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Histopathologic time course of myocardial infarct in rabbit hearts
Cardiovascular Pathology 11 (2002) 339 – 345
Histopathologic time course of myocardial infarct in rabbit hearts Celina Morales*, Germa´n E. Gonza´lez1, Manuel Rodrı´guez, Carlos A. Bertolasi, Ricardo J. Gelpi2 Laboratorio de Fisiopatologı´a Cardiovascular, Departamento de Patologı´a, Facultad de Medicina, Universidad de Buenos Aires, Uriburu 950, 1114 Buenos Aires, Argentina Received 7 November 2001; received in revised form 18 April 2002; accepted 25 June 2002
Abstract Introduction: The histopathologic evolution of myocardial infarct and of remote zones in rabbit hearts was studied. Methods: The left coronary artery of 55 rabbits was ligated and rabbits were sacrificed at 2, 4, 6, 8, 12, 14, 16, 18, 26, 35 and 56 days post-ligature (n = 5 per group). Two rabbits were used as control and four were sham-operated. The hearts were excised, cut in slices and stained with hematoxylineosin, Masson’s trichrome and picrosirius red. The histological evaluation was semiquantitative (scale: 0 to ++). Results: At day 2, the presence of neutrophils was ++, decreasing suddenly at day 4 and disappearing completely at day 6. The proliferation of cells with features of fibroblasts increased from days 4 to 14 post-occlusion. Coagulation necrosis in mid-myocardium during the first week was ++. Subendocardial myocytolysis was evident from day 2 up to day 56 post-infarction. During the second week, proliferation of lymphocytes and macrophages (++), granulation tissue formation (++) and incipient traces of fibrosis that peaked at day 35 were observed. Scarring was complete at day 56 (++). In remote zones (right ventricle and septum), the proliferation of cells+ on Vimentin was observed at day 2, and perivascular, interstitial and endocardial fibrosis started to increase at day 6 and peaked at day 16. Conclusion: Although myocardial infarction in rabbits maintains the essence of the infarct chronology, some differences as the early presence of cells+ on Vimentin and subendocardial fibrosis in infarcted areas, and also the rapid increase and early disappearance of neutrophils appear when other species are considered. An interesting finding was the early proliferation of cells with features of fibroblasts in remote zones. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Myocardial infarct; Rabbit heart; Histopathology
1. Introduction The rabbit heart is frequently used in research laboratories to study cardiac diseases in general and myocardial ischemia in particular [1]. The reason is that the rabbit heart is similar in certain aspects to the human heart, particularly in that it presents scarce collateral coronary circulation under physiologic conditions [2] and also low levels of xanthine oxidase in the myocardium [3]. Thus, it has been suggested that the rabbit heart as with hearts of other species with low collateral flow [2] such as pigs, baboons and ferrets is a good experimental model for the study of infarction not preceded by angina [4]. Furthermore, Gallagher et al. [5] showed that the cells with features of * Corresponding author. Tel./fax: +54-11-4962-4945. E-mail address: [email protected] (C. Morales). 1 Fellow of the Faculty of Medicine at Buenos Aires University. 2 Member of the Investigator Career of the National Commission of Scientific Investigation and Technique (CONICET).
fibroblasts of rabbit hearts, similarly to human heart cells+ on Vimentin, do not express the AT1 receptors of angiotensin II neither in normal conditions nor in coronary postocclusion. This differentiates the rabbit heart from the rat heart. In the latter, AT1 receptors are abundantly expressed. These variations among species could underlie differences in the histopathologic evolution of myocardial infarct. Although there are publications that have described in detail the chronology of the myocardial infarct in human beings [6] and in rats [7], as far as we know, there are no published studies that describe the temporal evolution of the experimental infarct in rabbits. From the early stages of the inflammatory process to the late stage of cicatrization, there are only partial, temporal descriptions [8– 10]. Therefore, the purpose of this investigation was to study the temporal evolution of histopathologic findings in myocardial infarct of rabbits without reperfusion, considering regional differences among the subendocardium, the midmyocardium and the subepicardium. Since another important element to be considered in the time course of the
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infarct in other species is the structural change in remote zones [11 –13], an important additional objective was to detect histopathologic alterations in these zones and their temporal evolution.
