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Myocardial viability after transient ischemia in primates
Myocardial viability after transient ischemia in primates Myocardial viability after acute occlusion of the left anterior descending coronary artery (LA D) for periods of from 1 to 5 hours was studied in 48 primates with the following results: First, sudden death after acute occlusion is related to the anatomic distribution of the LAD. Second, myocardial function is improved (ventriculography) and infarct size reduced (histology and unipolar electrocardiographic [ECG] mapping) after release of LAD occlusion through 3 hours of ligation. Similarly, 7 of 12 animals subjected to LAD ligation for 4 hours benefited from ligature release. Third, surface myocardial pH is indicative primarily of the presence or absence of myocardial blood flow. It returns promptly to normal following ligature release even in areas of established infarction.
J. Judson McNamara, M.D., Gregory T. Smith, B.S., Glenn T. Suehiro, B.S., John R. Soeter, M.D., Richard J. Anema, Andrew L. Morgan, Jr., and S. K. Liao, M.D., Honolulu, Hawaii
The surgical treatment of patients with acute myocardial infarction is now a clinical reality.>:' As yet, there is no definition of the limits of myocardial viability after the onset of clinical symptoms of myocardial infarction. The present study attempts to evaluate the time limits within which revascularization may salvage myocardium in a normal primate heart after acute occlusion of the left anterior descending coronary artery (LAD). Materials and methods
Fifty-two Macaca species primates were anesthetized with Sernylan intramuscularly (1 mg. per kilogram) and small doses of sodium Pentothal intravenously (12.5 mg. per dose). Ventilation was maintained via an endotracheal tube with a mechanical ventilator. A polyethylene catheter was placed in the left femoral artery and blood pressure From the Department of Surgery, Queen's Medical Center, and University of Hawaii School of Medicine, Honolulu, Hawaii 96808. Received for publication Jan. 4, 1974.
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was monitored continuously. Blood gases (pH, Pc0 2 , and Po 2 ) were determined at hourly intervals to monitor adequacy of respiration. The pericardium was entered via a left anterior thoracotomy, and a snare was placed around the LAD just distal to the first diagonal branch. The left ventricle was then mapped with an epicardial unipolar electrocardiographic (ECG) lead" at points outlined in Fig. 1. A small diagram was constructed of each heart, showing the points mapped, to assure that subsequent mappings were in nearly the same points. Myocardial surface pH was also measured* at two points (Fig. 1, A and B). The snare was then tightened, completely occluding the LAD. Ligatures were left in place for 1 hour (2 animals), 2 hours (3 animals), 3 hours (12 animals), 4 hours (12 animals), 5 hours (2 animals), and permanently (7 animals) . Fourteen animals died of intractable ventricular fibrillation less than 1 hour after ligation. "Instrumentation Laboratory Model 245 pH/EV Electrometer.
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Table I. Ventriculography in 21 animals
Duration of ligation (hr.) 2 3 4 5 Permanent
No. studied before and immediately after ligature release
No. restudied again I week later
1
9
3 6
2 3*
3
6
-No ligature release.
Fig. 1. Diagram of primate heart as seen through left thoracotomy. Numbers indicate mapping site for surface unipolar electrocardiographic recording, and Letters A and B refer to site of surface pH recording.
Table II. Distribution of animals studied Duration of ligation (hr.)
1 2 3
4
Epicardial unipolar ECG mapping and myocardial pH determinations were performed just prior to ligature release and again 1/2 to 1 hour after ligature release. Cine-ventriculography was performed by transfemoral retrograde catheterization in 21 animals just prior to ligature release (Table I). Only 21 animals were studied because of the difficulties in scheduling primate angiography in the hospital's single clinical cineangiography room. We were pre-empted by the need for clinical studies after initiating the experiment in 17 of the 38 surviving animals. Eighteen animals were studied by ventriculography 30 minutes to an hour after ligature release ( 1 animal with a 2 hour ligation, 5 with 3 hour ligations, 9 with 4 hour ligations, and 2 with 5 hour ligations). Nine of these animals (3 with 3 hour ligations and 6 with 4 hour ligations) were permitted to survive for a week and were restudied by ventriculography. Ventriculography was evaluated on a subjective grading system: 0, no change; 1+, slight improvement in ventricular wall motion; 2+, moderate improvement in ventricular wall motion; and 3+, major improvement or return to normal. All animals were assigned an identifying code number and were judged by one
5 Permanent Immediate death -Figures in immediately tFigures in restudy over
I
Acute study" jChronic study t
2 (1) 3 (2) 12 (6) 12 (5 ) 2 (2)
1 1 4 (2) 7
4 (3)
14
parentheses indicate animals put to death after acute study for histologic examination. parentheses indicate animals dying prior to a week later.
individual (1. J. M.) with no knowledge of the ligation status of the animal whose ventriculogram was being reviewed. Serial sections of myocardium were made at the time of sacrifice in all the animals and were stained with hematoxylin and eosin and hematoxylin-basic fuchsin-picric acid (HBFP).G
One animal with 1 hour ligation and 2 with 2 hour ligations were put to death right after ligature release, and 1 from each group was allowed to live for a week and then restudied (Table II). Six animals (3 hour ligations) were put to death immediately. Two of the remaining 6 animals in this group died less than a week postoperatively. The other 4 were put to death a week to 10 days later, and serial sections of myocardium were taken for histologic study. Animals subjected to 4 hours of ligation were similarly either put to death right after study (5 animals) or were allowed to live, restudied, and killed a week
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]I
(MONKEY)
I
(MONKEY)
Fig. 2. Type II LAD distribution in primate, with vessel extending well up the posterior interventricular groove.
