Laser induced microvascular thrombosis in mice treated with oral contraceptives

Vol.

THROMBOSIS RESEARCH Printed in the United

States

LASER INDUCED MICROVASCULAR TREATED

THROMBOSIS IN MICE

WITH ORAL CONTRACEPTIVES

Iren B. KovPcs .and L. Otto Korvin

3, PP. 545-552,1973 Pergamon Press, Inc.

Hospital,

Budapest

Pathophysiology , Semmelweis Budapest,

Csalay and Department

Medical

of

University,

Hungary.

(Received 6.7.1973. Accepted by Editor S. Niewiarowski. Received by Executive Editorial Office 21.9.1973) ABSTRACT The effect of treatment with two types of oral contraceptives on laser-induced arterial and venous thrombus formation was studied in the mesenteric microcirculation of the mouse. In mice treated for three weeks with tenfold amounts of the average human dose both arterial and venous thrombus formation was markedly increased. In comparison of the effects of oral contraceptives containing estrogen in high and low proportions, no relationship could be detected between the estrogen content and the enhanced predisposition to thrombosis.

INTRODUCTION

According relationship incidence

to numerous

exists

myocardial

the eye ( 1,2 ). to predispose

hypothesis

infarction

heart disease

and after

increased

of “increased

embolism,

and thrombosis

disease surgery

studies,

contraceptives

pulmonary

Women using contraceptives

to thromboembolic

State With greatly

retrospective

between the use of oral

of deep vein thrombosis,

thrombosis,

rheumatic

controlled

545

and the celebral

in the vessels

and with conditions

such as collagen

of known

disorders,

may be in a hypercoagulable

risk of thromboembolism

risk” put forward

a causal

(3).

in retrospective

However, studies

this has

ORAL CONTRACEPTIVES

546

IN THROMBOSIS

Vo1.3,No.5

not been confirmed by recent prospective investigations (4). It is known that thromboembolic disease is underdiagnosed in a considerable number of cases,

therefore experimenml study is particularly

important. The effects of oral contraceptives on blood coagulation, on platelet behaviour and fibrinolysis

have been studied by a number of

workers ( 5,6,7 ) but little information is at present available concerning the action of such compounds in experimental thrombus formation. The aim of the present study was to investigate the effect of oral contraceptives containing different doses of estrogens on experimental thrombosis formation using a highly accurate laser procedure for induction of the thrombosis.

the

We believe that this experimental procedure

produces a localised lesion in the blood vessel wall which closely approximates the changes occuring in human thrombosis.

MATERIALS AND METHOD

Adult, 22-26 g female mice of CFW (Car-worth) strain have been used. The mice were treated with oral contraceptives using the factory tabloid product homogenized in a Potter glass homogenizer suspended in 1 % carboxymethylcellulose.

and

The suspension was stirred The

vigorously with a magnetic mixer and administered by gavage.

controls were administered suspension fluid only, calculated on the basis of 20 ml/kg

body weight.

Materials and, doses R -Richter (Budapest) contained 2.5 mg norethynodrel Infecundin R and 0.1 mg mestranol (Synonym: Enovid-E -Searle). Bisecurin R-Richter (Budapest) contained 1 mg ethynodiol diacetate and 0.05 mg estradiol (Synonym: Demulen R-Searle).

With regard to the average human body

weight of 70 kg the mice were given the equivalent of tenfold

the

human dose daily, six days a week for three weeks. The doses for the hhro oral contraceptives

were as follows expressed as fg/kg

weight per animal. Infecundin: 35.7 ,ug.norethynodrel

body

and 1.43 pg

mestranol ; Bisecurin: 14.3 /ug ethynodiol diacetate and 0.71 /.‘g

Vo1.3,No.5

ORAL

CONTRACEPTIVES

IN THROMBOSIS

547

estradiol. Preparation

of mouse

The technique Born (8).

Arterioles

diameter

were

mesentery used corresponded of 40-6O/um

irradiated.