2. Methods 2.1. Experimental model Fifty-five female New Zealand rabbits (1.8 – 2.0 kg) were studied. Subcutaneous administration of ketamine (75 mg/kg) and xylazine (0.75 mg/kg) was administered to anesthetize the animals. By means of an endotracheal tube, they were mechanically ventilated with room air, using a Harvard ventilator (current volume 25 ml and a rate of 32 –36 cycles/min). A 5% dextrose solution was infused (3 ml/min) through a marginal ear vein. The anesthesia was supplemented using the same vein to administer additional doses of ketamine and thiopental sodium, according to surgical requirements. After performing left lateral thoracotomy and pericardiectomy, a prominent branch of the left coronary artery was ligated using a curved needle and 6 – 0 silk suture. The regional pallor of the cardiac surface confirmed myocardial ischemia. The thoracotomy was closed respecting the different anatomical planes and antibiotic therapy was administered as prophylaxis against infection. The animals remained under special control during the first 24 h and then were placed in individual cages until completion of the respective protocol period. The animals were sacrificed with an overdose of thiopental sodium (35 mg/kg) at 2, 4, 6, 8, 12, 14, 16, 18, 26, 35 and 56 days post-infarct (n = 5 in each group). Six animals were used as control, including normal (n = 2) and sham operation rabbits (n = 4), in which thoracotomy and pericardiectomy were performed and, once the left coronary artery was identified, a suture was positioned around the artery, but left loose. These sham animals were sacrificed at 16 and 35 days post-surgery (n = 2 and n = 2, respectively). Before sacrificing the animals, recordings of blood pressure were obtained from the sham group and from the 35-days post-infarct group. To this end, a catheter was placed at the humeral artery and was connected to a pressure transducer (Deltram II, Utah Medical System). The present study complies with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2. Histopathologic examination Once removed, the hearts were weighed and fixed in 10% neutral-buffered formalin, and then sectioned transversely from apex to base in 3– 4-mm slices. All slices were completely immersed in paraffin and then stained with hematoxylin-eosin, Masson’s trichrome and Picrosirius red, a specific
technique for collagen fibers [14]. Immunolabelling of cells with features of fibroblasts was performed. In brief, the sections of paraffin-embedded tissue were incubated with antibody against Vimentin (M0725, DAKO, Carpinterı´a, CA; 1:200) and the avidin – biotin – peroxidase complex technique was used to perform immunolocalization [15]. The histopathologic evolution of MI was evaluated in all slices, considering the three sectors of the myocardium: subendocardium, mid-myocardium and subepicardium. The following histopathologic findings were evaluated in all sectors: coagulation necrosis, myocytolysis, accumulation of neutrophils, lymphocytes, cells with features of fibroblasts and macrophages, granulation tissue, fibrosis and scar tissue [16]. The histopathologic evaluation was not only performed in infarct areas, but also in remote zones, i.e., right ventricle and septum. The histopathologic findings were semiquantitatively evaluated by two blinded observers and graded as follows: (0) absent, (+) slight, (+) moderate and (++) intense [6,17].
Fig. 1. Temporal and regional evolution of coagulation necrosis (upper panel) and post-infarct myocytolysis (lower panel). The ordinate shows the semiquantitative histopathologic scale from 0 to 3 (+). The largest necrosis area can be observed in the mid-myocardium within the first few days post-infarct and the presence of myocytolysis continues until the chronic stages of infarct only in the subendocardium (yP < .05 vs. Endo; * P < .05 vs. Epi; #P < .05 vs. Mid). Endo: endocardium, Epi: epicardium, Mid: mid-myocardium.
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coxon’s signed rank test [19,20] was used. Differences were considered statistically significant when P < .05.
3. Results When the hearts of normal animals were analyzed, no histopathologic lesions were observed, whereas in the group with sham operation animals, sacrificed at 16 and 35 days post-surgery, a fibroblastsic reaction in the subepicardium was significant, possibly due to pericardiectomy. The infarct
Fig. 2. Temporal evolution of the acute and chronic cellular inflammatory response during infarct evolution. The ordinate shows the semiquantitative histopathologic scale from 0 to 3 (+). The early decrease of neutrophils and early increase of cells+ on Vimentin can be observed in the first few days post-infarct.
2.3. Quantitative determination of myocardial infarct size Myocardial infarct size was measured in the animals with infarcts of 35 and 56 days of evolution. After establishing the histologic area of myocardial infarct, each slice was processed by means of a digital image analyzer (ImagePro Plus 3.0). The areas corresponding to normal tissue and scar tissue of the left ventricle were all measured. With the values obtained from each normal area and from each infarcted area, the myocardial infarct size was calculated for each heart and expressed as a percentage of the infarcted area in relation to the left ventricular mass [18]. 2.4. Statistical analysis Data are presented throughout as mean ± S.E. Comparisons among groups were assessed using Kruskall–Wallis’ test. For post-hoc comparisons of mean paired data, Wil-
Fig. 3. Presence of numerous cells with features of fibroblasts at the subendocardial level on day 6 post-infarct. Hematoxylin-eosin stain, original magnification 400.
Fig. 4. Temporal and regional evolution of post-infarct reparative tissue. The semiquantitative histopathologic scale from 0 to 3 (+) is shown in the ordinate. While granulation tissue starts to decrease during the third week post-infarct, fibrous tissue increases progressively until day 35. Finally, infarcted myocardial tissue is totally replaced by scar tissue at day 56 postocclusion of the coronary artery.