Fig. 3. Diagram of primate heart with Type I LAD distribution: LAD ends at or on ventricular apex.
to 10 days later (7 animals). Both animals with 5 hour ligations were put to death right after study. Animals with permanent ligation were allowed to survive for I to 2 weeks, restudied, and killed. Three of the 7 animals in this latter group died within a week, prior to restudy. The remaining 4 animals were restudied (3 had ventriculography prior to sacrifice), and specimens stained with hematoxylin and eosin or HBFP were examined histologically.
utes of ligature placement. After ligature release, the area of significant S-T elevation decreased (at least two fewer points showing an S-T elevation of > 2 mv.) in all animals subjected to ligation for I and 2 hours and in 9 to 12 animals with ligation for 3 hours (Fig. 4). Of the 12 animals with 4 hour ligations, 7 had a clear decrease in infarct size; in the remaining 5, the change was negligible (Table Ill). In both of the animals with LAD ligation for 5 hours, epicardial ECG mapping showed a decrease in infarct size after ligature release. All animals studied at 1 week had a marked reduction in infarct size, although S-T changes were present in all. Animals with long-term ligation had persistent large infarcts after I week, although the area involved was frequently smaller than that seen shortly after ligation. The infarct size in this group was considerably larger than that in animals with 3 or 4 hour ligations which then survived for I week. pH. Surface pH measurements consistently decreased in Area A from a mean value of 7.38 (range 7.65 to 7.05) to 6.95 (range 7.45 to 6.60) (p < 0.05).
Results Fourteen animals died of ventricular fibrillation less than 1 hour after ligature placement. All of those surviving for an hour survived for the duration of the ligation. In 12 of these 14 early deaths, the LAD was "dominant"; that is, the vessel extended beyond the apex of the heart up the posterior interventricular sulcus (Fig. 2). Only 7 of the surviving 38 animals had a dominant LAD. The nondominant distribution ending on the ventricular apex is shown in Fig. 3. ECG. S-T elevation of more than 2 mv. occurred over a large area of the anterior left ventricle in all animals within 15 min-
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Fig. 4. Surface electrocardiographic recording shows minimal S-T changes prior to ligation probably secondary to slight electrode contact pressure. Dramatic S-T changes 10 minutes after ligation (8) are further accentuated after 3 hours of ligation (C). One hour after release of ligation, S-T elevations are back to normal in all but two leads (D). (A),
Table III. Results of electrocardiographic (ECG) mapping ECG study 1 week later
ECG study immediately Duration of ligation (hr.) 1 2 3 4 5 Permanent
Total
I Improvement after
I
Further decrease in infarct
ligature release
Total
2 3 9 7 2
1 4 7
1 4 7
4
4
2 3 12 12 2
Within 30 minutes after ligature release, all animals exhibited an increase in surface pH to near preligation levels (mean 7.28, range 7.55 to 6.90). In spite of the wide range in pH values, these differences were statistically significant (p < 0.0 I). pH changes in Area B, in this experiment, were not significant. Return to normal pH seemed to have no relationship to the duration of ligation.
Cine-ventriculography. Ventriculography showed that, within 2 hours of coronary ligation, all animals developed a large dyskinetic segment in the anterior wall and most developed huge anterior wall aneurysms (Fig. 5). These changes persisted but were significantly improved (2+ or 3+) in animals with permanent ligation restudied a week later (Table IV, Fig. 6). Following ligature release, significant im-
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Fig. 6. Ventriculogram at end systole from a permanently ligated LAD shows anterior wall bulge and increased end-systolic volume.
Fig. 5. End -systolic ventriculogram after 4 hOUTS of ligation shows large anterior wall aneurysm.
Table IV. Results of ventriculography Study before and after ligatur e release Duration of ligation (lir .)
Total
2 3
2 6 9 2 3*
4
5 Permanent
Significant improvement (2+ or 3+) 2 5 5 0
Study .again I week later
Total
Significant improvement (2+ or 3+)
3 6
4
3
3
3
"No immed iate restud y.
provement or return to normal (2+ or 3+) occurred within an hour in all with 2 hour ligation, 5 of 6 animals with 3 hour ligations, and 5 of 9 with 4 hour ligations. Most animals that survived for a week and were restudied showed marked improvement or returned to normal (3+): all animals subjected to 3 hours of ligation and 4 of 6 subjected to 4 hours of ligation. Ventriculography showed no improvement lh hour after ligature release in the animals with LAD ligation for 5 hours.
Histology. Early evidence of ischemic swelling of myofibrils was seen in several dogs with 4 hour ligations and in both with 5 hour ligations (Fig. 7). Shorter ligations (3 hours or less) produced no evidence of myocardial changes on hematoxylin and eosin sections. The HBFP stain was fuchsin positive, indicating early ischemia in all animals beyond I hour of ligation. Furthermore, 'the ischemic area seemed to increase up to 4 hours after ligation but appeared stable beyond that point (Fig. 8). Ligature
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release did not seem to affect the presence of HBFP fuchsin-staining muscle. All animals that survived for a week had definite histologic evidence of myocardial infarction even after 1 hour of ligation (Fig. 9) . Infarct size was smaller in the 4 animals with 3 hour ligations and in 5 of 7 animals with 4 hour ligations than in the 4 animals with permanent LAD ligation (Fig. 10) .
