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Acceleration of cell necrosis following reperfusion after ischemia in the pig heart without collateral circulation
Acceleration of Cell Necrosis Following Reperfusion After lschemia iir the Pig Heart Without Collateral Circulation Hisayoshi Fujiwara, MD, Tomoya Onodera, MD, Masaru Tanaka, MD, Shunichi Miyazaki, MD, Der-Jinn Wu, MD, Mitsuo M&suda, MD, Atsushi Kawamura, MD, Moriharu Ishida, MD, Genzou Takemura, MD, Yasunori Fujiwara, MD, Takako Fujiwara, MD, and Chuichi Kawai MD
A study of whether reperfusion accelerates cell death was performed in 35 pig hearts without collateral circulation.,ln 15 animals, the distal onethird of the left anterior descending coronary artery was occluded for 1 hour followed by I-, 3-, or 7hour reperfusion in 5 animals each. As controls, 5 hearts each were examined after 1,2,4 and 8 hours of occlusion of the artery without reperfusion. Heart rate and aortic pressure before and during occlusion and reperfusion did not change in any group. The subepicardial and subendocardial regional blood flow decreased to almost zero in all hearts afier occlusion (SS f 1 to 2 f 2) but recovered during reperfusioh (65 f 15 ml/i66 g/min). Specimens were histologically examined by an enzyme method using nitrotetrazolium blue, an immunohistochemical method using myoglobin antibody, by staining with hematoxylin-eosin and Masson’s trichrome. In the control hearts, clear demarcation of the infarct area was observed 4 hours after occlusion. However, in the reperfusion group, clear demarcation of the infarct was seen after lhour reperfusion, namely, 2 hours after the onset, of infarct. Demarcation was seen not only in the tissue with contraction band necrosis, but also in the tissue with coagulation necrosis. Therefore, it is concluded that reperfusion accelerates cell death due to both contraction band necrosis and coagulation necrosis. (Am J Cardiol 1989;63:14E-18E)
Frdm the Third Division, Department of Internal Medicine and Department of Cardiovascular Surgery, Faculty of Medicine, Kyoto University and Department of Food Science, Kyoto Women’s University, Kyoto, Japan. Address for reprints: Chuichi Kawai, MD, Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
14E
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VOLUME
63
urrent treatment for acute myocardial infarction is recanalization of the occlusive artery by coronary thrombolysisor angioplasty, or both. 1,2Therefore, there is rapidly increasing interest in reperfusion injury, such as contraction band necrosis and hemorrhage.3-6 Numerous reports have described reperfusion injury after ischemia. Figure 1 shows the relation among reversible and irreversible cellular damage, contraction band necrosis, coagulation necrosis and bleeding during ischemia and reperfusion. Although clinical data suggest that reperfusion accelerates cell death,7m9 it has not yet been histologically clarified. Therefore, the present study was performed to determine whether reperfusion accelerates cell death, using pig hearts that lack collateral circulation.iOJ1
C
METHODS
Thirty-five farm pigs, each weighing 20 to 30 kg, were used in this study. In 15 animals, the distal one-third of the left anterior descending coronary artery was occluded for 1 hour followed by l-, 3-, or 7-hour reperfusion in 5 animals each. In the other 20 pigs used as controls, 5 hearts each were occluded for 1,2,4 and 8 hours without reperfusion. The animals were sedated with ketamine (2 mg/kg) and anesthetized with sodium pentobarbital(20 mg/kg). Through a median sternotomy, the heart was exposed and suspended in a pericardial cradle. After the baseline measurement of the aortic pressure, the limb lead of the electrocardiogram, and epicardial and endocardial regional blood flow in the risk area of the occluded artery by the generated hydrogen gas clearance method, the left anterior descending coronary artery was completely occluded with a Vesseloops (Med General) rubber band. The electrocardiogram and blood pressure were continuously monitored throughout the procedure. Regional blood flow in the risk area was measured at 30-minute intervals. After the animal was killed, the left and right coronary arteries were cannulated with 6Fr polyethylene tubes. The same site on the distal one-third of the left anterior descending coronary artery was again completely ligated with a rubber band. Postmortem coronary angiography was performed to determine the risk area of the
