European Heart Journal (1991) 12 (Supplement D), 28-31
Modulation of the inflammatory response in experimental myocardial infarction R. M.
BOHLE*,
S. PiCHt
AND
H. H.
KLEIN||
Institute of Pathology, General Hospital of Detmold, Academic Teaching Hospital of the University of Munster*, Departments of Cardiology, University of Gottingen't and University of Marburg\\, Germany KEY WORDS: Experimental myocardial infarction, reperfused porcine hearts, inflammatory response, antiphlogistic therapy.
Introduction
The size of myocardial infarction is influenced by the localization of the coronary artery occlusion, duration of ischaemia, left ventricular oxygen consumption during ischaemia and collateral blood flow'1'31. Clinical studies have demonstrated that early coronary reperfusion can limit mortality and myocardial necrosis[4l5]. Experimental studies in dogs suggested that reperfusion per se might be responsible for myocardial injury163. The tissue-destructive potential of inflammatory cells is mediated by proteolytic lysosomal enzymes, active metabolites of arachidonic acid, free radicals or oxidative inhibition of proteinase inhibitors and is suspected to be a factor determining myocardial infarct size after reperfusion[7-8]. In dogs, experimental myocardial infarct size was significantly reduced by leucopenia, application of anti-neutrophil antibodies and antiphlogistic drugs; however the influence of collateral blood flow cannot be excluded in these experimental models19"1 n . Since porcine hearts have no collateral circulation they are a suitable experimental preparation for a morphological investigation of ischaemic reperfused myocardium1131. This study was designed to characterize and modulate inflammatory responses to ischaemic reperfused myocardium, and to investigate their influence on myocardial infarct size. The effects of anti-inflammatory pharmacological treatments with BW755C (Wellcome, Pirti of the studies were supported by grant of the DFG. Address for correspondence: Di Rainer M. Bohle, Medical Center of Pathology, Justus-Lieblg-University, Langhani str. 10, D-6300 GieBen.
0195-668X/91/0DOO28 + O4 $03.00/0
U.K.), a dual inhibitor of both the cyclo-oxygenase and lipoxygenase pathway of arachidonic acid metabolism[14], and iloprost (Schering, F.R.G.), a stable prostacyclin analogue1151, on reperfused myocardium were compared using qualitative and quantitative morphological methods. Methods
Medication, anaesthesia, general surgical procedures, general experimental design and measurement of infarct size have been described in detail in previous studies02-161. The experiments were performed on 39 farm pigs of either sex (35-54 kg). EXPERIMENTAL PROTOCOL
The 39 pigs were allocated to one of the following five groups: group A comprised seven pigs that received BW755C (10 mg kg~' i.v.) 10 min before occlusion of the LAD and 5 mg kg"1 i.v. after 4 h of reperfusion. Group B consisted of eight animals treated with BW755C (10 mg kg"1 i.v.) before reperfusion and with 5mgkg - l i.v. after 4h of reperfusion. Group C comprised eight pigs receiving iloprost (25 ng kg"1 min"1 i.v.) before occlusion and continuously for the next 72 h. Group D and E each consisted of eight control pigs. The LAD was occluded at the beginning of its distal third for 45 min followed by 24 h of reperfusion in groups A, B, and D, and by 72 h of reperfusion in groups C and E. 15 min after restoring blood supply, the chest was closed in layers and the animal was allowed to © 1991 The European Society of Cardiology
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The role of inflammation in reperfused, ischaemic myocardium was assessed morphologically in 39 porcine hearts after 24 h (n=23) and 72 h (n = 16) of reperfusion and after different antiphlogistic treatments. The left anterior descending coronary artery (LAD) was occluded distally for 45 min. Seven pigs received BW755C (10mgkg~') i.v. prior to ischaemia (A), eight pigs were given the same dose before 24 h of reperfusion (B), and eight pigs were treated with iloprost (25 ngkg~' min~') i.v. before occlusion and continuously for 72 h (C). Two groups of eight pigs each served as controls for 24 h (D) and 72 h (E) of reperfusion. Infarct sizes were determined, myocardium was investigated by light microscopy, and polymorphonuclear leucocytes (PMNs) and macrophages were quantitated after histo- and immunohistochemical staining. Jeopardized myocardium contained 129 neutrophils mm ~2 and 120 macrophages mm ~2 (D) vs 10neutrophils mm~2 and 290macrophages mm~2 (E). Neutrophils and infarct sizes were only significantly decreased in group A (68 neutrophils mm'2, 30% reduction of infarct size). Macrophage infiltration was not significantly affected for all treatment groups (A, B, C). It is concluded that myocardial infarct sizes are neutrophil-mediated postischaemic tissue injury can be reduced by BW755C applied prior to ischaemia. Neutrophil-mediated myocardial injury is unlikely to occur beyond 3 days of reperfusion.