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size in animals with coronary occlusion was of 22.4 ± 3.1% and 20.2 ± 4.2%, respectively, regarding the left ventricular mass in the group of animals with 35 and 56 days of postinfarct evolution. Before sacrificing the animals, blood pressure was recorded from the sham group and from the 35 days post-infarct group. Mean blood pressure values were 91.8 ± 10.4 and 95.3 ± 7.7 mm Hg, respectively (NS vs. sham). Fig. 1 shows the findings of coagulation necrosis (upper panel) and myocytolysis (lower panel). We observed that
Fig. 6. Remote zone (right septal myocardium) in a heart with myocardial infarct at day 6 of evolution: early appearance of cells+ on Vimentin accumulations. In the normal, non-infarcted hearts, the accumulations were not observed. Original magnification 400.
Fig. 5. Temporal and regional evolution of cells+ on Vimentin (upper panel) and of endocardial (middle panel), and interstitial and perivascular fibrosis (lower panel) in remote zones. The early presence of cells+ on Vimentin at 2 and 4 days post-infarct in septum and right ventricle (RV), as well as the increasing presence of reactive fibrosis in the normal zones of the myocardium can be observed.
coagulation necrosis was most significant in the middle area of the myocardium (++) during the first week post-infarct and, although it remained during the second week, its significance decreased, disappearing almost completely at the beginning of the third week. On the other hand, myocytolysis was only evident in the subendocardium and remained stable during the 56 days of infarct evolution. The acute and chronic inflammatory process is reflected in the different panels shown in Fig. 2. The acute inflammatory infiltrate (upper left panel) increased up to (++) after the first 48 h in the subendocardium, mid-myocardium and subepicardium, and then decreased abruptly, in all the thickness of the wall. On the other hand, lymphocytes increased slowly and peaked in the second week, remaining steady at (+) until 35 days post-infarct, but were not detectable at day 56. Macrophages increased in the first 6 days (+), remained constant until the end of the second week, when they slowly began to decrease. A similar evolution was observed in the cells with features of fibroblasts that increased early in the first week (Fig. 3). In contrast with macrophages, these cells remained high even at day 35 post-infarct, but were not detectable at day 56. Fig. 4 shows the evolution of post-necrosis reparative tissue. The upper panel shows a gradual increase of granulation tissue toward the end of the first week post-occlusion, peaking during the second week and disappearing completely in the course of the fourth week. At the same time, the granulation tissue began to decrease. Fibrous tissue, or fibrosis, increased progressively until 35 days post-infarct, then disappeared completely at day 56. Cicatricial areas became significant from day 18 in the form of ‘‘patches,’’ with total replacement of infarcted tissue at day 56. Fig. 5 shows the cells+ on Vimentin and fibrosis found in remote zones. The early appearance of the cells+ on Vimentin accumulations, both in the free wall of the right ventricle and in the right ventricular portion of the intraventricular septum, can also be seen in the upper panel of
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Fig. 7. Remote zone (right septal endocardium) in a heart with myocardial infarct at day 16 of evolution: endocardium focally augmented at the expense of collagen tissue (A, arrow). Right ventricular endocardium free of fibrosis in the same heart (B). Masson’s trichrome stain, original magnification 400.
Fig. 5 and in Fig. 6. These accumulations disappear completely at the end of the second week. Simultaneous with the decrease of cells with features of fibroblasts, the appearance of endocardial fibrosis can be observed in the right ventricular portion of the intra-ventricular septum (Fig. 7), remaining high until 56 days. Interstitial and perivascular fibrosis increased slowly, both in the septum and in the free wall of the right ventricle, starting at day 4 post-infarct, peaked during the third week and remained at similar values up to day 56.
