Sth European Conference on Microcirculation, Gothenburg 1968

Bibllotheca Anatomica, No. 10 Editor: H.Harders, Hamburg S. Karger - Basel/New York (Printed in Switzerland) SEPARATUM 5th Europ. Conf. Microcirculation, Gothenburg 1968. Bib1. anat., No. 10, pp. 202-207 (Karger, Basel/New York 1969) Beilinson Hospital, Petah Tiqva, Israel

Prevention of Sludge-Induced Myocardial Damage by an

Anti.Adhesive Drug1
H. I. BlcrmR In 1957, WILLIAMS et al. [9] described red cell agglutination, reduced blood flow and transient plugging of small vessels in the bulbar conjunctiva of patients with coronary artery disease, especially after the ingestion of high fat meals. They related coronary insufficiency with anginal pain to these changes. BLOCH [4] described similar changes in the microcirculation in patients with acute myocardial infarction. In previous reports, we described lasting myocardial damage in atherosclerotic rabbits when sludge was induced by the intravenous administration of high molecular weight dextran [3]. Substance 86 (2 methyl 2 tert-butyl ~ ketolactone) has been developed in our laboratory as a prototype antiadhesive drug. It has been shown to counteract platelet and red cell aggregation, both in vitro and in vivo, and to prevent or diminish thrombosis resulting from these processes [2]. In the present study we undertook to evaluate the possible preventive action of 86 in sludge-induced myocardial damage. Materials and Methods
Two different techniques were used to assess the effect of 2 methyl 2 tert-butyl d ketolactone in minimizing the insult to cardiac muscle provoked by intravascular red cell aggregation. 1. Dextran Sludge in the Atherosclerotic Rabbit The experiments were carried out according to the previously reported method [2]. Thirty-six male adult rabbits were used. Coronary atherosclerosis was produced in all animals by feeding cholesterol, i g/day, according to the method of A_NrrscRKOV and CHALATOV [1]. Intravascular red cell aggregation was induced by the intravenous injection of high molecular weight colloids possessing strong red cell aggregating power. Dextran (PharWork presented as partial fulfillment for a Ph.D. thesis, Tel Aviv University.

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macia) mean mol. wt. 150,000, 6.5 0/0 solution was used in doses of 2 g/kg/Sludge, observed by vital microscopy of the omentum, was always present after the dextran administration. Three standard ECG leads, and one precordial lead, were recorded simultaneously on a Grass Mark III electroencephalograph. ECGs were performed continuously during the first 30 min of each experiment, subsequently at 1, 2, 4 and 24 h, and then daily for 3 days. Animals surviving the acute experiment were sacrificed on the fourth day by a blow on the neck. All hearts were fixed in 4 % buffered formalin, and submitted to pathological examination. Microscopic slides were stained with Hematoxylin eosin. The animals were divided into 3 groups of 12 rabbits each. Group 1 was injected with high molecu~r weight dextran only. Groups 2 and 3 received an intravenous injection of substance 86 15 rain prior to the dextran injection. The dose of 86 injected to group 2 animals was 200 mg/kg, and to group 3 animals, 100 mg/kg. 2. Perfusion of the Isolated Cats Heart Coronary perfusion of the isolated cats heart was performed according to the Langendorff method using a perfusion fluid consisting of cat erythrocytes suspended in LockeRinger solution, as described elsewhere [2]. Dextran (mol. wt. 150,000), added to the perfusion fluid to a final concentration of 2 %, produced sludge of erythrocytes. After 20 min from the start of the perfusion, clear signs of the typical deterioration pattern (see results) resulting from the pathological effect of sludge on the myocardium were observed in the ECG and contraction force records. At this point, substance 86 at a concentration of 1 rag/co was added to the perfusion medium. Electrocardiograms were recorded using suitable stainless steel electrodes attached to the apex and base of the heart. Contraction force was assessed by a system of pulleys and thread connected to an adequate transducer. A mode 5 Grass polygraph was used for graphic registration. Substance 86 was prepared by TAtra and CAIs according to their method [8]. High molecular weight dextran was supplied by Pharmacia, Sweden.

