The exclusion of the cerebral circulation: Effect on the “in vivo” platelet aggregation

THROMBOSIS RESEARCH Printed in the United States

Vol. 9, PP* 319-327, 1976 Pergsmon Press, Inc.

THE EXCLUSION UP THE CEYEBHAL CIRCULATION: di.+il'L;CT Cl.1 THE "IN VIVO" PLATELYT ~LGGL~EGATION. R. ARAGNO and M.G. DON1 from the Institute of Human Physiology, University of Padua, Italy.

(Received 15.5.1976; in revised form 2.8.1976. Accepted by Editor E. Deutsch)

ABSTRACT We have studied, in the rat, the variation of the platelet concentration after the exclusion of the ce rebral circulation. It was found that. the operation induced a progressive thrombocytopenia and that PPP obtained from an animal so operated provoked a tran_ sient drop in the platelet count if injected in an intact rat. We observed the thrombocytopenic effect of ADP infusions performed at different time inter _ vals after the onset of the cerebral circulation blo ck: the thrombocytopenia was more prononced if ADP was infused immediately after the circulatory block than if infused 30 minutes later. A second dose of ADP (infused 30 minutes after the first one),.was less effective than the first dose, in the first ca_ se. It produced a more marked effect than the first dose if the first dose itself had been infused 30 rn& nutes after the circulatory exclusion.

INTRODUCTION In a previous paper (1) it was found that the i.v. infusion of ADP promptly reduced the platelet concentration in rats submit ted to the exclusion of one of the following circulatory di 319

_

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sfricts: splenic, renal, cerebral and of one hind-limb, as well as in intact rats. 30 minutes later the platelet number was nearly normal in all animals. The role of the operation a_ lone was evaluated by counting the number of circulating plate lets before and immediately after the operation, but no signi_ ficant change was found (1). It appeared however interesting to investigate the influence of the above mentioned operations on the basal number of piatelets, at a more advanced lapse of time after the operation itself.

MATERIALS AND METHODS The technique followed was the .one described by Kobayashi & Di disheim (2) and by Reyers-Degli Innocenti et al. (3), with so_ me modifications. Adult albino Wistar rats weighing 300-400 g. were anaestethized with Na-Nembutal and Ethyl-Urethan i.p.. Infusion of solutions: the femoral vein was exposed and cate _ ----------therized by means of a needle no 20 connected to an infusion pump (Harvard Apparatus Co. Millis, Massachusetts, U.S.A., MO_ de1 940), through a polystan tube. A solution of heparin in ss line (0.25 mg,/ml; Fluka AG, Chemische, Fabrik Buchs SG, Swit _ zerland) was infused at a speed of 0.051 ml/min. A solution of ADP (Na3ADP, C.F. Boehringer and Sdhne H-Mannheim) was infused at a speed of 0.786 ml/min. The ADP concentration in saline was 0.427 mg/ml, the total of the ADP infused was adjusted to 0.854 mg/Kg b.w.. Platelet poor plasma (PPP) obtained by ten _ trifuging the blood at 700 g/min for 20 minutes was infused at a speed of 0.786 ml/min. The volume injected was adjusted to 2 ml/Kg b.w.. Exclusion of various circulatog _---districts was obtained by ty_ _-_-----_----__ ing up the arterial and then the venous blood vessels as prev&

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ously described (1). Ihe exclusion of the cerebral circulation was obtained by means of the'ligation of the carotids and then of the jugulars. The respiration was provided by means of a small animal respiratory pump (S.R.I., Croydon, Surrey, United Kingdom). collecqion of blood samples ---____ -_ was obtained, at time intervals by disclumping one catetherized carotid. The blood was collected with Wnopette

Disposable Pipetting System" (Becton Dickinson,

France), after discarding the first eight drops. Platelet count was performed by contrast phase microscope. The --_---_ basal number resulted from the mean of two control blood sam _ ples. The percentage in platelet count was calculated for each rat in respect to the basal value, then the mean of the values was estimated. The data obtained were calculated for their sta tistical significance with the two sample t-test for the limi_ ting value of 0.05 probability.