Discussion Ligation of the LAD in monkeys produces a large anterior wall infarct similar to that observed with acute LAD occlusion in man .7 • 8 The immediate mortality rate with acute ligation in the present study was 27 per cent. Death was due to ventricular fibrillation. Twelve of the 14 animals that died had a large LAD, extending into the posterior interventricular groove. In contrast, only 7 of 38 surviving animals had this dominant LAD distribution. In animals with a dominant LAD distribution, more blood supply to the left ventricle is derived from the LAD , and the resulting higher mortality rate in this group is probably a consequence of the much larger area of ischemic myocardium." LAD distribution in primates is similar to that noted in human hearts." By inference, it seems likely that a patient with a dominant LAD distribution is at much greater risk of dying from coronary occlusion than a patient with an LAD ending at the ventricular apex. ECG proved a reliable method of mapping the area of ischemic muscle right after ligation and it assisted in determining infarct size. It has been used by others with similar success.P- 12 The present information on ECG mapping does not provide incontrovertible evidence of reduction in infarct size since we do not have serial tracings during the course of the ligation. It is quite possible that the apparent area of S-T elevation decreases as the area of actual muscle necrosis increases, because these changes reflect myocardial cell injury and not cell death. Confirmation of these ob-
Fig. 7. Interstitial edema and inflammatory infiltrate by hematoxylin and eosin stain after 5 hour ligation . This was the most significant lesion found on any animal put to death immediately. (Original magn ification x150.)
servations with serial quantitative ECG tracings are the subject of research work currently in progress in our laboratory. Nevertheless, the very acute reduction in S-T elevation noted in the present study is in all probability a reflection of reversal of actual tissue ischemia with the onset of coronary reperfusion, Although the surface pH dropped rapidly following ligation, the value routinely increased rapidly toward normal after ligature release , even with persistence of a subsequently proved infarct. It appears that pH reflects primarily the presence or absence of blood flow and is of little value in determining muscle viability. P Furthermore, it seems that blood flow returns rapidly to the area of ischemia even in the presence of established infarction. This is consistent with the findings of Krug.>' who utilized fluorescent dye in cat hearts after coronary ligations lasting up to 90 minutes .
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Fig. 8. Hematoxylin-basic fuchsin-picric acid stain demonstrates large area of anterior wall ischemia after 4 hour ligation (arrows). The apparent thinning of the posterior, noninvolved wall is a sectioning artifact.
Fig. 9. Specimen from animal subjected to 1 hour of LAD ligation (survived for a week) shows patchy infarction by hematoxylin and eosin stain.
The present study documents a profound functional defect associated with anterior wall ischemia. The observed large asynergic areas or, more frequently, acute aneurysms of the anterior wall were usually reversible in animals with 3 hour ligations, and they improved significantly in over half of the animals (5 of 9) with 4 hour ligations. Release of 5 hour ligation produced no evident immediate change in ventricular contraction in the 2 animals studied. The greatest functional improvement appeared less than an hour after ligature release, though some further improvement was evident in animals restudied at 1 week. Krug':' again noted significant decrease in infarct size in cats, even after 90 minute coronary ligations, within 30 minutes after ligature release. Similarly, Maroko and co-workers:" have reported a decrease in infarct size in dogs after release of occlusions of up to 3 hours' duration. Significant ischemic injury was evident by HBFP stain after only an hour of ligation in the present study. Jennings and co-workers-" have demonstrated some irreversible ischemic injury in dogs after only 25 minutes of coronary occlusion. The
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Fig. 10. Specimen from animal subjected to 4 hours of LAD ligation (surv ived for a week) shows grossly evident myocardial infarct (between arrows). (Hematoxylin-basic fuchsin-picric acid ; original magnification xI O.)
