENBURG

When a loop-shaped, polyethylene cannula, an “aorta loop”, is inserted into the abdominal aorta of rats, endothelial damage and probably flow disturbances result in the production and growth of a fibrin-poor, platelet-rich mural thrombus which reaches an occlusive state after about 5 days. This paper describes the technique applied and the histological appearance of the growing thrombus. Moreover, two examples are given of the application of this technique in demonstrating the antithrombotic effect of prostaglandin El (PGEi) and dietary sunflowerseed oil. MATERIALS

AND

METHODS

Preparation of the aorta loop

The aorta loop is bent from an approx. 20-cm piece of polyethylene cannula, bore 1.00 mm, external diameter 2.00 mm (Portex@ PP 120, Portland Plastics Ltd. Hythe, Kent, England) under a stream of hot water and fastened with a central ligature (Fig. la). On the day of use, the central ligature is untied and the loop is cut as shown in Fig. lb. Finally the loop is provided with another ligature (Fig. lc). After preparation the loop is siliconized with a solution of Siliclad@ (Clay Adams, B & D. Company, Parsippany, N.J., U.S.A.) 1:20 and filled with a heparin (Vitrum@, Apoteks varucentralen Vitrum A.B., Stockholm, Sweden) solution in physiological saline (500 I.U./ml). Animals and operation procedure

Male Wistar rats, specific pathogen-free, body weight approx. 300 g are anaesthetized by intraperitoneal administration of pentobarbital sodium (Nembutal@, Abbott, Saint RCmy sur Avre, France, 40 mg/kg body weight). To the left of the median line the ventral skin from the costal border to the thigh is shaved and disinfected with tincture of iodine. Approximately 5 mm to the left of the median line, starting at the costal border, a 3 cm long caudad incision is made. From the renal arteries to the common iliac arteries the aorta is detached from the surrounding tissue. Subsequently the part cm -4 -3 -2 -1 G_ a Fig. 1. Preparation

b of aorta loop.

0 C

INDUCTION

OF EXPERIMENTAL

ARTERIAL

OCCLUSIVE

THROMBI

IN RATS

371

of the aorta bounded by the spermatic and iliolumbar arteries is carefully cleaned. Into this part the loop is introduced, To this end the aorta is clamped above the spermatic arteries and below the iliolumbar arteries. The aorta is incised 3 mm below the spermatic arteries and one end of the loop is inserted craniadly and fastened with a previously applied, loose ligature. The aorta is incised again, caudally from the first incision, as near as possible to the inserted part of the loop, and provided with another loose ligature. The upper clamp is momentarily opened in order to replace the heparin solution by blood after which the free end of the cannula is inserted caudadly into the aorta and fastened. Care should be taken that the ends of the loop do not block the branches of the spermatic and iliolumbar arteries. Inclusion of air bubbles must be avoided. The two clamps are removed so that the bloodflow through the aorta, via the loop, is restored. The muscle layer is sutured and the skin clamped in such a way, that the loop partly projects from the body. Between the muscle layer and the skin, a little Terramycin@ ointment (Pfizer Corporation, Brussels, Belgium) is spread. To prevent the loop from being gnawed through, a plastic-coated cardboard collar4 is put round the neck of the animal. The outer diameter of the collar was approx. 12 cm, inner diameter 2.5-3.0 cm. Unless otherwise stated the animals were fed ad libitum a comercial pellet diet (Muracon l@, Trouw & Co, Amsterdam).

Determination of thrombus growth Aorta loops were inserted in 32 animals; 1,2, 3 and 4 days later 8 animals per day were killed. The aortas were perfused with saline, opened longitudinally and the thrombi, which were always located against the proximal and distal cannula tip, were removed, with the aid of a Zeiss binocular dissecting microscope (magn. 10 x). After drying under vacuum, for at least 24 h over silicagel, the thrombi were weighed, using a Cahn Electrobalance@, Model G (Cahn Instrument Company Paramount, California, U.S.A.). Experimental time was restricted to 4 days, as it was shown that in most animals after 5 days the aortas were totally obstructed. In these cases it was impossible to remove the affixed blood clot from the thrombus. Determination of the moment of total obstruction As the loop projects from the body and is made of translucent material, the blood flow is easy to check. If the flow is satisfactory, the colour of the blood is light red. After the loop has become blocked up, the colour rapidly changes from light red via dark red to blue or black. A more accurate determination of the obstruction point can be effected by measuring the temperature of the surface of the loop. To this end an Ellab thermocouple, type K 8, (Elektrolaboratoriet, Copenhagen) is bent in the same curve as the projecting part of the loop, after which they are wrapped together with small strips of Parafilm@ (American Can Company, Marathon Products, Neennah, Wisconsin, U.S.A.). The rats are restrained5 and loop and rectal (thermocouple

372

G. HORNSTRA, A. VENDELMANS-STARRENBURG

36 3432 -

30’

---+

I

15 Time(h)

I

I

16

17

Fig. 2. Course of rectal (a) and loop (b) temperature at obstruction of the loop (arrow).