occluded artery, as previously described.5 Then, the heart was sliced in l-cm serial sections in a plane parallel to the atrioventricular groove. The risk area was clearly demarcated by the absence of the contrast medium in the x-ray film. One slice, containing the risk area, was incubated for 15 to 30 minutes at 37’C in 0.01 M phosphate buffer solution at pH 7.4 containing nitrotetrazolium blue (NTB, Sigma: 50 mg/lOO ml).‘* The infarct area was identified by the absence of a dark blue color as observed by the naked eye. Then, the slice was fixed with 10% formalin, embedded in paraffin and cut serially into 4-pm thickness. These were stained with hematoxylin-eosin and Masson’s trichrome, and examined immunohistochemically. Immunohistochemical analysis using myoglobin antibody was performed as previously described.t3 The degree of demarcation of infarct area was histologically classified into slight and clear (Fig. 2). The degree of demarcation wasbased on the intensity of negative staining in the immunohistochemical method macroscopically and microscopically and in the nitrotetrazolium blue stain macroscopically. In the hematoxylin-eosin and Masson’s trichrome stains, the demarcation was microscopically based on the intensity of deep redness with granular-banded cytoplasm (Fig. 2). Quantification of infarct size, contraction band necrosis and coagulation necrosis (Fig. 3) were done as reported previously.5
Late irreversible cellular damage
Early
irreversible
cellular
damage
Reversible cellular
damage
FIGURE 1. The relation among reversible and irreversible cellular damage, contraction band necrosis (CBN), coagulation necrosis (CN) and bleeding (6) during ischemia and reperfusion. Depending on the duration of &hernia, the degree of ischemic myocardial cellular damage increases and finally progresses to coagulation necrosis. The degree is classified into reversfble, early irreversible and late irreversible cellular damage. No necrosis is observed when reperfusion is performed at the stage of reversible cellular damage. However, at the stage of early irreversible cellular damage, reperfusion is associated with contraction band necrosis, and at the stage of late irreversible cellular damage with coagulation necrosis and bleeding. The present study revealed that reperfusion rapidly accelerated myocardial cell necrosis. Note that the acute curve of reperfusion indicates acceleration of cell necrosis. If it is possible to reduce the infarct areas by controlled reperfusion, the target tissue is myocytes with early irreversible cellular damage. However, it is unknown whether controlled reperfusion can salvage the myocytes with ear& irreversible cellular damage. Dotted arrows indicate reperfusion.
FIGURE 2. Clear demarcation of cell necrosis. Top, enzyme method using nitrotetrazolium blue. Note the absence of staining, indicating myocardial cell necrosis, by nitrotetrazolium blue on the center of the heart. The area is tocalized within the risk area. Middle, immunohistochemical method using myoglobin antibody. Note no staining by myoglobin, indicating myocardial cell necrosis, on the left side. (Magnification X 40.) Boftom, note deop redness with contraction band necrosis, indicating myocardial cell necrosis, on the left side. (Masson’s trichrome stain, X 200.)
THE AMERICAN
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MARCH
7, 1989
15E
A SYMPOSIUM:
FIGURE
TABLE
MYOCARDIAL
3. Contraction
band
I Hemodynamics
ISCHEMIA
necrosis
(A),and
and Regional
coagulation
necrosis
Blood Flow
;,
Heart Rate (beats/min) DO DR
115% 13 110 f 13
113&15 105f15
116f15 120f15
Mean Aortic Pressure DO DR
9Of15 90f16
95f12 92f12
I 120f13 115f14
114~12 116f13
(mm Hg)
95f15 90f15
Regional Blood Flow (ml/KID
9Odcl5 88f 15
88% 13 90f 12
g/min)
<: * No. of pigs. Data indicate mean f standard deviations. DO = duration of occlusion at the distal one-third of the left anterior Coronary artery; DR = duration of reperfusion after 1 hour occlusion anterior descending coronary artery. End = endocardial; Epi = epicardial.
descending at the left
Statistical analysis: Statistical analysis was performed by repeated-measures analysis for hemodynamic and blood flow data. The comparison between hearts with and without reperfusion in the degree of demarcation of infarct area was analyzed by Mann-Whitney’s U-test. The histologically determined percent area was analyzed by a l-way analysis of variance and the multiple comparison test. The level of significance was taken as p cO.05.