4. Discussion This study has shown experimental evidence that the time course of experimental myocardial infarct without reperfusion in rabbits is similar in many aspects to that of other species, including human beings. Nevertheless, we have observed some differences: (1) an early increase of neutrophils at day 2 post-occlusion, which rapidly disappeared in the three layers of the ventricular wall 48 h postocclusion, (2) early appearance of the cells+ on Vimentin in the subendocardium of the infarcted area and (3) early
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presence of cells with features of fibroblasts (day 2 postocclusion) in remote zones, such as the free wall of the right ventricle and septum. The latter is an interesting finding that has not been described in any other species. We think that several important differences can be found in our work with regard to previous studies. In the first place, no previous study, at least to our knowledge, evaluated the time course of the infarct in rabbit hearts within a similar sequence of time. This allows us to understand in detail the histologic evolution of myocardial infarction in rabbits. In our study, animals were sacrificed every other day (with the exception of day 10) up to day 18 and studied at 26, 35 and 56 days post-infarct. This time sequence in days used in the experimental protocol allowed us to study in detail the different cellular sequences in the resolution of myocardial infarction. Another difference is that the histologic evolution was studied so far considering regional changes between subendocardium, mid-myocardium and subepicardium. The fact that in certain important pathologies [21] such as heart failure ventricle dysfunction begins in the subendocardial layers stresses the importance of studying the time course of infarcts from the regional point of view. A third difference is that we evaluated the time course in hearts without reperfusion. In those studies where the time course of rabbit infarct has been described, reperfused infarcted hearts were used. It is believed that, in the last few years, the degree of reperfusion in infarcts has increased [22] due to the progress achieved with different reperfusion techniques in the practice of cardiology. Nevertheless, still a very large number of patients with acute myocardial infarcts can not be reperfused, hence, the importance of understanding the time course of the infarct in hearts that have not been reperfused. In other animal species, including human beings, important differences in the time course of infarcts with and without reperfusion have been described. Two frequently studied alterations in reperfused infarcts are the massive intra-myocardial hemorrhage and the presence of contraction bands [23]. In the studied hearts, although we found some intra-myocardial hemorrhagic foci, they were not of the magnitude of those observed in reperfused infarcts. Also, contraction bands were not found. Another important difference regarding previous studies was the early appearance of histologic alterations in remote zones. At day 2 post-occlusion, the presence of the cells with features of fibroblasts was detected. We also observed the late presence of reactive perivascular and interstitial fibrosis in the septum and the right ventricle, and endocardial fibrosis in the right sector of the septum two weeks post infarct. These findings are different from the collagen deposits that form a replacement fibrosis in the infarct area [24,25]. Although the mechanisms that caused reactive fibrosis in the infarct remote zones are beyond the objectives of this study, some speculation may be appropriate. One possibility could be that fibrosis was caused by the neurohumoral activation that occurs in cardiac failure [26].
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However, this is improbable as in our animals infarcts were not so extensive so as to induce cardiac failure. Another interesting observation was the presence of fibrosis in areas of normal myocardium by activation of the renin angiotensin system as in arterial hypertension [27]. However, we did not favor this observation because when we measured the blood pressure of our animals with 35 days of infarct, they were normal. One probable cause for the presence of fibrosis in the normal zones of our infarct hearts could be the regional changes in the stress and the strain that occur in regional pathologies. A recent study [28] showed that the asynchronous contractions of infarcted ventricles could modify the regional stress and strain, leading to a modification of collagen remodeling. It is important to point out that, according to information obtained from studies with rats, the positive area for collagen in remote zones becomes evident starting from day 7 post-infarct and remains constant until day 35 [12,29]. Thus, works that have described some kind of activity in remote zones have indicated the presence of fibrosis, but have not found early proliferation of cells with features of fibroblasts at day 4 post-occlusion. In our study with rabbits used as an experimental model of infarct, it is possible that the early presence of these cells with features of fibroblasts may be due to a specific characteristic of this specie. The coagulation necrosis observed in our animals peaked in the middle layer of the myocardium during the first week post-infarct. This type of necrosis was smaller in the subendocardium, while myocytolysis was evident after 48 h, remaining constant until day 56 post-infarct. This finding is similar to that of patients in whom myocytolysis was detected 24 h after infarct and lasted until later stages [6]. To our knowledge, no myocytolysis was reported, either in rats [7] or in dogs [30] subjected to similar protocols of ischemia. In our study, the acute inflammatory response was more rapid and less prolonged than in human beings [31]. This is similar to other rodents, notably the rat [7]. The chronic inflammatory infiltrate was similar in the three areas of the myocardium. Both lymphocytes and macrophages reached their highest levels after 2 weeks, as in human beings [6]. After the previously described stage, the infarcted areas were gradually replaced by granulation tissue. It is important to mention that, in our experimental model, the induction of infarct by means of a coronary arterial ligation requires performing a pericardiectomy. The action of leaving the pericardium open, as in any open chest surgery, represents a greater fibrogenic stimulus for the subepicardial area. This was reflected very early in the appearance of fibrosis preceding the fibrosis that could later be observed in the subendocardium and in the mid-myocardium [13]. In our experimental model, there still are areas in the cicatrization process that show presence of cells+ on Vimentin 35 days after infarct, while total replacement by scar tissue was observed in rabbits sacrificed at day 56.
To conclude, the histopathologic study of the evolution of myocardial infarct without reperfusion in rabbit hearts allowed the observation of its similarity with human beings, although with some differences. This detailed analysis of the infarct resolution chronology provides a source of reference for subsequent studies related to ischemia and myocardial infarction.
Acknowledgments The authors wish to thank Drs. Martı´n Donato and Rube´n Laguens for their technical assistance with the statistics, and to Ms. Olivia Avila for her thorough language review.