RM-4) temperature are recorded on a Ellab Strip Chart Recorder, type 28. Fig. 2 shows the temperature recording at the moment a loop became obstructed. Histological investigation of growing thrombi

In 12 animals aorta-loops were inserted. Twenty-four hr up to 6 days after the insertion of the loop, each day 2 animals were killed and, after opening the abdomen longitudinally, placed in a formol
In 36 rats aorta-loops were inserted. Three days before and 1 to 4 days after the operation blood samples were taken from the tail of each animal. The hemoglobin content was determined according to the hemicyanin method. Total erythrocyte, thrombocyte and leukocyte counts were carried out with a Coulter counter, model “A”. For the differentiation of the leukocytes blood smears were stained after May Grtinwald-Giemsa and 200 cells were counted. Influence of Prostaglandin El (PGE1) on arterial thrombus formation

Rats were provided with aorta-loops and prepared for thermometric determination of obstruction. In 48 rats a cannula (Portex @ PP 50) was inserted into the right atrium via the right jugular vein* and in 14 animals, via the left carotic artery, a small cannula (Portex@ PP 25) was introduced into the aorta and pushed down until the

INDUCTION OF EXPERIMENTAL ARTERIAL OCCLUSIVE THROMBI IN RATS

313

cannula tip was positioned just between the left renal artery and the proximal tip of the aorta-loop. Via these cannulae the rats were infused (0.88 ml/h) with - group 1 (n = 28) phosphate-buffered saline (pH 7.42) intravenously - group 2 (n = 20) PGEi in saline (10 pug/h) intravenously - group 3 (n = 5) saline intra-arterially - group 4 (n = 9) PGEi in saline (10 /&g/h) intra-arterially. From the temperature recording the period between insertion and total obstruction of the aorta-loop (“Obstruction Time”, OT) was calculated. Influence of increasing amounts qf dietary sunflowerseed oil on arterial thrombus formation

Eight groups of 12 animals, Sweeks old, were fed adequate diets containing 23 cal. % protein (casein) and 77 cal. % saccharose, the latter being partly replaced by 2.5, 5, 10, 20, 30, 40, 50 and 60 cal. % sunflowerseed oil. After 4 months of feeding, aorta-loops were inserted. The loops were visually checked for obstruction (colour) twice daily. RESULTS

General

After some practice, mortality due to the operation does not occur. In each animal a thrombus developed at both tips of the aorta-loop. When the rats are fed a normal stock diet the obstruction time (OT) is about 5 days. Once the loops have become obstructed, the animals do not die as, in the mean time, collateral vessels have developed which take over the blood suply to the hind quarters of the animal. Therefore it remains possible to take blood samples for plasma and for blood cell analysis after OT determination. Within each experimental group the OT values showed a log-normal pattern so that for statistical evaluation of the results, logarithmic transformation of the OT was necessary. Food consumption decreases considerably after insertion of the loop. Restoration takes place rather slowly, as can be seen from Table 1. In sham-operated rats the food consumption is completely normal within 3 days. TABLE 1 POST-OPERATNE (%

FOOD CONSUMPTION

of preoperative

value + standard error of the mean)

Day

n

Food consumption

1

59 55 55 54

20.4 28.7 38.7 41.1

2 3 4

+ f f +

2.03 1.83 1.77 1.89

( %)

G. HORNSTRA, A. VENDELMANS-STARRENBURG

374 TABLE 2 WEIGHT(~~

h

standard error ofthe

mean)

OF THROMMXIC

MATERIAL FORMED

AT ROTH TIPS OF THE

AORTA-LOOP (n = 8)

Exp. period (days)

Weight (mg)

1 2 3 4

122.5 261.6 304.2 493.7

i f f f

21.8 21.4 45.9 33.1

Determination of thrombus-growth In Table 2 the average thrombus weight 1 to 4 days after loop insertion is given. Statistical evaluation of the results showed that thrombus weight increases significantly with time. The relationship between thrombus weight and experimental time was positive and did not deviate significantly from rectilinearity (see Fig. 3).

500-

400 -

300 -

6 E 200E .P t

$

loo-

E I+

1

-

2

3

4

Exp. period (days)

Fig. 3. Thrombus weight (mg f standard error of the mean) as a function of experimental time (days) (line: y = 118 x).