RESULTS No significant changes were detected in heart rate or aortic pressure before and during occlusion and reperfusion in any of the groups (Table I). The subendocardial and subepicardial regional blood flow was 87 f 11 and 88 f 10 ml/ 100 g/mm before occlusion, and 2 f 2 and 2 f 2 ml/ 100 g/min after ligation, and 70 f 15 and 65 f 15 ml/100 g/min during reperfusion after 1 hour of occlusion, respectively (Table I). In all hearts, the regional blood flow was within 7 ml/ 100 g/min during occlusion. There was no significant difference in regional blood flow 16E
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(B). (Masson’s
trichrome
stain,
X 400.)
among groups before and during occlusion and reperfusion. Table II lists the data of histology. In the group without reperfusion, no clear histologic differences were detected by the enzyme method using nitrotetrazolium blue, the immunohistochemical method using myoglobin antibody, and hematoxylin-eosin and Masson’s trichrome stains between risk and normal areas in hearts at 1 and 2 hours after occlusion. The demarcation varied between slight or clear 4 hours after occlusion but was clearly seen 8 hours after occlusion. However, in the reperfusion group, demarcation was clearly observed after 1 hour of reperfusion. Table III lists the percent infarct size, percent contraction band necrosis and percent coagulation necrosis in each group. There were no significant differences in percent infarct size, percent contraction band necrosis or percent coagulation necrosis among the l-, 3- and 7-hour reperfusion groups after l-hour occlusion. In each of the hearts, coagulation necrosis was found in the inner third of the left ventricular wall of the risk area and contraction band necrosis in the middle and outer thirds. The percent infarct size to the risk area was smaller in the reperfusion groups than in the groups with 8-hour occlusion. Most of the salvaged area was localized in the outer third of the risk area. Contraction band necrosis occupied most of the infarct tissues in the reperfusion groups, but was rarely seen in the occlusion groups. DISCUSSION In the present study, hemodynamic measurements before and during occlusion and reperfusion did not change, This was due to the small infarct after ligation at the distal one-third of the left anterior descending coronary artery. The subendocardial and subepicardial regional blood flow by the generated hydrogen gas clearance method was below the resolution of the method (<7 ml/ 100 g/min) during occlusion (transmurally, almost zero in all the pigs). Regional blood flow was restored during reperfusion. This confirmed the previous studies in the pig heart without collateral circulation.lO;” Thus, the present study is different from the model using the dog heart, which has rich collateral circulation, and the histologic differences are independent of regional blood flow and hemodynamics.
TABLE II Comparison Necrosis
Between
Hearts
DO
of the Degree of Demarcation of Cell with Occlusion and Reperfusion 2 hrs 5’
1 hr
5’ DR
4 hrs 5* 3 hrs 5”
1 hr
5* Enzyme
DO
DR
Method
No Slight Clear
Using
Nitrotetrazolium
5
4
0 0
1 0
No
0
Slight Clear
0 5
lmmunohistochemical
Method
Using
8 hrs
Infarct Size, Contraction Necrosis
5’ 5*
0 2 3 0
0 0 5 0
0
0
5
5
Between Duration Band Necrosis
% Infarct Size
7 hrs
Blue
Myoglobin
TABLE Ill Relation
8-hour occlusion l-hour reperfusion after l-hour occlusion 3-hour reperfusion
95 f 3
of lschemia and or Coagulation
% Contraction Band
Necrosis
2f2
% Coagulation Necrosis
98f3 30% 12
81 f 10
70f
12
78fll
67x1~ 11
33f
11
80f9
68Ztll
32f
12
after l-hour occlusion 7-hour reperfusion
after l-hour
Antibody
occlusion DO
DR
No Slight Clear
5
0
0
0
0 0
5 0
0
0
5 0
5 0
0
0
5
5
0 5
0 5
0 0
0 0
5
5
5
0
0
0
No
0
Slight Clear
0 5 Hematoxylin-Eosin
DO
DR
[\!o Slight Clear No Slight Clear
DO
DR
Stain
5
4
0 0
1 0 0
Masson’s
Trichrome
Stain
5
1
0
0
0 0
4
4
0
1
No
0
0
0 5 0
Slight Clear
0
0
0
5
5
5
No Slight Clear
Percent infarct area indicates the percentage of infarct area to the risk area, percent contraction band necrosis the percentage of contraction band necrosis to the infarct area, percent coagulation necrosis the percentage of coagulation necrms to the infarct area. Data indicate mean f standard deviations.
