0017_PREVENTION OF SLUDGE-INDICED MYOCARDINAL DAMAGE.pdf

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Sth European C onference on Microcirculation, Gothenb urg 1968Bibllotheca Anatomica No. 10Editor: H.Harders, HamburgS. Karg er - Basel /New York Printed in Switzerland)S E P A R A T U M

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 M yocardial Damage by anAnti .Adhes ive Dru g 1H. I. BlcrmR

I n 1 9 5 7 , W I L L I A M S et al. [9] described red cell agglutination, reduced bloodflow and transient plugging of small vessels in the bulbar conjunctiva ofpatients with coronary artery disease, especially after the ingestion of highfat meals. They related coronary insufficiency with anginal pain to thesechanges. BLOCH [4] described similar changes in the microcirculation inpatien ts with acute m yocardial infarction.In previous reports, we described lasting myocardial damage in athero-sclerotic rabbits when sludge was induced by the intravenous administrationof high molecular weight dextran [3].Substance 86 (2 methyl 2 tert-butyl ~ ketolactone) has been developedin our laboratory as a prototype antiadhesive drug. It has been shown tocounteract platelet and red cell aggregation, both in vitro and in vivo and toprevent or diminish thrombosis resultin g from these processes [2].In the present study we undertook to evaluate the possible preventiveaction of 86 in sludge-induced myocardial damage.

Materials and MethodsTwo different techniques were used to assess the effect of 2 methyl 2 tert-butyl d keto-lactone in minimizing the insult to cardiac muscle provoked by intravascular red cellaggregation. 1. Dextran Sludge in the Atherosclerotic RabbitThe experiments were carried out according to the previously reported method [2].Thirty-six male adult rabbits were used. Coronary atherosclerosis was produced in allanimals by feeding cholesterol, i g/day, according to the method of A_NrrscRKOV andCHALATO V [1].ntravascular redcell aggregation was induced by the intravenous injection of highmolecular weight colloids possessing strong red cell aggregating power. Dextran (Phar-

Work presented as partial fulfillment for a Ph.D. thesis, Tel Aviv University.


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BII-IER 20 3macia) mean mol. wt. 150,000, 6.5 0/0 solution was used in doses of 2 g/kg/Sludge, ob-served by vital microscopy of the omentum, was always present after the dextran ad-ministration.Three standard ECG leads, and one precordial lead, were recorded simultaneouslyon a Grass Mark III electroencephalograph. ECGs were performed continuously duringthe first 30 min of each experiment, subsequently at 1, 2, 4 and 24 h, and then daily for3 days. Animals surviving the acute experiment were sacrificed on the fourth day by ablow on the neck. All hearts were fixed in 4 buffered formalin, and submitted topathological examination. Microscopic slides were stained with Hematoxylin eosin. Theanimals were divided into 3 groups of 12 rabbits each. Group 1 was injected with highmolecu~r weight dextran only. Groups 2 and 3 received an intravenous injection ofsubstance 86 15 rain prior to the dextran injection. The dose of 86 injected to group2 animals was 200 mg/kg, and to group 3 animals, 100 mg/kg.

2. Perfusion of the Isolated Cats HeartCoronary perfusion of the isolated cats heart was performed according to the Langen-dorff method using a perfusion fluid consisting of cat erythrocytes suspended in Locke-Ringer solution, as described elsewhere [2]. Dextran (mol. wt. 150,000), added to theperfusion fluid to a final concentration of 2 , produced sludge of erythrocytes. After20 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 wereobserved in the ECG and contraction force records. At this point, substance 86 at aconcentration of 1 rag/co was added to the perfusion medium.Electrocardiograms were recorded using suitable stainless steel electrodes attachedto the apex and base of the heart. Contraction force was assessed by a system of pul-leys and thread connected to an adequate transducer. A mode 5 Grass polygraph wasused 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.

Results1 . Dextran - A therosclerotie Rabbits

Group 1Of the 12 cholesterol-fed rabbits injected with high molecular weight dextranonly, 11 developed electrocardiographic signs of myocardial ischemia (STdepression , T in version , A -V block and extrasystoles). These signs developedgradually, starting 5-10min after dextran injection, then increasing instrength for another 15 min and lasting for 24 h or more. Four animals diedwithin 24 h after treatment, two of them with ventricular fibrillation.

Gross pathological examinations showed varying degrees of myocardialdamage. The heart muscle was pale and flabby, and occasionally sub-endo-cardial hemorrhages were observed. Microscopic examination revealedkaryorhexis and fragmentation of the myofibrils. In some animals there wascalcification and necrosis. Clumps of red cells were often present in smallvessels. Perinecrotic inflammatory infiltration w as present in some an imals.


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20 4 BICHER Prevention of Sludge-InducedTable I. Prevention of sludge-induced myocardial damage by 86

Atherosclerotic rabbitsSubstance injected Evidence of myocardial ischemiaElectrocardiographic Pathological Death

Group 1 Dextran 1 1 / 1 2 1 1 / 1 2 4 / 12Group 2 86 200 mg/kg 3/1 2 4 /1 2 1 / 1 2DextranGroup 3 86 100 mg/kg 9 /1 2 9 /1 2 3/12Dextran

Group 2Only 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 showedpathological evidence of organ ic tissue damage (see table I). O n e an imal diedduring the period of experimentation, because of ventricular fibrillation.