Evans Blue (50 mg/kg) Thrombus

formation

was exposed

could also be observed

occluded first

in detail

irradiation vessel

to be irradiated

time of administration

output coupled

in our earlier

to the laser

microscopically

the first

the observed

vessel

to the laser

of 10 m W power

By the aid of an interference

from

of 80-100pm

intravenously.

apparatus

The vessel

elapsed

exposure

and

by laser

was described

minute.

and venules

before

was injected

with a microscope

every

diameter

Five min.

The He -Ne gas -laser

chosen

to the method of Atherton

report

beam a period filter

of 5 sec.

the preparation

during the irradiation.

until the platelet

was registered. and 30 min.

The time

thrombus

The arteriole

later

(9).

(calculated

completely was the

from

the

of the dye) the venule in the preparation

was

then irradiated.

RESULTS

Arterial

thrombosis

After

the first

irradiation,

platelet

adhesion

at the site of the endothelial

lesion.

size

on the vascular

of the platelet

thrombus

Part of the trombus the bulk adhered formed

closely

an increasing

after the first constriction

of the initial

Upon subsequent

detached

invagination

the arteriole

an embolus,

from

but

irradiations

Two or three minutes

began to narrow

set in at five to seven minutes

the

increased.

wall and upon repeated

into the lumen.

visible

irradiation

wall steadily

and constituted

to the vessel

irradiation

The obstruction,

the first

and

maximal

irradiation.

at the site of the injury was often as great

as 50 %

diameter.

The platelet occluded

became

was clearly

thrombus

the badly narrowed

increased lumen.

by repeated As a rule,

irradiation

complete

and finally

occlusion

lasted

ORAL CONTRACEPTIVES IN THROMBOSIS

548 only a few seconds,

Vo1.3,No.5

then the thrombus was dislodged and flow resumed.

TABLE 1 Laser induced arterial and venous thrombus formation in the mesentery of mice treated with oral contraceptives.

Arteriole No. of Flow -stop cases’ time (min.+ S.E.)

Venule No. OF Flow -stop cases time (min.2 S. E.)

46

46

9.16 + 1.32

37

lnfecundin

32

32

4.17 +_ 1.8s

26

5.70 + 2.25’

Bisecurin

38

38

3.75 +_ 2. OSx

27

5.25 2 1.5Sx

Treatment

zl,”

Control

+ x

10.82 +_ 3.75

The cases were fewer than the no. of animals, because no venules adequate diameter were found in some preparations. P < 0.01 compared to results with vehicle-treated controls. The differences between the effects of Infecundin and Bisecurin were not significant, either in arteriolar or in venous “flow-stop time” (P>O.OS).

The above described sequence of events observed in controls was slightly modified in the animals pretreated with oral contraceptives. the drug treated animals the arteriolar

Typically in

constriction was slight or did not

appear at all, the developed thrombus actually obliterated the vascular lumen at a diameter corresponding to the initial diameter. Animals treated with either of the two contraceptives all showed this accelerated arteriolar

thrombus formation occuring in advance of significant constriction.

As demonstrated in the Table 1,

with either of the two drugs the

difference from controls was highly significant,

but there was

no

significant difference between the two oral contraceptives in promoting the thrombus formation. Venous thrombus formation In the control animal the first irradiation was followed immediately by the onset of thrombus formation on the vessel wall. With repeated

Vo1.3,No.5

irradiation

ORAL

CONTRACEPTIVES

the thrombus

greatly

IN THROMBOSIS

increased

in size

because,

- detachment

was minimal

due to a slower

of the lumen usually

took a longer

time (sometimes

artery,

In animals

treated

was strongly flow-stop

with oral contraceptives

enhanced.

No significant

time” between

animals

549

- unlike the

blood flow. Occlusion several

venous

thrombi

minutes). formation

change was found in the “venous

given Infecundin or Bisecurin.