nl.l.“^l,i^l:^^* ^r i^Td.l^ I
In the control hearts without reperfusion, the degree of defect of nitrotetrazolium blue staining in the enzyme method and of myoglobin in the immunohistochemical method was none in hearts with l-hour occlusion, slight or none in hearts with a-hour occlusion, clear or slight in hearts with 4-hour occlusion, but clear after 8 hours of occlusion. Our previous studies revealed that, in the pig heart, irreversible cellular damage occurs in the inner third of the left ventricular wall 30 to 40 minutes after occlusion of the coronary artery, in the inner, middle and a part of the outer third after l-hour occlusion and in the entire ventricular wall after a-hour occlusion.5,10,1 l Therefore, slight and clear demarcation in the enzyme and histochemical methods indicates the process of cell necrosis in the infarct area. The present data revealed that l-hour reperfusion after l-hour occlusion caused a clear defect of nitrotetrazolium blue and myoglobin staining in the inner, middle and a part of the outer third of the risk area. No staining was found in the tissues with contraction band necrosis and in the tissues with coagulation necrosis. This indicates that reperfusion rapidly accelerates cell necrosis of the myocardium. Such rapid progression of cell necrosis is proba-
bly due to the washout effect of reperfusion. Many clinical reports have shown early peaking creatine kinase-MB and lactate dehydrogenase or early accumulation of technetium-99m pyrophosphate in patients with acute myocardial infarction in whom recanalization was achieved by coronary thrombolysis. 7-9 These phenomena are explained by the acceleration of cell death after reperfusion. It is well known that various microscopic methods require 6 to 8 hours after the start of infarction to distinguish infarct myocardium. l4 However, the present data revealed that acute myocardial infarction could be detected within 2 hours after the onset, when the hearts were reperfused. Probably, microscopic detection of acute myocardial infarction in human hearts within 2 hours after the onset may indicate reperfusion after ischemia. Thus, we conclude that reperfusion follows both acceleration of cell death and the salvage of the ischemic myocytes, depending on the degree of ischemic cellular damage before reflow. cknowledgment: We thank Daniel Mrozek for criticism of the manuscript, and Satoko Tomita and Michie Jinnai for assistance with preparation of the manuscript.
REFERENCES 1. Simoons ML, Serruys PW, van der Brand M, Res J, Verhcugt FWA, Kraus XH, Remme WJ, Bar F, de Zwaan C, van der Laarse A, Vermeer F, Lubsen J. Early thrombolysis in acute myocardial infarction: limitation of infarct size and improved survival. JACC 1986;7:717-723. 2. Hart&r GO, Rutherford BD, McConakay DR. Percutaneous transluminal coronary angioplasty: application for acute myocardial infarction. Am J Cardiol 1984;53:117C-121 C. 3. Jennings RB, Reimer KA. Factors involved in salvaging ischemic myocardium: effect of repcrfusion of arterial blood. Circulation 1983;68:suppl H-25-1-36. 4. Fujiwara H, Onodera T, Tanaka M, Fujiwara T, Wu DJ, Kawai C, Hamashima Y. A ciinicopathologic study of patients with hemorrhagic myocardial infarction treated with selective coronary thrombolysis with urokinase. Circulation 1986:73:749-757. 5. Miyazaki S, Fujiwara H, Onodera T, Kihara Y, Matsuda M, Wu DJ, Nakamura Y, Kumada T, Sasayama S, Kawasi C, Hamashima Y. Quantitative analysis of contraction band and coagulation necrosis after ischemia and reperfusion in the porcine heart. Circulation 1987;75:1074-1082.
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JOURNAL
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7. 1989
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MYOCARDIAL
ISCHEMIA
6. Matsuda M, Fujiwara H, Onodera T, Tanaka M, Wu DJ, Fujiwara T, Hamashima Y, Kawasi C. Quantitative analysis of infarct size, contraction band necrosis and coagulation necrosis in human autopsied hearts with acute myocardial infarction after treatment with selective intiacoronary thrombolysis. Circulation 1987;76:981-989.