Microscopic examination of the living microcirculation after the dextraninjection in animals of this group revealed a mild degree of intravascular redcell aggregation, present only in venules. In contradistinction, in animals ofgroup 1, red cell aggregates were much bigger, sludge was present also inarterioles and capillaries, and the flow was stagnant.Group 3The lower dose of 86 (100 mg/sg) was ineffective in preventing the path-ogenic effect of sludge on cardiac muscle. Nine of the 12 animals of thisgroup exhibited electrocardiographic and pathological evidence of myocardialischemic damage (see table I). The pattern of the peripheral microcirculationalso corresponded well with that of group 1 .

2. Perfusion of the Isolated C at s H eartPerfusion of the isolated heart with sludged erythrocytes causes a deteriora-tion pattern in the heart s performan ce, characterized by an increasing degreeof arrhythmia. The process usually begins with ectopic beats, arising firstfrom one, and then from many, ventricular foci. Contraction force becomesirregular until the gradually increasing ventricular ectopic beats result interminal ventricular fibrillation.W hen substance 86 was added to the perfusion fluid the above describedsigns were dramatically reversed. The most striking effect was achieved whenthe drug was administered shortly after the onset of ventricular fibrillation


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Myocardial Damage by an Anti-Adhesive Drug 20 5

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

(fig. 1). Defibrillation usually followed immediately. In some experimentsthis procedure (alternating sludged and de-sludgedperfusion)could be repeat-ed 2 or 3 times, and the heart alternated between fibrillation and organizedbeats, according to the agglutinated or dispersed status of the red cells in theperfusate. This beneficial effect was limited, however, an d 1 5 to 35 min afterthe initial administration of86, most hearts went into irreversible ventricularfibrillation. When substance 86 was applied at an earlier stage of thesludged-erythrocyte perfusion, the ectopic beats tended to disappear, andthen the cardiac activity ended with progressive bradycardia leading tocardiac standstill, in contradistinction to the results of sludged-erythrocyteperfusion that resulted in ventricular fibrillation.

DiscussionHIGGINBOTHAM et al [5] reported increased red cell aggregation, reducedblood flow and transient plugging of vessels in the bulbar conjunctivae of fivepatients with coronary artery disease following fat-enriched meals, and alsothe occurrence of angina during the time blood cells were agglutinated. In alater publication [9] they suggested that these changes may be a contributingcausative mechanism in the production of coronary insufficiency, cardiacischemia and anginal pain.BLOCH [4] described similar changes in the microcirculation of patientshospitalized after acute myocardial infarction and considered the rationale ofadequate anti-sludge treatment in this condition.


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20 6 BIt;HER Prevention of Sludge-InducedOBRIEN [7] described a group of substances that shared the commonproperty of preven tin g blood platelet aggregation . H e called these substan ces,

that included different categories of drugs such as anti-malarials, anti-his-tamines, and imipramine derivatives, the an ti-adhesive drugs.We reported previously that the anti-adhesive drugs were able to preventboth types of blood aggregation that seem to be related to thrombus forma-

tion [2], namely, red cell as well as platelet aggregation, by what seems to bea generalized type of membrane activity [6]. Substance 86 is the firstcompound developed as a prototype for an anti-adhesive drug, devoid ofany other significant pharmacologic activity.In the present study we demonstrated that anti-adhesive drugs are able toprevent sludge-induced myocardial ischemic damage. This is true for theorganic tissue insult that Dextran-induced intravascular red cell aggregationprovokes in the atherosclerotic rabbit and for the impairment of function andarrhythmia that perfusion with aggregated red cells causes in the isolatedheart.In view of these fin dings, the use of drugs of this type should be consideredin the therapy of ischemic atherosclerotic heart disease.

Summary2 methyl 2 tert-butyl 15 ketolactone (substance 86), a prototype anti-adhesive drugwhich counteracts red cell and platelet aggregatic,n was found to prevent functional andorganic sludge-induced myocardial damage. The rationale of atherosclerotie ischemicheart disease treatment with drugs preventing intravascular red cell aggregation is dis-cussed.

References1. A N r rs cI ~ K O V , N . u n d CHALATOV,S.: Ober experimentelle cholesterinsteatose undihre Bedeutung ffir die Entstehung einiger pathologischer Prozesse. Zbl. allg. Path.

path. Anat. 24:1 (1913). BICI~ER, H.I.: The anti-adhesive drugs as potential anti-thrombotic compoundspreventing red cell and platelet aggregation; screening and properties of ketolactonederivates. Nature (in press).3. BICI~ER, H.I. and BEEMER, A. M.: The role of sludge in the production of experi-mental ischemic myocardial damage; in H. HARDERS 4th Europ. Conf. Microcircula-don, 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 experi-mental animals as they are related to thrombosis and embolism. Angiology, Balti-

more 10:6 (1959).5. HI~Gn~OTHAM, A.C.; W~LLIAMS, A.V. and KNIS~., M.H.: Some acute effects ofdietary fat on intravascular agglutination in cardiac patients. Anat. Rec. 121:310(1 9 5 5 ) .6 OBrien, J.R.: Changes in platelet membrane possibly associated with platelet stick-

iness.Nature, Lond. 212:1057 (1966).


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Myocardial Damage by an Anti-Adhesive Drug 20 77. OBRIEN, J. R.: Platelet aggregation. Part II. Some results from a new method ofstudy. J. din. Path. 15:452 (1962).8. TAtm, L. and CMs, M.: The synthesis of ketolactones with potential pharmacody-namic 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 cellagglutination 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 Collegeof South Carolina, Charleston, S. C. 29401 (USA).

0017_PREVENTION OF SLUDGE-INDICED MYOCARDINAL DAMAGE.pdf

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