Results 1. Dextran - Atherosclerotie Rabbits Group 1 Of the 12 cholesterol-fed rabbits injected with high molecular weight dextran only, 11 developed electrocardiographic signs of myocardial ischemia (ST depression, T inversion, A-V block and extrasystoles). These signs developed gradually, starting 5-10min after dextran injection, then increasing in strength for another 15 min and lasting for 24 h or more. Four animals died within 24 h after treatment, two of them with ventricular fibrillation. Gross pathological examinations showed varying degrees of myocardial damage. The heart muscle was pale and flabby, and occasionally sub-endocardial hemorrhages were observed. Microscopic examination revealed karyorhexis and fragmentation of the myofibrils. In some animals there was calcification and necrosis. Clumps of red cells were often present in small vessels. Perinecrotic inflammatory infiltration was present in some animals.

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BICHER Prevention of Sludge-Induced Table I. Prevention of sludge-induced myocardial damage by 86 Atherosclerotic rabbits Substance injected Evidence of myocardial ischemia Electrocardiographic Pathological 11/12 3/12 9/12 11/12 4/12 9/12

Death 4/12 1/12 3/12

Group 1 Group 2 Group 3

Dextran 86 200 mg/kg Dextran 86 100 mg/kg Dextran

Group 2 Only 3 of the 12 animals pre-treated with the higher dose of 86 (200 mg/kg) showed electrocardiographic signs of myocardial anoxia, and only 4 showed pathological evidence of organic tissue damage (see table I). One animal died during the period of experimentation, because of ventricular fibrillation. Microscopic examination of the living microcirculation after the dextran injection in animals of this group revealed a mild degree of intravascular red cell aggregation, present only in venules. In contradistinction, in animals of group 1, red cell aggregates were much bigger, sludge was present also in arterioles and capillaries, and the flow was stagnant. Group 3 The lower dose of 86 (100 mg/sg) was ineffective in preventing the pathogenic effect of sludge on cardiac muscle. Nine of the 12 animals of this group exhibited electrocardiographic and pathological evidence of myocardial ischemic damage (see table I). The pattern of the peripheral microcirculation also corresponded well with that of group 1. 2. Perfusion of the Isolated Cats Heart Perfusion of the isolated heart with sludged erythrocytes causes a deterioration pattern in the hearts performance, characterized by an increasing degree of arrhythmia. The process usually begins with ectopic beats, arising first from one, and then from many, ventricular foci. Contraction force becomes irregular until the gradually increasing ventricular ectopic beats result in terminal ventricular fibrillation. When substance 86 was added to the perfusion fluid the above described signs were dramatically reversed. The most striking effect was achieved when the drug was administered shortly after the onset of ventricular fibrillation

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205

Fig. 1. Reversal of sludge-induced ventricular fibrillation by 86 in the isolated perfused cat heart. Upper record: Electrocardiogram - Lower record: Contraction force. At arrow, 86 added to the perfusion fluid. Then organized contraction of the heart is reinstated, as shown in both records. For explanation, see text.

(fig. 1). Defibrillation usually followed immediately. In some experiments this procedure (alternating sludged and de-sludgedperfusion)could be repeated 2 or 3 times, and the heart alternated between fibrillation and organized beats, according to the agglutinated or dispersed status of the red cells in the perfusate. This beneficial effect was limited, however, and 15 to 35 min after the initial administration of86, most hearts went into irreversible ventricular fibrillation. When substance 86 was applied at an earlier stage of the sludged-erythrocyte perfusion, the ectopic beats tended to disappear, and then the cardiac activity ended with progressive bradycardia leading to cardiac standstill, in contradistinction to the results of sludged-erythrocyte perfusion that resulted in ventricular fibrillation.
Discussion HIGGINBOTHAM et al. [5] reported increased red cell aggregation, reduced blood flow and transient plugging of vessels in the bulbar conjunctivae of five patients with coronary artery disease following fat-enriched meals, and also the occurrence of angina during the time blood cells were agglutinated. In a later publication [9] they suggested that these changes may be a contributing causative mechanism in the production of coronary insufficiency, cardiac ischemia and anginal pain. BLOCH [4] described similar changes in the microcirculation of patients hospitalized after acute myocardial infarction and considered the rationale of adequate anti-sludge treatment in this condition.