Inflammatory modulation in MI
did not differ significantly from the control groups MORPHOLOGY
Infarct sizes of the control groups (D: 72±13%; E: 68 ± 18%) did not differ significantly. Group A treatment reduced the infarct size significantly to 50-9± 12% (P< 0005). Infarct sizes of group B (65 ±16%) and group C (74 ± 14%) did not differ from the control experiments. The salvaged myocardium was mainly the subepicardial layer at risk of necrosis. The results indicate that lipoxygenase-cyclo-oxygenase inhibition is only able to limit infarct size in this preparation when BW755C was administered before and after ischaemia. Application of BW755C at the time of reperfusion was ineffective in reducing infarct size. Iloprost applied before occlusion did not limit infarct size. LIGHT MICROSCOPY
After 24 h of reperfusion, ischaemic myocardium contained interstitial oedema, focal haemorrhages, and interstitial inflammatory cells. Myofibres showed predominantly contraction band necrosis (Fig. 1). After 72 h of reperfusion, necrosis type changed into partly calcified coagulation necrosis bordered by large numbers of inflammatory cells.
STATISTICS
All results are presented as mean ± standard deviation (sd). Standard comparisons among the groups were performed with the Kruskal-Wallis H-Test[20]. When this test demonstrated a significant difference, the WilcoxonMann-Whitney rank-sum test (U-test) was used to compare the results of the groups. The U-test was also applied to assess significance between two groups. Probabilities
Modulation of the inflammatory response in experimental myocardial infarction R. M.
BOHLE*,
S. PiCHt
AND
H. H.
KLEIN||
Institute of Pathology, General Hospital of Detmold, Academic Teaching Hospital of the University of Munster*, Departments of Cardiology, University of Gottingen't and University of Marburg\\, Germany KEY WORDS: Experimental myocardial infarction, reperfused porcine hearts, inflammatory response, antiphlogistic therapy.
Introduction
The size of myocardial infarction is influenced by the localization of the coronary artery occlusion, duration of ischaemia, left ventricular oxygen consumption during ischaemia and collateral blood flow'1'31. Clinical studies have demonstrated that early coronary reperfusion can limit mortality and myocardial necrosis[4l5]. Experimental studies in dogs suggested that reperfusion per se might be responsible for myocardial injury163. The tissue-destructive potential of inflammatory cells is mediated by proteolytic lysosomal enzymes, active metabolites of arachidonic acid, free radicals or oxidative inhibition of proteinase inhibitors and is suspected to be a factor determining myocardial infarct size after reperfusion[7-8]. In dogs, experimental myocardial infarct size was significantly reduced by leucopenia, application of anti-neutrophil antibodies and antiphlogistic drugs; however the influence of collateral blood flow cannot be excluded in these experimental models19"1 n . Since porcine hearts have no collateral circulation they are a suitable experimental preparation for a morphological investigation of ischaemic reperfused myocardium1131. This study was designed to characterize and modulate inflammatory responses to ischaemic reperfused myocardium, and to investigate their influence on myocardial infarct size. The effects of anti-inflammatory pharmacological treatments with BW755C (Wellcome, Pirti of the studies were supported by grant of the DFG. Address for correspondence: Di Rainer M. Bohle, Medical Center of Pathology, Justus-Lieblg-University, Langhani str. 10, D-6300 GieBen.