7. Schafer J, Mathey DG, Montz R, Bleifeld W, Stritzke P. Use of dual intracoronary scintigraphy with thallium-201 and technetium-99 m pyrophosphate to predict improvement in left ventricular wall motion immediately after intracoronary thrombolysis in acute myocardial infarction. JACC 1983;2:737-742. 8. Wheelen K, Wolfe C, Corbett J, Rude RE, Winniford M, Parkey RW, Buja LM, Willerson JT. Early positive technetium-99 m stannous pyrophosphate images as a marker of reperfusion after thrombolytic therapy for acute myocardial infarction. Am J Cardiol 1985;56:252-258. 9. Kondo M, Takahashi M, Matsuda T, Kume N, Yuzuki Y, Shimono Y, Fujiwara H. Clinical significance of early myocardial To99 m pyrophosphate uptake in patients with acute myocardial infarction. Am Heart J 1987;113:250256.
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10. Fujiwara H, Ashraf M, Sato S, Millard RW. Transmural cellular damage and blood flow distribution in early ischemia in pig hearts. CircRes 1982;51:683693. 11. Fujiwara H, Ashraf M, Millard RW, Sato S, Schwarz A. Effects of diltiazem, a calcium channel inhibitor, in retarding cellular damage produced during early myocardial ischemia in pigs: a morphometric and ultrastructural analysis. JACC
1984:3:1427-1437.
12. Anderson KR, Popple A, Parker DJ, Sayer R, Trickey RJ, Davies MJ. An experimental assessment of macroscopic enzyme techniques for the autopsy demonstration of myocardial infarction. J Pathol 1979;127:93-98. 13. Fujiwara H, Fujiwara T, Tanaka M, Miyazaki S, Wu DJ, Matsuda M, Sasayama S, Kawai C. Detection of early myocardial infarction in formalintixsation-paraffin-embedded tissue by immunohistochemical method using myoglobin antibody. Am J Cardiouasc Pathol 1988:2:26-32. 14. Alpert JS, Braunwald E. Pathological and clinical manifestations of acute myocardial infarction. In: Braunwald E, ed. Heart Disease. Philadelphia: WB Saunders, 1980:1309-1311.
A study of whether reperfusion accelerates cell death was performed in 35 pig hearts without collateral circulation.,ln 15 animals, the distal onethird of the left anterior descending coronary artery was occluded for 1 hour followed by I-, 3-, or 7hour reperfusion in 5 animals each. As controls, 5 hearts each were examined after 1,2,4 and 8 hours of occlusion of the artery without reperfusion. Heart rate and aortic pressure before and during occlusion and reperfusion did not change in any group. The subepicardial and subendocardial regional blood flow decreased to almost zero in all hearts afier occlusion (SS f 1 to 2 f 2) but recovered during reperfusioh (65 f 15 ml/i66 g/min). Specimens were histologically examined by an enzyme method using nitrotetrazolium blue, an immunohistochemical method using myoglobin antibody, by staining with hematoxylin-eosin and Masson’s trichrome. In the control hearts, clear demarcation of the infarct area was observed 4 hours after occlusion. However, in the reperfusion group, clear demarcation of the infarct was seen after lhour reperfusion, namely, 2 hours after the onset, of infarct. Demarcation was seen not only in the tissue with contraction band necrosis, but also in the tissue with coagulation necrosis. Therefore, it is concluded that reperfusion accelerates cell death due to both contraction band necrosis and coagulation necrosis. (Am J Cardiol 1989;63:14E-18E)
Frdm the Third Division, Department of Internal Medicine and Department of Cardiovascular Surgery, Faculty of Medicine, Kyoto University and Department of Food Science, Kyoto Women’s University, Kyoto, Japan. Address for reprints: Chuichi Kawai, MD, Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
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THE AMERICAN
JOURNAL
OF CARDIOLOGY
VOLUME
63
urrent treatment for acute myocardial infarction is recanalization of the occlusive artery by coronary thrombolysisor angioplasty, or both. 1,2Therefore, there is rapidly increasing interest in reperfusion injury, such as contraction band necrosis and hemorrhage.3-6 Numerous reports have described reperfusion injury after ischemia. Figure 1 shows the relation among reversible and irreversible cellular damage, contraction band necrosis, coagulation necrosis and bleeding during ischemia and reperfusion. Although clinical data suggest that reperfusion accelerates cell death,7m9 it has not yet been histologically clarified. Therefore, the present study was performed to determine whether reperfusion accelerates cell death, using pig hearts that lack collateral circulation.iOJ1