INDUCTION

OF EXPERIMENTAL

ARTERIAL

OCCLUSIVE

THROMBI

Fig. 4. Adhesion of platelets to denuded aortic wall. a: erythrocytes; wall. (Sudan-black B, phase contrast, 2000 x).

IN RATS

375

b: platelets; c: denuded aortic

Histological investigation of growing thrombi The inserted aorta-loop causes endothelial damage: the underlying tissue is denuded and platelets adhere to the affected sites (Fig. 4). Twenty-four h after the insertion of the aorta-loop large thrombi are already found at both ends of the loop and platelet aggregates radiate out from the juncture of the vessel wall and the lumen. The platelets nearest to the vessel wall are swollen and have lost their internal structure. In the interstices of the platelet thrombus numerous erythrocytes are trapped, while accumulations of polynuclear leukocytes are observed on the surface of the aggregates (Fig. 5). Strands of fibrin are seen between the platelets. The thrombus is not homogeneous, but contains several histologically different layers. The vessel wall is degenerated by the pressure of the loop and damaged by the tapered end of the loop. After 2 and 3 experimental days the thrombi, the area of attachment to the vessel wall and the amount of fibrin have increased (Fig. 6). The thrombus material is invaded by polynuclear leukocytes and mononuclear cells. The trailing ends of the thrombi can be followed for a considerable distance into th.e lumen. After 4 experimental days the vessel wall beneath the thrombus is necrotic and in adjacent parts the smooth muscle cells in the media show proliferation and mitosis, while the endothelial cells of the aorta are swollen and pyknotic. After 5 and 6 experimental days smooth muscle cells, fibroblasts and macrophages penetrate into the thrombus and endothelization of the thrombus starts (Fig. 7). The presence of some hemosiderin and calcium can be demonstrated. Conclusion: the structure of the thrombi which are induced by the insertion of an aorta-loop in rats is similar, in comparative phases of development, to that of natural arterial thrombi in man9 and animalslo.

376

Fig. 5. Polynuclear neutrophils

G. HORNSTRA, A. VENDELMANS-STARRENBURG

(arrows) on the surface of a one-day thrombus

(Masson, 240 x).

Fig. 6. Thrombus after 3 days: (a) necrotic vessel wall; (b) strands of fibrin. (PTAH-fibrin,

60 x).

377

INDUCTION OF EXPERIMENTAL ARTERIAL OCCLUSIVE THROMBI IN RATS

Fig, 7. Smooth muscle cells and mitosis (arrow) in endothelized aortic wall. (Masson, 600 x).

Hematological

6-day thrombus:

(a) thrombus;

(b)

changes after loop insertion

The hematological data (Table 3) show the normal postoperative change@. The decrease in hemoglobin content and the number of erythrocytes is due to the loss of blood during and after the operation. Surgical trauma and thrombosis cause an increase of neutrophils, whereas the number of thrombocytes tends to decrease during the first postoperative day and subsequently increases again.

Influence of prostaglandin

El (PGE1) on arterial thrombus formation

The influence of PGEr on arterial thrombus formation is shown in Table 4. The two control groups did not differ significantly from each other; therefore they were combined to one common control group (log OT = 2, 1026 f 0.0316 OT = 126.6 h, n = 33). From statistical treatment it appeared that intravenous infusion of 10 ,ug PGEr per animal per hour resulted in a significant increase of the OT (~2 < 0.05). When the same amount of PGEl was infused intra-arterially, the increase in OT was also significant and moreover significantly higher than on intravenous administration (pz < 0.01).

Influence formation

of increasing

amounts

of dietary

sunflowerseed

oil on arterial

thrombus

Table 5 shows the influence of sunflowerseed oil. A significantly positive, linear

3

OF RATS,

3

DAYS

BEFORE

AND

1 TO

4 DAYS

AFTER

LOOP

INSERTION

Hemoglobin (mmole)

10.1 8.3 8.4 8.4 8.4

Exp. period in days

3 before 1 2 3(34) 4(18)

7.41 6.39 6.24 6.23 6.08

Erythrocytes (10=/l)

874 709 713 845 964

(We)

Thrombocytes

13.6 15.6 17.2 18.1 20.7

(l@+ll)

leukocytes

Leukocytes

Average data of 36 animals unless otherwise indicated (in parentheses)

HEMATOLOGY

TABLE

1.5 0.6 1.4 1.3 1.1

eosinophiIs

differential

10.6 51.2 41.8 35.7 40.4

neutrophils

count ( %)

86.1 46.5 54.9 60.9 57.1

lymphocytes

1.8 1.7 1.9 2.1 1.4

monocytes

INDUCTION

OF EXPERIMENTAL

ARTERIAL

OCCLUSIVE

THROMBI

319

IN RATS

TABLE 4 INFLUENCE

OF

OBSTRUCTION

PGEl(l0

pg/animal. h)

GIVEN INTRAVENOUSLY

(i.v.)