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Histopathologic time course of myocardial infarct in rabbit hearts Celina Morales*, Germa´n E. Gonza´lez1, Manuel Rodrı´guez, Carlos A. Bertolasi, Ricardo J. Gelpi2 Laboratorio de Fisiopatologı´a Cardiovascular, Departamento de Patologı´a, Facultad de Medicina, Universidad de Buenos Aires, Uriburu 950, 1114 Buenos Aires, Argentina Received 7 November 2001; received in revised form 18 April 2002; accepted 25 June 2002
Abstract Introduction: The histopathologic evolution of myocardial infarct and of remote zones in rabbit hearts was studied. Methods: The left coronary artery of 55 rabbits was ligated and rabbits were sacrificed at 2, 4, 6, 8, 12, 14, 16, 18, 26, 35 and 56 days post-ligature (n = 5 per group). Two rabbits were used as control and four were sham-operated. The hearts were excised, cut in slices and stained with hematoxylineosin, Masson’s trichrome and picrosirius red. The histological evaluation was semiquantitative (scale: 0 to ++). Results: At day 2, the presence of neutrophils was ++, decreasing suddenly at day 4 and disappearing completely at day 6. The proliferation of cells with features of fibroblasts increased from days 4 to 14 post-occlusion. Coagulation necrosis in mid-myocardium during the first week was ++. Subendocardial myocytolysis was evident from day 2 up to day 56 post-infarction. During the second week, proliferation of lymphocytes and macrophages (++), granulation tissue formation (++) and incipient traces of fibrosis that peaked at day 35 were observed. Scarring was complete at day 56 (++). In remote zones (right ventricle and septum), the proliferation of cells+ on Vimentin was observed at day 2, and perivascular, interstitial and endocardial fibrosis started to increase at day 6 and peaked at day 16. Conclusion: Although myocardial infarction in rabbits maintains the essence of the infarct chronology, some differences as the early presence of cells+ on Vimentin and subendocardial fibrosis in infarcted areas, and also the rapid increase and early disappearance of neutrophils appear when other species are considered. An interesting finding was the early proliferation of cells with features of fibroblasts in remote zones. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Myocardial infarct; Rabbit heart; Histopathology
1. Introduction The rabbit heart is frequently used in research laboratories to study cardiac diseases in general and myocardial ischemia in particular [1]. The reason is that the rabbit heart is similar in certain aspects to the human heart, particularly in that it presents scarce collateral coronary circulation under physiologic conditions [2] and also low levels of xanthine oxidase in the myocardium [3]. Thus, it has been suggested that the rabbit heart as with hearts of other species with low collateral flow [2] such as pigs, baboons and ferrets is a good experimental model for the study of infarction not preceded by angina [4]. Furthermore, Gallagher et al. [5] showed that the cells with features of * Corresponding author. Tel./fax: +54-11-4962-4945. E-mail address: [email protected] (C. Morales). 1 Fellow of the Faculty of Medicine at Buenos Aires University. 2 Member of the Investigator Career of the National Commission of Scientific Investigation and Technique (CONICET).
fibroblasts of rabbit hearts, similarly to human heart cells+ on Vimentin, do not express the AT1 receptors of angiotensin II neither in normal conditions nor in coronary postocclusion. This differentiates the rabbit heart from the rat heart. In the latter, AT1 receptors are abundantly expressed. These variations among species could underlie differences in the histopathologic evolution of myocardial infarct. Although there are publications that have described in detail the chronology of the myocardial infarct in human beings [6] and in rats [7], as far as we know, there are no published studies that describe the temporal evolution of the experimental infarct in rabbits. From the early stages of the inflammatory process to the late stage of cicatrization, there are only partial, temporal descriptions [8– 10]. Therefore, the purpose of this investigation was to study the temporal evolution of histopathologic findings in myocardial infarct of rabbits without reperfusion, considering regional differences among the subendocardium, the midmyocardium and the subepicardium. Since another important element to be considered in the time course of the
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infarct in other species is the structural change in remote zones [11 –13], an important additional objective was to detect histopathologic alterations in these zones and their temporal evolution.