C
METHODS
Thirty-five farm pigs, each weighing 20 to 30 kg, were used in this study. In 15 animals, the distal one-third of the left anterior descending coronary artery was occluded for 1 hour followed by l-, 3-, or 7-hour reperfusion in 5 animals each. In the other 20 pigs used as controls, 5 hearts each were occluded for 1,2,4 and 8 hours without reperfusion. The animals were sedated with ketamine (2 mg/kg) and anesthetized with sodium pentobarbital(20 mg/kg). Through a median sternotomy, the heart was exposed and suspended in a pericardial cradle. After the baseline measurement of the aortic pressure, the limb lead of the electrocardiogram, and epicardial and endocardial regional blood flow in the risk area of the occluded artery by the generated hydrogen gas clearance method, the left anterior descending coronary artery was completely occluded with a Vesseloops (Med General) rubber band. The electrocardiogram and blood pressure were continuously monitored throughout the procedure. Regional blood flow in the risk area was measured at 30-minute intervals. After the animal was killed, the left and right coronary arteries were cannulated with 6Fr polyethylene tubes. The same site on the distal one-third of the left anterior descending coronary artery was again completely ligated with a rubber band. Postmortem coronary angiography was performed to determine the risk area of the
occluded artery, as previously described.5 Then, the heart was sliced in l-cm serial sections in a plane parallel to the atrioventricular groove. The risk area was clearly demarcated by the absence of the contrast medium in the x-ray film. One slice, containing the risk area, was incubated for 15 to 30 minutes at 37’C in 0.01 M phosphate buffer solution at pH 7.4 containing nitrotetrazolium blue (NTB, Sigma: 50 mg/lOO ml).‘* The infarct area was identified by the absence of a dark blue color as observed by the naked eye. Then, the slice was fixed with 10% formalin, embedded in paraffin and cut serially into 4-pm thickness. These were stained with hematoxylin-eosin and Masson’s trichrome, and examined immunohistochemically. Immunohistochemical analysis using myoglobin antibody was performed as previously described.t3 The degree of demarcation of infarct area was histologically classified into slight and clear (Fig. 2). The degree of demarcation wasbased on the intensity of negative staining in the immunohistochemical method macroscopically and microscopically and in the nitrotetrazolium blue stain macroscopically. In the hematoxylin-eosin and Masson’s trichrome stains, the demarcation was microscopically based on the intensity of deep redness with granular-banded cytoplasm (Fig. 2). Quantification of infarct size, contraction band necrosis and coagulation necrosis (Fig. 3) were done as reported previously.5
Late irreversible cellular damage
Early
irreversible
cellular
damage
Reversible cellular
damage
FIGURE 1. The relation among reversible and irreversible cellular damage, contraction band necrosis (CBN), coagulation necrosis (CN) and bleeding (6) during ischemia and reperfusion. Depending on the duration of &hernia, the degree of ischemic myocardial cellular damage increases and finally progresses to coagulation necrosis. The degree is classified into reversfble, early irreversible and late irreversible cellular damage. No necrosis is observed when reperfusion is performed at the stage of reversible cellular damage. However, at the stage of early irreversible cellular damage, reperfusion is associated with contraction band necrosis, and at the stage of late irreversible cellular damage with coagulation necrosis and bleeding. The present study revealed that reperfusion rapidly accelerated myocardial cell necrosis. Note that the acute curve of reperfusion indicates acceleration of cell necrosis. If it is possible to reduce the infarct areas by controlled reperfusion, the target tissue is myocytes with early irreversible cellular damage. However, it is unknown whether controlled reperfusion can salvage the myocytes with ear& irreversible cellular damage. Dotted arrows indicate reperfusion.