AND INTRA

ARTERIALLY (i.a.) ON THE

TIME OF AORTA-LOOPS

Group

Control

PGEl

Route

n

Log OT Standard error of mean OT (h)

i.v.

i.a.

i.v.

i.a.

28 2.1074 0.0351 128.1

5 2.0755 0.0776 119.0

20 2.2043 0.0305 160.1

9 2.4078 0.0700 255.7

TABLE 5 OBSTRUCTION

TIME

(OT)

IN RATS,

FED DIETS CONTAINING

DIFFERENT

AMOUNTS

OF SUNFLOWERSEED

OIL

log OT f standard error of the mean and OT (h) Sunflower-seed oil

n

Log OT

12 11 10 12 12 12 11 10

1.975 1.988 2.083 2.176 2.172 2.212 2.229 2.303

OT (h)

(Cal. %! 2.5 5

10 20 30 40 50 60

& 0.045 f 0.041 =t 0.065 i 0.044 i 0.039 + 0.064 + 0.030 f 0.054

94.4 97.3 121.1 150.0 148.6 162.9 169.4 200.9

9-

B-

-l-

6-

5-

i3LL 2.5

_j

5

10

20

40

60

Oil (Cal %. log scale)

Fig. 8. Influence of the dietary amount of sunflowerseed loop. log OT = 0.481 + 0.2258 (log Cal.% SO).

oil on thrombotic

obstruction

of the aorta-

380

G. HORNSTRA, A. VENDELMANS-STARRENBURG

Fig. 9. A: Scanning electron microscopic picture of early thrombus formation (2200 x). B: Blood platelet, spread onto a red cell (1). Pseudopodia radiate out to other red cell (2) and other platelet (3). Higher magnification of A (5500 x).

relationship exists between the amount of sunflowerseed oil in the diet and the OT (see Fig. 8). DISCUSSION

The described technique is simple, the thrombosis frequency is 100% and mortality - after some practice - is negligible. The method is very suitable for the investigation of antithrombotic drugs and diets. An example of both possibilities has been given by demonstrating the antithrombotic effect of Prostaglandin Er (Table 4) and by showing the effective prophylactic treatment with dietary sunflowerseed oil (Table 5). Other effects of dietary treatments (different fats and carbohydrates) have been published recentlyrsJ3. Endothelial damage seems to be the initiating stimulus for thrombus formation in this model. In rabbits it was shown, however, that endothelial damage alone does not produce thrombosis, but results in the formation of a platelet “pseudo-endothelium” followed by layering of leucocytes, disappearance of platelets and formation of a new endothelial layerid. It is most probable that the turbulent flow, which will also occur as a result of aorta cannulation, is an important contribution to the formation of the obstructing thrombus. The red blood cells should also be considered as a contributing factor in thrombus formation. This is suggested from scanning electron microscopy of the early stage of thrombus formation after loop insertion (Fig. 9). Platelets adhering to the inner arterial wall form pseudopodia in which red cells seem to be trapped (A). Sometimes platelets adherent to erythrocytes are seen (B). It might be

INDUCTION

OF EXPERIMENTAL

ARTERIAL

OCCLUSIVE

THROMBI

IN RATS

381

assumed that the adhered platelets together with the trapped red cells, are the source of ADP that causes passing blood platelets to aggregate on the adhered mass. An important role of erythrocytes in early thrombus formation was also suggested by Kloezels, who showed that venous platelet thrombi induced by electrical stimuli generally have settled around a central plug of red blood cells. It seems, therefore, important to pay more attention to the role of erythorcytes in thrombogenesis. As endothelial injury may also trigger the local formation of fibrinls, thrombus growth in the present model might be enhanced by platelet aggregation, induced by fibrin polymerization and the subsequent association of platelets with the polymerizing fibrin, which was recently shown to occur in vitrol7. Because of the presence of strands of fibrin in the thrombus (Fig. 6) it might be expected that anticoagulants also increase the obstruction time. Indeed this was shown for heparin, which, when infused intravenously in an amount of 7 I.U./animal. h, lengthened the OT to 172.2 h (log OT = 2.236 & 0.0461, n = 15). As PGEi, at least in vitro, has no effect on blood coagulation18 its effect on thrombus formation is most probably solely due to its inhibiting activity on platelet stickiness, both in vitro19 and in viv020,2~.The mechanism by which sunflowerseed oil affects thrombus formation is not known. This is now under investigation. The aorta-loop appeared to be not only a suitable device in thrombosis research but it also proved to be very useful in the measurement of platelet aggregation in circulating rat blood22, m . rapid cross transfusions and in perfusion of isolated organs by means of a blood donor. ACKNOWLEDGEMENT

For technical assistance we thank Mrs. W. C. G. Maarseveen-Stienstra, Mr. D. F. van Wichen, Mr. J. A. Don, Mr E. Haddeman and Mr R. N. Lussenburg. The SEM-pictures were taken by Dr J. Holst.