2. Methods 2.1. Experimental model Fifty-five female New Zealand rabbits (1.8 – 2.0 kg) were studied. Subcutaneous administration of ketamine (75 mg/kg) and xylazine (0.75 mg/kg) was administered to anesthetize the animals. By means of an endotracheal tube, they were mechanically ventilated with room air, using a Harvard ventilator (current volume 25 ml and a rate of 32 –36 cycles/min). A 5% dextrose solution was infused (3 ml/min) through a marginal ear vein. The anesthesia was supplemented using the same vein to administer additional doses of ketamine and thiopental sodium, according to surgical requirements. After performing left lateral thoracotomy and pericardiectomy, a prominent branch of the left coronary artery was ligated using a curved needle and 6 – 0 silk suture. The regional pallor of the cardiac surface confirmed myocardial ischemia. The thoracotomy was closed respecting the different anatomical planes and antibiotic therapy was administered as prophylaxis against infection. The animals remained under special control during the first 24 h and then were placed in individual cages until completion of the respective protocol period. The animals were sacrificed with an overdose of thiopental sodium (35 mg/kg) at 2, 4, 6, 8, 12, 14, 16, 18, 26, 35 and 56 days post-infarct (n = 5 in each group). Six animals were used as control, including normal (n = 2) and sham operation rabbits (n = 4), in which thoracotomy and pericardiectomy were performed and, once the left coronary artery was identified, a suture was positioned around the artery, but left loose. These sham animals were sacrificed at 16 and 35 days post-surgery (n = 2 and n = 2, respectively). Before sacrificing the animals, recordings of blood pressure were obtained from the sham group and from the 35-days post-infarct group. To this end, a catheter was placed at the humeral artery and was connected to a pressure transducer (Deltram II, Utah Medical System). The present study complies with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.2. Histopathologic examination Once removed, the hearts were weighed and fixed in 10% neutral-buffered formalin, and then sectioned transversely from apex to base in 3– 4-mm slices. All slices were completely immersed in paraffin and then stained with hematoxylin-eosin, Masson’s trichrome and Picrosirius red, a specific
technique for collagen fibers [14]. Immunolabelling of cells with features of fibroblasts was performed. In brief, the sections of paraffin-embedded tissue were incubated with antibody against Vimentin (M0725, DAKO, Carpinterı´a, CA; 1:200) and the avidin – biotin – peroxidase complex technique was used to perform immunolocalization [15]. The histopathologic evolution of MI was evaluated in all slices, considering the three sectors of the myocardium: subendocardium, mid-myocardium and subepicardium. The following histopathologic findings were evaluated in all sectors: coagulation necrosis, myocytolysis, accumulation of neutrophils, lymphocytes, cells with features of fibroblasts and macrophages, granulation tissue, fibrosis and scar tissue [16]. The histopathologic evaluation was not only performed in infarct areas, but also in remote zones, i.e., right ventricle and septum. The histopathologic findings were semiquantitatively evaluated by two blinded observers and graded as follows: (0) absent, (+) slight, (+) moderate and (++) intense [6,17].
Fig. 1. Temporal and regional evolution of coagulation necrosis (upper panel) and post-infarct myocytolysis (lower panel). The ordinate shows the semiquantitative histopathologic scale from 0 to 3 (+). The largest necrosis area can be observed in the mid-myocardium within the first few days post-infarct and the presence of myocytolysis continues until the chronic stages of infarct only in the subendocardium (yP < .05 vs. Endo; * P < .05 vs. Epi; #P < .05 vs. Mid). Endo: endocardium, Epi: epicardium, Mid: mid-myocardium.
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coxon’s signed rank test [19,20] was used. Differences were considered statistically significant when P < .05.
3. Results When the hearts of normal animals were analyzed, no histopathologic lesions were observed, whereas in the group with sham operation animals, sacrificed at 16 and 35 days post-surgery, a fibroblastsic reaction in the subepicardium was significant, possibly due to pericardiectomy. The infarct
Fig. 2. Temporal evolution of the acute and chronic cellular inflammatory response during infarct evolution. The ordinate shows the semiquantitative histopathologic scale from 0 to 3 (+). The early decrease of neutrophils and early increase of cells+ on Vimentin can be observed in the first few days post-infarct.
2.3. Quantitative determination of myocardial infarct size Myocardial infarct size was measured in the animals with infarcts of 35 and 56 days of evolution. After establishing the histologic area of myocardial infarct, each slice was processed by means of a digital image analyzer (ImagePro Plus 3.0). The areas corresponding to normal tissue and scar tissue of the left ventricle were all measured. With the values obtained from each normal area and from each infarcted area, the myocardial infarct size was calculated for each heart and expressed as a percentage of the infarcted area in relation to the left ventricular mass [18]. 2.4. Statistical analysis Data are presented throughout as mean ± S.E. Comparisons among groups were assessed using Kruskall–Wallis’ test. For post-hoc comparisons of mean paired data, Wil-
Fig. 3. Presence of numerous cells with features of fibroblasts at the subendocardial level on day 6 post-infarct. Hematoxylin-eosin stain, original magnification 400.
Fig. 4. Temporal and regional evolution of post-infarct reparative tissue. The semiquantitative histopathologic scale from 0 to 3 (+) is shown in the ordinate. While granulation tissue starts to decrease during the third week post-infarct, fibrous tissue increases progressively until day 35. Finally, infarcted myocardial tissue is totally replaced by scar tissue at day 56 postocclusion of the coronary artery.