FIGURE 2. Clear demarcation of cell necrosis. Top, enzyme method using nitrotetrazolium blue. Note the absence of staining, indicating myocardial cell necrosis, by nitrotetrazolium blue on the center of the heart. The area is tocalized within the risk area. Middle, immunohistochemical method using myoglobin antibody. Note no staining by myoglobin, indicating myocardial cell necrosis, on the left side. (Magnification X 40.) Boftom, note deop redness with contraction band necrosis, indicating myocardial cell necrosis, on the left side. (Masson’s trichrome stain, X 200.)
THE AMERICAN
JOURNAL
OF CARDIOLOGY
MARCH
7, 1989
15E
A SYMPOSIUM:
FIGURE
TABLE
MYOCARDIAL
3. Contraction
band
I Hemodynamics
ISCHEMIA
necrosis
(A),and
and Regional
coagulation
necrosis
Blood Flow
;,
Heart Rate (beats/min) DO DR
115% 13 110 f 13
113&15 105f15
116f15 120f15
Mean Aortic Pressure DO DR
9Of15 90f16
95f12 92f12
I 120f13 115f14
114~12 116f13
(mm Hg)
95f15 90f15
Regional Blood Flow (ml/KID
9Odcl5 88f 15
88% 13 90f 12
g/min)
<: * No. of pigs. Data indicate mean f standard deviations. DO = duration of occlusion at the distal one-third of the left anterior Coronary artery; DR = duration of reperfusion after 1 hour occlusion anterior descending coronary artery. End = endocardial; Epi = epicardial.
descending at the left
Statistical analysis: Statistical analysis was performed by repeated-measures analysis for hemodynamic and blood flow data. The comparison between hearts with and without reperfusion in the degree of demarcation of infarct area was analyzed by Mann-Whitney’s U-test. The histologically determined percent area was analyzed by a l-way analysis of variance and the multiple comparison test. The level of significance was taken as p cO.05.
RESULTS No significant changes were detected in heart rate or aortic pressure before and during occlusion and reperfusion in any of the groups (Table I). The subendocardial and subepicardial regional blood flow was 87 f 11 and 88 f 10 ml/ 100 g/mm before occlusion, and 2 f 2 and 2 f 2 ml/ 100 g/min after ligation, and 70 f 15 and 65 f 15 ml/100 g/min during reperfusion after 1 hour of occlusion, respectively (Table I). In all hearts, the regional blood flow was within 7 ml/ 100 g/min during occlusion. There was no significant difference in regional blood flow 16E
THE AMERICAN
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63
(B). (Masson’s
trichrome
stain,
X 400.)
among groups before and during occlusion and reperfusion. Table II lists the data of histology. In the group without reperfusion, no clear histologic differences were detected by the enzyme method using nitrotetrazolium blue, the immunohistochemical method using myoglobin antibody, and hematoxylin-eosin and Masson’s trichrome stains between risk and normal areas in hearts at 1 and 2 hours after occlusion. The demarcation varied between slight or clear 4 hours after occlusion but was clearly seen 8 hours after occlusion. However, in the reperfusion group, demarcation was clearly observed after 1 hour of reperfusion. Table III lists the percent infarct size, percent contraction band necrosis and percent coagulation necrosis in each group. There were no significant differences in percent infarct size, percent contraction band necrosis or percent coagulation necrosis among the l-, 3- and 7-hour reperfusion groups after l-hour occlusion. In each of the hearts, coagulation necrosis was found in the inner third of the left ventricular wall of the risk area and contraction band necrosis in the middle and outer thirds. The percent infarct size to the risk area was smaller in the reperfusion groups than in the groups with 8-hour occlusion. Most of the salvaged area was localized in the outer third of the risk area. Contraction band necrosis occupied most of the infarct tissues in the reperfusion groups, but was rarely seen in the occlusion groups. DISCUSSION In the present study, hemodynamic measurements before and during occlusion and reperfusion did not change, This was due to the small infarct after ligation at the distal one-third of the left anterior descending coronary artery. The subendocardial and subepicardial regional blood flow by the generated hydrogen gas clearance method was below the resolution of the method (<7 ml/ 100 g/min) during occlusion (transmurally, almost zero in all the pigs). Regional blood flow was restored during reperfusion. This confirmed the previous studies in the pig heart without collateral circulation.lO;” Thus, the present study is different from the model using the dog heart, which has rich collateral circulation, and the histologic differences are independent of regional blood flow and hemodynamics.