RESULTS 1. In a previous work we have observed that the basal number of the circulating platelets did not change immediately af_ ter ligation of the splenic or renal or cerebral or of one hind-limb.blood vessels (1). By counting the circulating platelets until 30 minutes after the end of the operation, we confirmed, in the present experiments, our previous re _ suits, except in the case of the cerebral circulation block where we observed a reduction (-18.1@)

of the mean plate _

let number. The results obtained are shown in the Table, while the time course of the drop of the platelet count after exclusion of the cerebral circulation is shown in Fig. 1 (curve b). Con_ trol rats (Fig. l-curve a) submitted for 30 minutes to art&

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,fj.cialrespiration, as the animals submitted to the carotid ligations, did not show any variation in platelet count. TABLE ?umber If exp. 21

Mean values and S.D. 839523+63858

t for p=O.O5

Exclusion of the splenic circulation

6

825167*53420

0.523342.060

Exclusion of the renal circulation

5

862400t53612

0.7100<2.064

Exclusion of the circulation of a lim1

5

752600*79122

1.4848~2.064

Control

Exclusion of the cerebral circulation

10

5.0995>2.045

Effect of the exclusion, from the general circulation, of different circulatory districts on the platelet concentra tion, 30 minutes after the onset of the circulatory block.

FIG. 1 Platelet count in: a) control rats artificially ventila 2' ted, b) rats with cerebral circulation block and artifi _ cially ventilated (Mean and S.D, of 9 experiments).

2. In the light of the just reported results we turned our i2 terest to the platelet count after exclusion of cerebral circulation with the following results:

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a) we found that the plasma of operated rats is able to re_ duce the platelet concentration in the intact animal. In fact the i.v. infusion (2 ml/Kg b.w.), into the intact rat, of PPP obtained from a rat 30 minutes after the ex_ elusion of the cerebral circulation, induces a thromboq topenia, which reaches its maximum (-15.67*4.25$) at 2 minutes from the beginning of the infusion, being almost completely reversible within 15 minutes (Fig. 2-curve b). On the contrary PPP of the intact rat is without any si_ gnificant effect (Fig. 2-curve a).

a b

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2

4

;5 min FIG. 2

Platelet count in the intact rat after i;v. infusion of PPP ob tained from: a) control rats, b) rats 30 minutes after block of the cerebral circulation. (Mean and S.D. of 5 experiments). b) platelet responsiveness to ADP after the exclusion of the cerebral circulation: we found (Fig. 3) that the in_ jection of ADP, performed immediately after the exclusi_ on of the cerebral circulation, was followed by a drop of the platelet count; recovery was almost complete wi _ thin 30 minutes. A second dose of ADP was then followed by a small reduction of the platelet concentration. In

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the controls, the dro-pof the platelet concentrationaf_ ter ADP was always more extensive compared with the one observed in rats with the exclusion of the cerebral cir_ culation (Fig. 3-curve a).

60-

1

.

0

1

4I

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I 30

32I

34I

min

FIG. 3 Effect of ADP infusions (0.854 mg/Kg b.w,) on the platelet co_ unt: a) control rats: the maximum of the first drop is 42.14i 3.71s and that of the second one is 29.54t4.22$. (Mean and S.D. of 9 experiments).b) R&S with cerebral circulationblo_ ck:,the maximum of the first drop is 28.14+2.81$ and that of the second one is 18.12&3.01$. (Mean and S.D. of 5 experimen _ ta). In another group of experiments (Fig. 4) the first injection of ADP was performed 30 minutes after the block of the cerebrs 1 circulation and the second one, half an hour later. Therefo_ re the first injection of ADP was performed at the end of a t& me lapse following the carotid ligations, during which the:& telet concentrationwas reduced in respect to its basal value as reported in Fig. 2. The injection of ADP was followed by a