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size in animals with coronary occlusion was of 22.4 ± 3.1% and 20.2 ± 4.2%, respectively, regarding the left ventricular mass in the group of animals with 35 and 56 days of postinfarct evolution. Before sacrificing the animals, blood pressure was recorded from the sham group and from the 35 days post-infarct group. Mean blood pressure values were 91.8 ± 10.4 and 95.3 ± 7.7 mm Hg, respectively (NS vs. sham). Fig. 1 shows the findings of coagulation necrosis (upper panel) and myocytolysis (lower panel). We observed that
Fig. 6. Remote zone (right septal myocardium) in a heart with myocardial infarct at day 6 of evolution: early appearance of cells+ on Vimentin accumulations. In the normal, non-infarcted hearts, the accumulations were not observed. Original magnification 400.
Fig. 5. Temporal and regional evolution of cells+ on Vimentin (upper panel) and of endocardial (middle panel), and interstitial and perivascular fibrosis (lower panel) in remote zones. The early presence of cells+ on Vimentin at 2 and 4 days post-infarct in septum and right ventricle (RV), as well as the increasing presence of reactive fibrosis in the normal zones of the myocardium can be observed.
coagulation necrosis was most significant in the middle area of the myocardium (++) during the first week post-infarct and, although it remained during the second week, its significance decreased, disappearing almost completely at the beginning of the third week. On the other hand, myocytolysis was only evident in the subendocardium and remained stable during the 56 days of infarct evolution. The acute and chronic inflammatory process is reflected in the different panels shown in Fig. 2. The acute inflammatory infiltrate (upper left panel) increased up to (++) after the first 48 h in the subendocardium, mid-myocardium and subepicardium, and then decreased abruptly, in all the thickness of the wall. On the other hand, lymphocytes increased slowly and peaked in the second week, remaining steady at (+) until 35 days post-infarct, but were not detectable at day 56. Macrophages increased in the first 6 days (+), remained constant until the end of the second week, when they slowly began to decrease. A similar evolution was observed in the cells with features of fibroblasts that increased early in the first week (Fig. 3). In contrast with macrophages, these cells remained high even at day 35 post-infarct, but were not detectable at day 56. Fig. 4 shows the evolution of post-necrosis reparative tissue. The upper panel shows a gradual increase of granulation tissue toward the end of the first week post-occlusion, peaking during the second week and disappearing completely in the course of the fourth week. At the same time, the granulation tissue began to decrease. Fibrous tissue, or fibrosis, increased progressively until 35 days post-infarct, then disappeared completely at day 56. Cicatricial areas became significant from day 18 in the form of ‘‘patches,’’ with total replacement of infarcted tissue at day 56. Fig. 5 shows the cells+ on Vimentin and fibrosis found in remote zones. The early appearance of the cells+ on Vimentin accumulations, both in the free wall of the right ventricle and in the right ventricular portion of the intraventricular septum, can also be seen in the upper panel of
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Fig. 7. Remote zone (right septal endocardium) in a heart with myocardial infarct at day 16 of evolution: endocardium focally augmented at the expense of collagen tissue (A, arrow). Right ventricular endocardium free of fibrosis in the same heart (B). Masson’s trichrome stain, original magnification 400.
Fig. 5 and in Fig. 6. These accumulations disappear completely at the end of the second week. Simultaneous with the decrease of cells with features of fibroblasts, the appearance of endocardial fibrosis can be observed in the right ventricular portion of the intra-ventricular septum (Fig. 7), remaining high until 56 days. Interstitial and perivascular fibrosis increased slowly, both in the septum and in the free wall of the right ventricle, starting at day 4 post-infarct, peaked during the third week and remained at similar values up to day 56.