size of fuchsin-staining (ischemic) area increased with time up to 4 hours after LAD occlusion. Furthermore, specimens examined I week after ligature release demonstrated some areas of actual infarction even with a I hour ligation. In animals that survived for 1 week after sustaining LAD occlusion for 4 hours or less, the infarct appeared patchy and significantly smaller than in those animals sustaining permanent occlusion that were put to death at I week. The significance of observations of myocardial ischemia based on the HBFP stain are not entirely clear. Though fuchsin staining was evident at very low-power magnification over the area of expected ischemia, the staining was frequently very patchy under higher power. Furthermore, fuchsin staining was occasionally noted in areas well beyond any possible LAD distribution. We have attempted to verify the HBFP stain in rat hind limbs with blood supply totally occluded for varying lengths of time. It appears that the HBFP stain is reliable, when positive, in mapping zones of known ischemia. Its capricious propensity to occasionally stain normal muscle makes it of questionable value in identifying early
ischemic zones where the presence and distribution of the ischemia are not certain. After considering the observations of Krug, H Maroko ," Jennings.!" and Herd son" in light of the present data, we have formulated the following hypothesis relevant to the evolution of the primate myocardial infarct. After acute occlusion for periods of 1 hour (present study) or less (Jennings' ") , areas of irreversible myocardial necrosis occur. The ischemic area increases in size for 3 to 4 hours after occlusion. A resulting large area of functional impairment is evident even at I hour. It appears that early muscle necrosis extends concentrically from a small, centrally located area to progressively involve an initially larger peripheral zone of ischemic, nonfunctional, but potentially viable myocardium. Furthermore, in primates , much of this peripheral zone remains potentially viable 3 hours and, in many animals, for at least 4 hours after acute occlusion. Consequently, in primates, reperfusion of an area of acute LAD occlusion for periods of up to 4 hours will produce a significant reduction in infarct size and immediate improvement in ventricular performance. This improvement
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was not evident functionally after 5 hours of occlusion, although reduction by EeG mapping was noted in the 2 animals studied. The present study then demonstrates a large zone of marginal ischemia around an acute infarct in primates. This experimental situation is not directly analogous to acute myocardial infarct in man, most notably because it does not allow for long-standing ischemia with attendent collateral development or for the possible presence of antecedent myocardial damage. Nevertheless, it suggests that a significant volume of muscle, rendered nonfunctional and doomed to gradual infarction, may be salvaged by prompt restoration of blood supply. These observations support continued investigation into the potential benefits of emergency myocardial revascularization following massive acute myocardial infarction. REFERENCES Scanlon, P. J., Nemickas, R., Tobin, J. R., Jr., Anderson, W., Montoya, A., and Pifarre, R.: Myocardial Revascularization During Acute Phase of Myocardial Infarction, J. A M. A 218, 207, 1971. 2 Hill, J. D., Kerth, W. J., Kelly, J. J., Jr., Selzer, A, Armstrong, W., Popper, R., and Cohn, K.: Emergency Aorto-coronary Bypass for Impending or Extending Myocardial Infarction (Abstr.), Circulation 42: 106, 1970 (Supp!. III).
3 Lambert, C. J., Adam, M., Geisler, G. F., Verzosa, E., Nazarian, M., and Mitchel, B. F., Jr.: Emergency Myocardial Revascularization for Impending Infarctions and Arrhythmias, J. THORAC. CARDIOVASC. SURG. 62: 522, 1971. 4 Mundth, E. D., Buckley, M. H., Daggett, W. M., Sander, C. A, and Austen, W. G.: Surgery for Complications of Acute Myocardial Infarction, Circulation 45: 1279, 1972. 5 Maroko, P. R., Libby, P., Covell, J. W., Sobel, B. E., Ross, J., Jr., and Braunwald, E.: Precordial ST Segment Elevation Mapping: An Atraumatic Method for Assessing Alterations in the Extent of Myocardial Ischemic injury. The Effects of Pharmacologic and Hemodynamic Interventions, Am. J. Cardio!' 29: 223, 1972. 6 Lie, J. T., Holley, K. E., Kampa, W. R., and
Titus, J. L.: New Histochemical Method for Morphologic Diagnosis of Early Stages of Myocardial Ischemia, Mayo Clin. Proc. 46: 319, 1971. 7 Hill, J. D., Malinow, M. R., McNalty, W. P., and Ochsner, A J.: Experimental Myocardial Infarction in Unanesthetized Monkeys, Am. Heart J. 84: 82, 1972. 8 Grayson, 1., and Irvine, M.: Myocardial Infarction in Monkeys: Studies on the Collateral Circulation After Acute Coronary Occlusion, Cardiovasc. Res. 2: 170, 1968. 9 McNamara, J. J., Fergusson, D., Yamase, H., Suehiro, G., and Soeter, J. R.: Significant Variations in Distribution of the Anterior Descending Coronary Artery in Primates, J. THORAC. CARDIOVASC. SURG. 65: 639, 1973. 10 James, T. N.: Anatomy of the Coronary Arteries, New York, 1961, Paul B. Hoeber, Inc. II Maroko, P. R., Kjeksus, J. K., Sobel, B: E., Watanabe, T., Covell, J. W., Ross, 1., Jr., and Braunwald, E.: Factors Influencing Infarct Size Following Experimental Coronary Artery Occlusion, Circulation 43: 67, 1971. 12 Maroko, P. R., Bernstein, E. F., Libby, P., DeLaria, G. A., Covell, J. W., Ross, J., Jr., and Braunwald, E.: Effects of Intra-aortic Balloon Counterpulsation on the Severity of Myocardial Ischemic Injury Following Acute Coronary Occlusion, Circulation 45: 1150, 1972. 13 McNamara, J. J., Soeter, J. R., Suehiro, G. T., Anema, R. J., and Smith, G. T.: Surface pH Changes During and After Myocardial Ischemia in Primates, 1. THORAC. CARDIOVASC. SURG. 67: 191, 1973. 14 Krug, A.: The Extent of Ischemic Damage in the Myocardium of the Cat After Permanent and Temporary Coronary Occlusion, J. THORAC. CARDIOVASC. 60: 242, 1970. 15 Maroko, P. R., Ginks, W. R., Libby, P., Sobel, B. E., Shell, W. E., and Ross, J. Jr.: Salvage of Myocardial Tissue by Coronary Artery Reperfusion Following Acute Coronary Occlusion, Am. J. Cardio!. 29: 278, 1972. 16 Jennings, R. B., Sommers, H. M., Smyth, G. A., Flack, H. A, and Linn, H.: Myocardial Necrosis Induced by Temporary Occlusion of a Coronary Artery in the Dog, Arch. Patho!. 70: 68, 1960. 17 Herdson, P. B., Sommers, H. M., and Jennings, R. B.: A Comparative Study of the Five Structures of Normal and Ischemic Dog Myocardium With Special Reference to Early Changes Following Temporary Occlusion of a Coronary Artery, Am. J. Patho!. 46: 367, 1965.