0195-668X/91/0DOO28 + O4 $03.00/0
U.K.), a dual inhibitor of both the cyclo-oxygenase and lipoxygenase pathway of arachidonic acid metabolism[14], and iloprost (Schering, F.R.G.), a stable prostacyclin analogue1151, on reperfused myocardium were compared using qualitative and quantitative morphological methods. Methods
Medication, anaesthesia, general surgical procedures, general experimental design and measurement of infarct size have been described in detail in previous studies02-161. The experiments were performed on 39 farm pigs of either sex (35-54 kg). EXPERIMENTAL PROTOCOL
The 39 pigs were allocated to one of the following five groups: group A comprised seven pigs that received BW755C (10 mg kg~' i.v.) 10 min before occlusion of the LAD and 5 mg kg"1 i.v. after 4 h of reperfusion. Group B consisted of eight animals treated with BW755C (10 mg kg"1 i.v.) before reperfusion and with 5mgkg - l i.v. after 4h of reperfusion. Group C comprised eight pigs receiving iloprost (25 ng kg"1 min"1 i.v.) before occlusion and continuously for the next 72 h. Group D and E each consisted of eight control pigs. The LAD was occluded at the beginning of its distal third for 45 min followed by 24 h of reperfusion in groups A, B, and D, and by 72 h of reperfusion in groups C and E. 15 min after restoring blood supply, the chest was closed in layers and the animal was allowed to © 1991 The European Society of Cardiology
Downloaded from by guest on November 12, 2014
The role of inflammation in reperfused, ischaemic myocardium was assessed morphologically in 39 porcine hearts after 24 h (n=23) and 72 h (n = 16) of reperfusion and after different antiphlogistic treatments. The left anterior descending coronary artery (LAD) was occluded distally for 45 min. Seven pigs received BW755C (10mgkg~') i.v. prior to ischaemia (A), eight pigs were given the same dose before 24 h of reperfusion (B), and eight pigs were treated with iloprost (25 ngkg~' min~') i.v. before occlusion and continuously for 72 h (C). Two groups of eight pigs each served as controls for 24 h (D) and 72 h (E) of reperfusion. Infarct sizes were determined, myocardium was investigated by light microscopy, and polymorphonuclear leucocytes (PMNs) and macrophages were quantitated after histo- and immunohistochemical staining. Jeopardized myocardium contained 129 neutrophils mm ~2 and 120 macrophages mm ~2 (D) vs 10neutrophils mm~2 and 290macrophages mm~2 (E). Neutrophils and infarct sizes were only significantly decreased in group A (68 neutrophils mm'2, 30% reduction of infarct size). Macrophage infiltration was not significantly affected for all treatment groups (A, B, C). It is concluded that myocardial infarct sizes are neutrophil-mediated postischaemic tissue injury can be reduced by BW755C applied prior to ischaemia. Neutrophil-mediated myocardial injury is unlikely to occur beyond 3 days of reperfusion.
Inflammatory modulation in MI
did not differ significantly from the control groups MORPHOLOGY
Infarct sizes of the control groups (D: 72±13%; E: 68 ± 18%) did not differ significantly. Group A treatment reduced the infarct size significantly to 50-9± 12% (P< 0005). Infarct sizes of group B (65 ±16%) and group C (74 ± 14%) did not differ from the control experiments. The salvaged myocardium was mainly the subepicardial layer at risk of necrosis. The results indicate that lipoxygenase-cyclo-oxygenase inhibition is only able to limit infarct size in this preparation when BW755C was administered before and after ischaemia. Application of BW755C at the time of reperfusion was ineffective in reducing infarct size. Iloprost applied before occlusion did not limit infarct size. LIGHT MICROSCOPY
After 24 h of reperfusion, ischaemic myocardium contained interstitial oedema, focal haemorrhages, and interstitial inflammatory cells. Myofibres showed predominantly contraction band necrosis (Fig. 1). After 72 h of reperfusion, necrosis type changed into partly calcified coagulation necrosis bordered by large numbers of inflammatory cells.
STATISTICS
All results are presented as mean ± standard deviation (sd). Standard comparisons among the groups were performed with the Kruskal-Wallis H-Test[20]. When this test demonstrated a significant difference, the WilcoxonMann-Whitney rank-sum test (U-test) was used to compare the results of the groups. The U-test was also applied to assess significance between two groups. Probabilities