TABLE II Comparison Necrosis
Between
Hearts
DO
of the Degree of Demarcation of Cell with Occlusion and Reperfusion 2 hrs 5’
1 hr
5’ DR
4 hrs 5* 3 hrs 5”
1 hr
5* Enzyme
DO
DR
Method
No Slight Clear
Using
Nitrotetrazolium
5
4
0 0
1 0
No
0
Slight Clear
0 5
lmmunohistochemical
Method
Using
8 hrs
Infarct Size, Contraction Necrosis
5’ 5*
0 2 3 0
0 0 5 0
0
0
5
5
Between Duration Band Necrosis
% Infarct Size
7 hrs
Blue
Myoglobin
TABLE Ill Relation
8-hour occlusion l-hour reperfusion after l-hour occlusion 3-hour reperfusion
95 f 3
of lschemia and or Coagulation
% Contraction Band
Necrosis
2f2
% Coagulation Necrosis
98f3 30% 12
81 f 10
70f
12
78fll
67x1~ 11
33f
11
80f9
68Ztll
32f
12
after l-hour occlusion 7-hour reperfusion
after l-hour
Antibody
occlusion DO
DR
No Slight Clear
5
0
0
0
0 0
5 0
0
0
5 0
5 0
0
0
5
5
0 5
0 5
0 0
0 0
5
5
5
0
0
0
No
0
Slight Clear
0 5 Hematoxylin-Eosin
DO
DR
[\!o Slight Clear No Slight Clear
DO
DR
Stain
5
4
0 0
1 0 0
Masson’s
Trichrome
Stain
5
1
0
0
0 0
4
4
0
1
No
0
0
0 5 0
Slight Clear
0
0
0
5
5
5
No Slight Clear
Percent infarct area indicates the percentage of infarct area to the risk area, percent contraction band necrosis the percentage of contraction band necrosis to the infarct area, percent coagulation necrosis the percentage of coagulation necrms to the infarct area. Data indicate mean f standard deviations.
nl.l.“^l,i^l:^^* ^r i^Td.l^ I
In the control hearts without reperfusion, the degree of defect of nitrotetrazolium blue staining in the enzyme method and of myoglobin in the immunohistochemical method was none in hearts with l-hour occlusion, slight or none in hearts with a-hour occlusion, clear or slight in hearts with 4-hour occlusion, but clear after 8 hours of occlusion. Our previous studies revealed that, in the pig heart, irreversible cellular damage occurs in the inner third of the left ventricular wall 30 to 40 minutes after occlusion of the coronary artery, in the inner, middle and a part of the outer third after l-hour occlusion and in the entire ventricular wall after a-hour occlusion.5,10,1 l Therefore, slight and clear demarcation in the enzyme and histochemical methods indicates the process of cell necrosis in the infarct area. The present data revealed that l-hour reperfusion after l-hour occlusion caused a clear defect of nitrotetrazolium blue and myoglobin staining in the inner, middle and a part of the outer third of the risk area. No staining was found in the tissues with contraction band necrosis and in the tissues with coagulation necrosis. This indicates that reperfusion rapidly accelerates cell necrosis of the myocardium. Such rapid progression of cell necrosis is proba-
bly due to the washout effect of reperfusion. Many clinical reports have shown early peaking creatine kinase-MB and lactate dehydrogenase or early accumulation of technetium-99m pyrophosphate in patients with acute myocardial infarction in whom recanalization was achieved by coronary thrombolysis. 7-9 These phenomena are explained by the acceleration of cell death after reperfusion. It is well known that various microscopic methods require 6 to 8 hours after the start of infarction to distinguish infarct myocardium. l4 However, the present data revealed that acute myocardial infarction could be detected within 2 hours after the onset, when the hearts were reperfused. Probably, microscopic detection of acute myocardial infarction in human hearts within 2 hours after the onset may indicate reperfusion after ischemia. Thus, we conclude that reperfusion follows both acceleration of cell death and the salvage of the ischemic myocytes, depending on the degree of ischemic cellular damage before reflow. cknowledgment: We thank Daniel Mrozek for criticism of the manuscript, and Satoko Tomita and Michie Jinnai for assistance with preparation of the manuscript.
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