J. Judson McNamara, M.D., Gregory T. Smith, B.S., Glenn T. Suehiro, B.S., John R. Soeter, M.D., Richard J. Anema, Andrew L. Morgan, Jr., and S. K. Liao, M.D., Honolulu, Hawaii
The surgical treatment of patients with acute myocardial infarction is now a clinical reality.>:' As yet, there is no definition of the limits of myocardial viability after the onset of clinical symptoms of myocardial infarction. The present study attempts to evaluate the time limits within which revascularization may salvage myocardium in a normal primate heart after acute occlusion of the left anterior descending coronary artery (LAD). Materials and methods
Fifty-two Macaca species primates were anesthetized with Sernylan intramuscularly (1 mg. per kilogram) and small doses of sodium Pentothal intravenously (12.5 mg. per dose). Ventilation was maintained via an endotracheal tube with a mechanical ventilator. A polyethylene catheter was placed in the left femoral artery and blood pressure From the Department of Surgery, Queen's Medical Center, and University of Hawaii School of Medicine, Honolulu, Hawaii 96808. Received for publication Jan. 4, 1974.
248
was monitored continuously. Blood gases (pH, Pc0 2 , and Po 2 ) were determined at hourly intervals to monitor adequacy of respiration. The pericardium was entered via a left anterior thoracotomy, and a snare was placed around the LAD just distal to the first diagonal branch. The left ventricle was then mapped with an epicardial unipolar electrocardiographic (ECG) lead" at points outlined in Fig. 1. A small diagram was constructed of each heart, showing the points mapped, to assure that subsequent mappings were in nearly the same points. Myocardial surface pH was also measured* at two points (Fig. 1, A and B). The snare was then tightened, completely occluding the LAD. Ligatures were left in place for 1 hour (2 animals), 2 hours (3 animals), 3 hours (12 animals), 4 hours (12 animals), 5 hours (2 animals), and permanently (7 animals) . Fourteen animals died of intractable ventricular fibrillation less than 1 hour after ligation. "Instrumentation Laboratory Model 245 pH/EV Electrometer.
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Table I. Ventriculography in 21 animals
Duration of ligation (hr.) 2 3 4 5 Permanent
No. studied before and immediately after ligature release
No. restudied again I week later
1
9
3 6
2 3*
3
6
-No ligature release.
Fig. 1. Diagram of primate heart as seen through left thoracotomy. Numbers indicate mapping site for surface unipolar electrocardiographic recording, and Letters A and B refer to site of surface pH recording.
Table II. Distribution of animals studied Duration of ligation (hr.)
1 2 3
4
Epicardial unipolar ECG mapping and myocardial pH determinations were performed just prior to ligature release and again 1/2 to 1 hour after ligature release. Cine-ventriculography was performed by transfemoral retrograde catheterization in 21 animals just prior to ligature release (Table I). Only 21 animals were studied because of the difficulties in scheduling primate angiography in the hospital's single clinical cineangiography room. We were pre-empted by the need for clinical studies after initiating the experiment in 17 of the 38 surviving animals. Eighteen animals were studied by ventriculography 30 minutes to an hour after ligature release ( 1 animal with a 2 hour ligation, 5 with 3 hour ligations, 9 with 4 hour ligations, and 2 with 5 hour ligations). Nine of these animals (3 with 3 hour ligations and 6 with 4 hour ligations) were permitted to survive for a week and were restudied by ventriculography. Ventriculography was evaluated on a subjective grading system: 0, no change; 1+, slight improvement in ventricular wall motion; 2+, moderate improvement in ventricular wall motion; and 3+, major improvement or return to normal. All animals were assigned an identifying code number and were judged by one
5 Permanent Immediate death -Figures in immediately tFigures in restudy over
I
Acute study" jChronic study t
2 (1) 3 (2) 12 (6) 12 (5 ) 2 (2)
1 1 4 (2) 7
4 (3)
14
parentheses indicate animals put to death after acute study for histologic examination. parentheses indicate animals dying prior to a week later.
individual (1. J. M.) with no knowledge of the ligation status of the animal whose ventriculogram was being reviewed. Serial sections of myocardium were made at the time of sacrifice in all the animals and were stained with hematoxylin and eosin and hematoxylin-basic fuchsin-picric acid (HBFP).G
One animal with 1 hour ligation and 2 with 2 hour ligations were put to death right after ligature release, and 1 from each group was allowed to live for a week and then restudied (Table II). Six animals (3 hour ligations) were put to death immediately. Two of the remaining 6 animals in this group died less than a week postoperatively. The other 4 were put to death a week to 10 days later, and serial sections of myocardium were taken for histologic study. Animals subjected to 4 hours of ligation were similarly either put to death right after study (5 animals) or were allowed to live, restudied, and killed a week
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]I
(MONKEY)
I
(MONKEY)
Fig. 2. Type II LAD distribution in primate, with vessel extending well up the posterior interventricular groove.