4. Discussion This study has shown experimental evidence that the time course of experimental myocardial infarct without reperfusion in rabbits is similar in many aspects to that of other species, including human beings. Nevertheless, we have observed some differences: (1) an early increase of neutrophils at day 2 post-occlusion, which rapidly disappeared in the three layers of the ventricular wall 48 h postocclusion, (2) early appearance of the cells+ on Vimentin in the subendocardium of the infarcted area and (3) early
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presence of cells with features of fibroblasts (day 2 postocclusion) in remote zones, such as the free wall of the right ventricle and septum. The latter is an interesting finding that has not been described in any other species. We think that several important differences can be found in our work with regard to previous studies. In the first place, no previous study, at least to our knowledge, evaluated the time course of the infarct in rabbit hearts within a similar sequence of time. This allows us to understand in detail the histologic evolution of myocardial infarction in rabbits. In our study, animals were sacrificed every other day (with the exception of day 10) up to day 18 and studied at 26, 35 and 56 days post-infarct. This time sequence in days used in the experimental protocol allowed us to study in detail the different cellular sequences in the resolution of myocardial infarction. Another difference is that the histologic evolution was studied so far considering regional changes between subendocardium, mid-myocardium and subepicardium. The fact that in certain important pathologies [21] such as heart failure ventricle dysfunction begins in the subendocardial layers stresses the importance of studying the time course of infarcts from the regional point of view. A third difference is that we evaluated the time course in hearts without reperfusion. In those studies where the time course of rabbit infarct has been described, reperfused infarcted hearts were used. It is believed that, in the last few years, the degree of reperfusion in infarcts has increased [22] due to the progress achieved with different reperfusion techniques in the practice of cardiology. Nevertheless, still a very large number of patients with acute myocardial infarcts can not be reperfused, hence, the importance of understanding the time course of the infarct in hearts that have not been reperfused. In other animal species, including human beings, important differences in the time course of infarcts with and without reperfusion have been described. Two frequently studied alterations in reperfused infarcts are the massive intra-myocardial hemorrhage and the presence of contraction bands [23]. In the studied hearts, although we found some intra-myocardial hemorrhagic foci, they were not of the magnitude of those observed in reperfused infarcts. Also, contraction bands were not found. Another important difference regarding previous studies was the early appearance of histologic alterations in remote zones. At day 2 post-occlusion, the presence of the cells with features of fibroblasts was detected. We also observed the late presence of reactive perivascular and interstitial fibrosis in the septum and the right ventricle, and endocardial fibrosis in the right sector of the septum two weeks post infarct. These findings are different from the collagen deposits that form a replacement fibrosis in the infarct area [24,25]. Although the mechanisms that caused reactive fibrosis in the infarct remote zones are beyond the objectives of this study, some speculation may be appropriate. One possibility could be that fibrosis was caused by the neurohumoral activation that occurs in cardiac failure [26].
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However, this is improbable as in our animals infarcts were not so extensive so as to induce cardiac failure. Another interesting observation was the presence of fibrosis in areas of normal myocardium by activation of the renin angiotensin system as in arterial hypertension [27]. However, we did not favor this observation because when we measured the blood pressure of our animals with 35 days of infarct, they were normal. One probable cause for the presence of fibrosis in the normal zones of our infarct hearts could be the regional changes in the stress and the strain that occur in regional pathologies. A recent study [28] showed that the asynchronous contractions of infarcted ventricles could modify the regional stress and strain, leading to a modification of collagen remodeling. It is important to point out that, according to information obtained from studies with rats, the positive area for collagen in remote zones becomes evident starting from day 7 post-infarct and remains constant until day 35 [12,29]. Thus, works that have described some kind of activity in remote zones have indicated the presence of fibrosis, but have not found early proliferation of cells with features of fibroblasts at day 4 post-occlusion. In our study with rabbits used as an experimental model of infarct, it is possible that the early presence of these cells with features of fibroblasts may be due to a specific characteristic of this specie. The coagulation necrosis observed in our animals peaked in the middle layer of the myocardium during the first week post-infarct. This type of necrosis was smaller in the subendocardium, while myocytolysis was evident after 48 h, remaining constant until day 56 post-infarct. This finding is similar to that of patients in whom myocytolysis was detected 24 h after infarct and lasted until later stages [6]. To our knowledge, no myocytolysis was reported, either in rats [7] or in dogs [30] subjected to similar protocols of ischemia. In our study, the acute inflammatory response was more rapid and less prolonged than in human beings [31]. This is similar to other rodents, notably the rat [7]. The chronic inflammatory infiltrate was similar in the three areas of the myocardium. Both lymphocytes and macrophages reached their highest levels after 2 weeks, as in human beings [6]. After the previously described stage, the infarcted areas were gradually replaced by granulation tissue. It is important to mention that, in our experimental model, the induction of infarct by means of a coronary arterial ligation requires performing a pericardiectomy. The action of leaving the pericardium open, as in any open chest surgery, represents a greater fibrogenic stimulus for the subepicardial area. This was reflected very early in the appearance of fibrosis preceding the fibrosis that could later be observed in the subendocardium and in the mid-myocardium [13]. In our experimental model, there still are areas in the cicatrization process that show presence of cells+ on Vimentin 35 days after infarct, while total replacement by scar tissue was observed in rabbits sacrificed at day 56.
To conclude, the histopathologic study of the evolution of myocardial infarct without reperfusion in rabbit hearts allowed the observation of its similarity with human beings, although with some differences. This detailed analysis of the infarct resolution chronology provides a source of reference for subsequent studies related to ischemia and myocardial infarction.
Acknowledgments The authors wish to thank Drs. Martı´n Donato and Rube´n Laguens for their technical assistance with the statistics, and to Ms. Olivia Avila for her thorough language review.
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