DISCUSSION

Since the appearance ship between

the risk

disorders,

Numerous

the literature

studies

of thromboembolic

ceptives.

reports

the use of oral contraceptives

thromboembolic extensively.

of the first

Extensive

disease

prospective

the lack of a cause-effect of an increased of increased

risk

appear

on the subject

to justify

and in fact,

(4, lo),

by retrospective

surveys.

extensive

has been observed

thrombosis Sweden,

and coronary

Denmark)

most widely

embolism,

but no significant

The hypothesis

compounds

the estrogen

content

predisposition

to thrombosis

rests

doses

of estrogen

are clearly

in women who have received who received

estrogens

between

estrogen

content celebral

difference

was found between the

ethynilestradiol

largely

thrombogenic,

therapeutically

of

countries

(Great-Britain,

and mestranol

estrogen/progestogen

should be indicted

estrogens

studies of

in several

that in the combined

contraceptives

attention to

would otherwise

deep vein thrombosis,

thrombosis

used estrogen

diagnostic

in which the diagnosis

Positive

of pulmonary

,The hypothesis

of many cases

or not have been made (11).

and the risk

have demonstrated

of having directed

have been uncertain

correlation

that

and do not support the hypothesis

has led to more

complications

has grown

with the use of oral contra-

women who are taking the pill and the discovery thromboembolic

of

the assumption

however

studies,

risk has the great advantage

side effects

with the relation-

and the incidence

increases

relationship,

indicated

dealing

on clinical

(2).

oral

for increased experiences.

High

as has been demonstrated

to suppress for prostatic

lactation, carcinoma

and in men and for

550

ORAL

CONTRACEFTIVES

vo1.3,No.5

IN THROMBOSIS

the treatment of hyperlipidemia and coronary atherosclerosis combined oral contraceptives

(12). The

contains only small amounts of estrogen,

therefore the thrombogenic role of estrogen in these studies is not selfevident. From the retrospective

studies the conclusion have been drawn

that “high estrogen type preparations” ( 100 pg estrogen or more ) are associated with a greater risk of’thrombotic

complications (12).

Normal haemostasis is considerably influenced by oral contraceptives. It has been demonstrated that in women taking the pill, coagulation factors II, VII and X as well as factors VII-X antithrombins decreased,

(P - P complex) were increased,

antiplasmin content was increased,

while

fibrinolysis

activator cant ent of the vascular wall was greatly decreased.

Acceleration

of Chandler’s

tube platelet aggregation and platelet adhesiveness

to glass was observed and hypercoagulable thromboelastographic have been recorded

patterns

(5, 6, 7 ).

In women on oral contraceptives vascular abnormalities may develop which are likely to play a role in the appearance of thromboembolic complications (J3). Angioma, endothelial hyperplasia and intimal thickening have been observed,

although there is no direct evidence that these vascular

changes predispose to thrombosis,

an interaction between changes in the

blood and those in the intima of the vessels,

- especially

arteries,

- may

provide an explanation of the increased risk of thromboembolism with oral contraceptives

(14).

Selection of the species of animal was one of our problems in the present study. Of the known microcirculation microvasculature

preparations the mesenteric

of the mouse was chosen for the following reasons:

1. / This method of preparation is exceedingly rapid, which makes

it

possible to use great number of animals; 2./

Menstrual cycle occurs in the mouse at a short intervals of 5 days

and lasts approximately 12hr. In such a way a minimum of three cycles occured within the three week’s

period of treatment. Hence any variations

arising from the animals being in different phases of the cycle

were

negligible. Platelets play a decisive role in the formation of a laser

induced

Vo1.3,No.5

ORAL

thrombotic causes

lesion.

With the aid of an energy-absorbing

an endothelial

Humoral further

substances

“micro-burn” released

aggregation

explained

by an altered

endothelial

treated

behaviour

lesions

than in the controls, occurs

between

injured

endothelial

The oral

containing

platelets

thrombus

by changes

formation

with oral

of the

contraceptives

that an increased

and the collagen

be

more

produced

used in this study were

interaction

fibres

of the

chosen

because

for use in Hungary, and they contain different

of the estrogen

component.

any significant

“high’ or “low”

to thrombosis

and/or

treated

possible

may induce

in this study may

Perhaps the laser

but it is also

contraceptives

have not revealed

platelet

adhere.

surface.

both are registered and amounts

animals

of platelets

in animals

the circulating

dye - the laser

platelets

The increased

wall due to treatment.

damaging

these adhering

and adhesion.

with the contraceptive

55

injury to which the platelets

from

observed

vascular

IN THROMBOSIS

CONTRACEPTIVES

The results

difference

estrogen

obtained,

types

however,

between combinations

content in increasing

the predisposition

in mice.

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