Fig. 3. Diagram of primate heart with Type I LAD distribution: LAD ends at or on ventricular apex.
to 10 days later (7 animals). Both animals with 5 hour ligations were put to death right after study. Animals with permanent ligation were allowed to survive for I to 2 weeks, restudied, and killed. Three of the 7 animals in this latter group died within a week, prior to restudy. The remaining 4 animals were restudied (3 had ventriculography prior to sacrifice), and specimens stained with hematoxylin and eosin or HBFP were examined histologically.
utes of ligature placement. After ligature release, the area of significant S-T elevation decreased (at least two fewer points showing an S-T elevation of > 2 mv.) in all animals subjected to ligation for I and 2 hours and in 9 to 12 animals with ligation for 3 hours (Fig. 4). Of the 12 animals with 4 hour ligations, 7 had a clear decrease in infarct size; in the remaining 5, the change was negligible (Table Ill). In both of the animals with LAD ligation for 5 hours, epicardial ECG mapping showed a decrease in infarct size after ligature release. All animals studied at 1 week had a marked reduction in infarct size, although S-T changes were present in all. Animals with long-term ligation had persistent large infarcts after I week, although the area involved was frequently smaller than that seen shortly after ligation. The infarct size in this group was considerably larger than that in animals with 3 or 4 hour ligations which then survived for I week. pH. Surface pH measurements consistently decreased in Area A from a mean value of 7.38 (range 7.65 to 7.05) to 6.95 (range 7.45 to 6.60) (p < 0.05).
Results Fourteen animals died of ventricular fibrillation less than 1 hour after ligature placement. All of those surviving for an hour survived for the duration of the ligation. In 12 of these 14 early deaths, the LAD was "dominant"; that is, the vessel extended beyond the apex of the heart up the posterior interventricular sulcus (Fig. 2). Only 7 of the surviving 38 animals had a dominant LAD. The nondominant distribution ending on the ventricular apex is shown in Fig. 3. ECG. S-T elevation of more than 2 mv. occurred over a large area of the anterior left ventricle in all animals within 15 min-
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Fig. 4. Surface electrocardiographic recording shows minimal S-T changes prior to ligation probably secondary to slight electrode contact pressure. Dramatic S-T changes 10 minutes after ligation (8) are further accentuated after 3 hours of ligation (C). One hour after release of ligation, S-T elevations are back to normal in all but two leads (D). (A),
Table III. Results of electrocardiographic (ECG) mapping ECG study 1 week later
ECG study immediately Duration of ligation (hr.) 1 2 3 4 5 Permanent
Total
I Improvement after
I
Further decrease in infarct
ligature release
Total
2 3 9 7 2
1 4 7
1 4 7
4
4
2 3 12 12 2
Within 30 minutes after ligature release, all animals exhibited an increase in surface pH to near preligation levels (mean 7.28, range 7.55 to 6.90). In spite of the wide range in pH values, these differences were statistically significant (p < 0.0 I). pH changes in Area B, in this experiment, were not significant. Return to normal pH seemed to have no relationship to the duration of ligation.
Cine-ventriculography. Ventriculography showed that, within 2 hours of coronary ligation, all animals developed a large dyskinetic segment in the anterior wall and most developed huge anterior wall aneurysms (Fig. 5). These changes persisted but were significantly improved (2+ or 3+) in animals with permanent ligation restudied a week later (Table IV, Fig. 6). Following ligature release, significant im-
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Fig. 6. Ventriculogram at end systole from a permanently ligated LAD shows anterior wall bulge and increased end-systolic volume.
Fig. 5. End -systolic ventriculogram after 4 hOUTS of ligation shows large anterior wall aneurysm.
Table IV. Results of ventriculography Study before and after ligatur e release Duration of ligation (lir .)
Total
2 3
2 6 9 2 3*
4
5 Permanent
Significant improvement (2+ or 3+) 2 5 5 0
Study .again I week later
Total
Significant improvement (2+ or 3+)
3 6
4
3
3
3
"No immed iate restud y.
provement or return to normal (2+ or 3+) occurred within an hour in all with 2 hour ligation, 5 of 6 animals with 3 hour ligations, and 5 of 9 with 4 hour ligations. Most animals that survived for a week and were restudied showed marked improvement or returned to normal (3+): all animals subjected to 3 hours of ligation and 4 of 6 subjected to 4 hours of ligation. Ventriculography showed no improvement lh hour after ligature release in the animals with LAD ligation for 5 hours.
Histology. Early evidence of ischemic swelling of myofibrils was seen in several dogs with 4 hour ligations and in both with 5 hour ligations (Fig. 7). Shorter ligations (3 hours or less) produced no evidence of myocardial changes on hematoxylin and eosin sections. The HBFP stain was fuchsin positive, indicating early ischemia in all animals beyond I hour of ligation. Furthermore, 'the ischemic area seemed to increase up to 4 hours after ligation but appeared stable beyond that point (Fig. 8). Ligature
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release did not seem to affect the presence of HBFP fuchsin-staining muscle. All animals that survived for a week had definite histologic evidence of myocardial infarction even after 1 hour of ligation (Fig. 9) . Infarct size was smaller in the 4 animals with 3 hour ligations and in 5 of 7 animals with 4 hour ligations than in the 4 animals with permanent LAD ligation (Fig. 10) .