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r;m:Ct.l drop

325

OP t!lo ~~L.lol.ctcolln-1;. ,\n shown

in Fi,n:.1,

this drop was followed by a marked increase; the rise of the curve above lOO$ is due,/to the recovery following the ADP in _ jection and obviously to the resolution of platelet aggregates which have been formed after exclusion of the cerebral circula tion alone. The second injection of ADP was followed by a mar_ ked drop of the platelet count. Consequently, (see

Fig. 3 and

Fig. 4) it appears that the thrombocytopenia following a sin _ gle dose of ADP is more

prononced when it is infused just af _

ter the exclusion of the cerebral circulation than 30 minutes ADP 7

ADP

I

I I I

I

4’5

8’0

6’2

8’4

min

FIG. 4 Effect of ADP infusions (0.854 mg/Kg b.w.) on the platelet co_ unt: a) control rats as in Fig. 3; b) rats with cerebral circ; lation block: the maximum of the first drop is 10.43*4.22$~ and that of the second one is 32.45&1.94$. (Mean and S.D. of 6 ex_ periments). For further explanations see text.

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3IXiJSSION .Yhe present results show that, in the rat,thrombocytopenia de_ velops within 30 minutes following the exclusion of the cere _ bra1 circulation, while the exclusion of the circulation of the kidney, spleen or of one hind-limb is ineffective in this regard. As the thrombocytopenia is usually referred to plate _ let aggregation and to their trapping in the vascular bed, it seems probable that the plasma after the block oft/the cerebral circulation contains some substance (or condition) which indu_ ces the platelets to aggregate "in vivol@. This hypothesis is supported by the thrombocytopenic effect of the dnfusion

of

plasma obtained from operated animals into intact ones and by the different degree of the thrombocytopenic effect of ADP in_ fusion performed immediately or 30 minutes after the exclusion of the cerebral circulation. However the responsiveness of the platelets to ADP returns normal within one hour. The mechani _ sms involved in the post-aggregative platelet desaggregation after ADP, interact with the spontaneous platelet aggregation induced by the exclusion of the cerebral circulation. The ex& stence of an interaction between the two phenomena is supper_ ted by our experimental observations. In fact, the spontaneo_ us lowering of the platelet count do not appear if the infusl on of ADP is performed immediately after the cerebral circula tion block (Fig. 3). Therefore, in this case, we can suppose that the induced thrombocytopenia is prevented by the ADP in_ fusion itself. On the contrary, a very small thrombocytopenia occurs if the ADP infusion is performed 30 minutes after the end of the ape ration (Fig. 4). The scarce effect observed in this case can be referred to the ineffectiveness of ADP on that portion of platelets which is under the influence of the aggregating con

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subsequent to the circulatory block. The observation,

that in this last case the desaggregated platelets after ADP are more responsive to a second dose of ADP, can support the hypothesis according to the ADP itself could prevent and also abolish the spontaneous platelet aggregation occuring after the cerebral circulation block. This finding coming from the a nalysis of the results reported in Fig. 4 shows that the well known (4).reduction of the aggregative effect of a second dose of ADP in respect to the first one is not verified "in viva" if the first dose is infused 30 minutes after the exclusion of the cerebral circulation.

We are grateful to Prof. P. Zatti for valuable help and discuE sion throughout the work and constructive criticism of the te_ xt. REFERENCES

1. DONI, M.G. and ARAGNO, R. ADP-induced platelet aggregation in vivo after exclusion of different circulatory districts. Experientia 31, 1224, 1975. 2. KOBAYASHI, J. and DIDISHEIM, P. Systemic effect of ADP indc ted platelet aggregation and their modification by aspirin and by pyridinolcarbemate. Thromb. Diath. Haemorrh. 30, 178, 1973.

3. REYERS-DEGLI INNOCENTI, I., POGGI, A. and DE GAETANO, G. Platelet aggregation and haemolysis induced in rats by in _ travenous infusion of adenosine diphosphate. Effect of po _ tentially antithrombotic ,drugs. Stand. J. Haematol. 13,

331, 1974. 4. BORN, G.V.R. and CROSS, M.J. The aggregation of blood plate lets. J. Physiol. (London) 168, 178,' 1963.