References HARLAND,W. A., Thepathogenesis of myocardialinfarct and coronary thrombosis. In: S. SHERRY, K. M. BRINKHOUS,E. GENTONAND J. M. STENCLE(Eds.), Thrombosis, National Academy of Sciences, Washington, D.C., 1969, p. 126. DUGUID, J. B., Thrombosis as a factor in the pathogenesis of coronary atherosclerosis, J. Pathol. Bacterial., 58 (1946) 207. HEGGTVEIT,H. A., Atheromatous transformation of intracardiac mural thrombi, Amer. J. Pathol., 52 (1968) 70a. EINHEBER,A., WREN, R. E., CARTER,D., ANLIROSE,L. R., A simple collar device for the protection of skin grafts in mice, Lab. Animal Care, 17 (1967) 345. VAN DE GRAAF, H. M., A method for limiting the activity of rats, Z. Versuchstierk., 8 (1966) 246. EVER~~NPEARSE,A. G., Histochemistry. Churchill, London, 1968. ROMEIS,B., Mikorskopische Technik, Oldenbourg, Munich, Vienna, 1968. POPOVIC,V., ANDPOPOVIC,P., Permanent cannulation of aorta and vena cava in rats and ground squirrels, J. Appl. Physiol., 15 (1960) 727. GOULD, S. E., Pathology of the Heart and Blood Vessels, Thomas, Springfield, Ill., 1968.

382

G. HORNSTRA, A. VENDELMANS-STARRENBURG

10 SMITH, H. A., AND JONES,TH. E., Veterinary Pathology, Lea and Febiger, Philadelphia, Penn., 1968. 11 EASTHAM,R. D., Clinical Haematology, Wright, Bristol, 1966. 12 HORNSTRA, G., The influence of dietary sunflowerseed oil and hardened coconutoil on intraarterial occlusive thrombosis in rats, Nutr. Metabolism, 13 (1971) 140. 13 HOR~YSTRA,G., The influence of dietary fats on experimental arterial thrombosis in viva in rats, Acta Univ. Carolinae Med., To be published. 14 STEMERMAN,M. B., COBEN, D. C., AND SPEAT, T. H., Thrombogenic response of twice injured rabbit arteries, Lecture, given at the 2nd Symp. of the Intern. Sot. on Thrombosis and Haemostasis,

Oslo, 1971. 15 KLOEZE,J., Prostaglandins and platelet aggregation in vivo, Part 2 (Influence of PGEi and PGFi, on platelet thrombus formation induced by an electric stimulus in veins on the rat brain surface),

Thromb. Diath. Haemorrh., 33 (1970) 293. 16 ASHFORD,T. P., AND FREIMAN,D. G., The role of the endothelium in the initial phases of thrombosis. An electron microscopic study, Amer. J. Pathol., 50 (1967) 257. 17 NIEWIAROWSKI,S., REGOECZI,E., STEWART, G. J., SENYI, A. F., AND MUSTARD, J. F., Platelet interaction with polymerizing fibrin, J. Clin. Invest., 51 (1972) 685. 18 KLOEZE, J., Influence of prostaglandins El and Ez on coagulation of rat blood, Experientia, 26 (1970) 307. 19 KLOEZE, J., Influence of prostaglandins on platelet adhesiveness and platelet aggregation. In: S. BERGSTROMAND B. SAMUELSSON (Eds.), Prostaglandins, Almqvist and Wiksell, Stockholm, 1967, p. 241. 20 KLOEZE,J., Prostaglandins

and platelet aggregation in vivo, Thromb. Diath. Haemorrh., 33 (1970)

286. 21 HORNSTRA,G., Degree and duration ofprostaglandin El-induced inhibition of platelet aggregation in the rat, Eur. J. Pharmacol., 15 (1971) 343. 22 HoRhwru, G., Method to determine the degree of ADP-induced platelet aggregation in circulating rat blood (Filter-loop technique), Bit. J. Haematol., 19 (1970) 321.