Discussion Ligation of the LAD in monkeys produces a large anterior wall infarct similar to that observed with acute LAD occlusion in man .7 • 8 The immediate mortality rate with acute ligation in the present study was 27 per cent. Death was due to ventricular fibrillation. Twelve of the 14 animals that died had a large LAD, extending into the posterior interventricular groove. In contrast, only 7 of 38 surviving animals had this dominant LAD distribution. In animals with a dominant LAD distribution, more blood supply to the left ventricle is derived from the LAD , and the resulting higher mortality rate in this group is probably a consequence of the much larger area of ischemic myocardium." LAD distribution in primates is similar to that noted in human hearts." By inference, it seems likely that a patient with a dominant LAD distribution is at much greater risk of dying from coronary occlusion than a patient with an LAD ending at the ventricular apex. ECG proved a reliable method of mapping the area of ischemic muscle right after ligation and it assisted in determining infarct size. It has been used by others with similar success.P- 12 The present information on ECG mapping does not provide incontrovertible evidence of reduction in infarct size since we do not have serial tracings during the course of the ligation. It is quite possible that the apparent area of S-T elevation decreases as the area of actual muscle necrosis increases, because these changes reflect myocardial cell injury and not cell death. Confirmation of these ob-
Fig. 7. Interstitial edema and inflammatory infiltrate by hematoxylin and eosin stain after 5 hour ligation . This was the most significant lesion found on any animal put to death immediately. (Original magn ification x150.)
servations with serial quantitative ECG tracings are the subject of research work currently in progress in our laboratory. Nevertheless, the very acute reduction in S-T elevation noted in the present study is in all probability a reflection of reversal of actual tissue ischemia with the onset of coronary reperfusion, Although the surface pH dropped rapidly following ligation, the value routinely increased rapidly toward normal after ligature release , even with persistence of a subsequently proved infarct. It appears that pH reflects primarily the presence or absence of blood flow and is of little value in determining muscle viability. P Furthermore, it seems that blood flow returns rapidly to the area of ischemia even in the presence of established infarction. This is consistent with the findings of Krug.>' who utilized fluorescent dye in cat hearts after coronary ligations lasting up to 90 minutes .
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Fig. 8. Hematoxylin-basic fuchsin-picric acid stain demonstrates large area of anterior wall ischemia after 4 hour ligation (arrows). The apparent thinning of the posterior, noninvolved wall is a sectioning artifact.
Fig. 9. Specimen from animal subjected to 1 hour of LAD ligation (survived for a week) shows patchy infarction by hematoxylin and eosin stain.
The present study documents a profound functional defect associated with anterior wall ischemia. The observed large asynergic areas or, more frequently, acute aneurysms of the anterior wall were usually reversible in animals with 3 hour ligations, and they improved significantly in over half of the animals (5 of 9) with 4 hour ligations. Release of 5 hour ligation produced no evident immediate change in ventricular contraction in the 2 animals studied. The greatest functional improvement appeared less than an hour after ligature release, though some further improvement was evident in animals restudied at 1 week. Krug':' again noted significant decrease in infarct size in cats, even after 90 minute coronary ligations, within 30 minutes after ligature release. Similarly, Maroko and co-workers:" have reported a decrease in infarct size in dogs after release of occlusions of up to 3 hours' duration. Significant ischemic injury was evident by HBFP stain after only an hour of ligation in the present study. Jennings and co-workers-" have demonstrated some irreversible ischemic injury in dogs after only 25 minutes of coronary occlusion. The
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Fig. 10. Specimen from animal subjected to 4 hours of LAD ligation (surv ived for a week) shows grossly evident myocardial infarct (between arrows). (Hematoxylin-basic fuchsin-picric acid ; original magnification xI O.)
size of fuchsin-staining (ischemic) area increased with time up to 4 hours after LAD occlusion. Furthermore, specimens examined I week after ligature release demonstrated some areas of actual infarction even with a I hour ligation. In animals that survived for 1 week after sustaining LAD occlusion for 4 hours or less, the infarct appeared patchy and significantly smaller than in those animals sustaining permanent occlusion that were put to death at I week. The significance of observations of myocardial ischemia based on the HBFP stain are not entirely clear. Though fuchsin staining was evident at very low-power magnification over the area of expected ischemia, the staining was frequently very patchy under higher power. Furthermore, fuchsin staining was occasionally noted in areas well beyond any possible LAD distribution. We have attempted to verify the HBFP stain in rat hind limbs with blood supply totally occluded for varying lengths of time. It appears that the HBFP stain is reliable, when positive, in mapping zones of known ischemia. Its capricious propensity to occasionally stain normal muscle makes it of questionable value in identifying early
ischemic zones where the presence and distribution of the ischemia are not certain. After considering the observations of Krug, H Maroko ," Jennings.!" and Herd son" in light of the present data, we have formulated the following hypothesis relevant to the evolution of the primate myocardial infarct. After acute occlusion for periods of 1 hour (present study) or less (Jennings' ") , areas of irreversible myocardial necrosis occur. The ischemic area increases in size for 3 to 4 hours after occlusion. A resulting large area of functional impairment is evident even at I hour. It appears that early muscle necrosis extends concentrically from a small, centrally located area to progressively involve an initially larger peripheral zone of ischemic, nonfunctional, but potentially viable myocardium. Furthermore, in primates , much of this peripheral zone remains potentially viable 3 hours and, in many animals, for at least 4 hours after acute occlusion. Consequently, in primates, reperfusion of an area of acute LAD occlusion for periods of up to 4 hours will produce a significant reduction in infarct size and immediate improvement in ventricular performance. This improvement
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was not evident functionally after 5 hours of occlusion, although reduction by EeG mapping was noted in the 2 animals studied. The present study then demonstrates a large zone of marginal ischemia around an acute infarct in primates. This experimental situation is not directly analogous to acute myocardial infarct in man, most notably because it does not allow for long-standing ischemia with attendent collateral development or for the possible presence of antecedent myocardial damage. Nevertheless, it suggests that a significant volume of muscle, rendered nonfunctional and doomed to gradual infarction, may be salvaged by prompt restoration of blood supply. These observations support continued investigation into the potential benefits of emergency myocardial revascularization following massive acute myocardial infarction. REFERENCES Scanlon, P. J., Nemickas, R., Tobin, J. R., Jr., Anderson, W., Montoya, A., and Pifarre, R.: Myocardial Revascularization During Acute Phase of Myocardial Infarction, J. A M. A 218, 207, 1971. 2 Hill, J. D., Kerth, W. J., Kelly, J. J., Jr., Selzer, A, Armstrong, W., Popper, R., and Cohn, K.: Emergency Aorto-coronary Bypass for Impending or Extending Myocardial Infarction (Abstr.), Circulation 42: 106, 1970 (Supp!. III).