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BIt;HER Prevention of Sludge-Induced

OBRIEN [7] described a group of substances that shared the common property of preventing blood platelet aggregation. He called these substances, that included different categories of drugs such as anti-malarials, anti-histamines, and imipramine derivatives, the anti-adhesive drugs. We reported previously that the anti-adhesive drugs were able to prevent both types of blood aggregation that seem to be related to thrombus formation [2], namely, red cell as well as platelet aggregation, by what seems to be a generalized type of membrane activity [6]. Substance 86 is the first compound developed as a prototype for an anti-adhesive drug, devoid of any other significant pharmacologic activity. In the present study we demonstrated that anti-adhesive drugs are able to prevent sludge-induced myocardial ischemic damage. This is true for the organic tissue insult that Dextran-induced intravascular red cell aggregation provokes in the atherosclerotic rabbit and for the impairment of function and arrhythmia that perfusion with aggregated red cells causes in the isolated heart. In view of these findings, the use of drugs of this type should be considered in the therapy of ischemic atherosclerotic heart disease.
Summary
2 methyl 2 tert-butyl 15 ketolactone (substance 86), a prototype anti-adhesive drug which counteracts red cell and platelet aggregatic,n was found to prevent functional and organic sludge-induced myocardial damage. The rationale of atherosclerotie ischemic heart disease treatment with drugs preventing intravascular red cell aggregation is discussed.

References
1. ANrrscI~KOV, N. und CHALATOV, S.: Ober experimentelle cholesterinsteatose und ihre Bedeutung ffir die Entstehung einiger pathologischer Prozesse. Zbl. allg. Path. path. Anat. 24:1 (1913). 2. BICI~ER, H.I.: The anti-adhesive drugs as potential anti-thrombotic compounds preventing red cell and platelet aggregation; screening and properties of ketolactone derivates. Nature (in press). 3. BICI~ER, H.I. and BEEMER, A. M.: The role of sludge in the production of experimental ischemic myocardial damage; in H. HARDERS 4th Europ. Conf. Microcirculadon, Cambridge 1966, Bibl. anat., No 9, p. 116 (Karger, Basel/New York 1967). 4. B~.ocI~, E. H.: Visual changes in the living microvascular system in man and experimental animals as they are related to thrombosis and embolism. Angiology, Baltimore 10:6 (1959). 5. HI~Gn~OTHAM, A.C.; W~LLIAMS, A.V. and KNIS~., M.H.: Some acute effects of dietary fat on intravascular agglutination in cardiac patients. Anat. Rec. 121:310 (1955). 6. OBrien, J.R.: Changes in platelet membrane possibly associated with platelet stickiness.Nature, Lond. 212:1057 (1966).

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207

7. OBRIEN, J. R.: Platelet aggregation. Part II. Some results from a new method of study. J. din. Path. 15:452 (1962). 8. TAtm, L. and CMs, M.: The synthesis of ketolactones with potential pharmacodynamic properties. Bull. Res. Coun. Israel, II. A., 18 (1962). 9. WmL~MS, A.V.; HIGGI~mOTr~M, A.C. and KN~SELY, M.H.: Increased blood cell agglutination following ingestion of fat. A factor contributing to cardiac ischemia, coronary insufficiency and anginal pain. Angiology, Baltimore 8:19 (1957).

Authors present address: Dr. I-I. I. BIcl-mR, Department of Anatomy, Medical College of South Carolina, Charleston, S. C. 29401 (USA).