3 Lambert, C. J., Adam, M., Geisler, G. F., Verzosa, E., Nazarian, M., and Mitchel, B. F., Jr.: Emergency Myocardial Revascularization for Impending Infarctions and Arrhythmias, J. THORAC. CARDIOVASC. SURG. 62: 522, 1971. 4 Mundth, E. D., Buckley, M. H., Daggett, W. M., Sander, C. A, and Austen, W. G.: Surgery for Complications of Acute Myocardial Infarction, Circulation 45: 1279, 1972. 5 Maroko, P. R., Libby, P., Covell, J. W., Sobel, B. E., Ross, J., Jr., and Braunwald, E.: Precordial ST Segment Elevation Mapping: An Atraumatic Method for Assessing Alterations in the Extent of Myocardial Ischemic injury. The Effects of Pharmacologic and Hemodynamic Interventions, Am. J. Cardio!' 29: 223, 1972. 6 Lie, J. T., Holley, K. E., Kampa, W. R., and
Titus, J. L.: New Histochemical Method for Morphologic Diagnosis of Early Stages of Myocardial Ischemia, Mayo Clin. Proc. 46: 319, 1971. 7 Hill, J. D., Malinow, M. R., McNalty, W. P., and Ochsner, A J.: Experimental Myocardial Infarction in Unanesthetized Monkeys, Am. Heart J. 84: 82, 1972. 8 Grayson, 1., and Irvine, M.: Myocardial Infarction in Monkeys: Studies on the Collateral Circulation After Acute Coronary Occlusion, Cardiovasc. Res. 2: 170, 1968. 9 McNamara, J. J., Fergusson, D., Yamase, H., Suehiro, G., and Soeter, J. R.: Significant Variations in Distribution of the Anterior Descending Coronary Artery in Primates, J. THORAC. CARDIOVASC. SURG. 65: 639, 1973. 10 James, T. N.: Anatomy of the Coronary Arteries, New York, 1961, Paul B. Hoeber, Inc. II Maroko, P. R., Kjeksus, J. K., Sobel, B: E., Watanabe, T., Covell, J. W., Ross, 1., Jr., and Braunwald, E.: Factors Influencing Infarct Size Following Experimental Coronary Artery Occlusion, Circulation 43: 67, 1971. 12 Maroko, P. R., Bernstein, E. F., Libby, P., DeLaria, G. A., Covell, J. W., Ross, J., Jr., and Braunwald, E.: Effects of Intra-aortic Balloon Counterpulsation on the Severity of Myocardial Ischemic Injury Following Acute Coronary Occlusion, Circulation 45: 1150, 1972. 13 McNamara, J. J., Soeter, J. R., Suehiro, G. T., Anema, R. J., and Smith, G. T.: Surface pH Changes During and After Myocardial Ischemia in Primates, 1. THORAC. CARDIOVASC. SURG. 67: 191, 1973. 14 Krug, A.: The Extent of Ischemic Damage in the Myocardium of the Cat After Permanent and Temporary Coronary Occlusion, J. THORAC. CARDIOVASC. 60: 242, 1970. 15 Maroko, P. R., Ginks, W. R., Libby, P., Sobel, B. E., Shell, W. E., and Ross, J. Jr.: Salvage of Myocardial Tissue by Coronary Artery Reperfusion Following Acute Coronary Occlusion, Am. J. Cardio!. 29: 278, 1972. 16 Jennings, R. B., Sommers, H. M., Smyth, G. A., Flack, H. A, and Linn, H.: Myocardial Necrosis Induced by Temporary Occlusion of a Coronary Artery in the Dog, Arch. Patho!. 70: 68, 1960. 17 Herdson, P. B., Sommers, H. M., and Jennings, R. B.: A Comparative Study of the Five Structures of Normal and Ischemic Dog Myocardium With Special Reference to Early Changes Following Temporary Occlusion of a Coronary Artery, Am. J. Patho!. 46: 367, 1965.