Of Mice and Dogs
2004; Elsevier BV; Volume: 164; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63154-9
ISSN1525-2191
AutoresOliver Dewald, Guofeng Ren, Georg Daniel Duerr, Martin Zoerlein, Christina Klemm, Christine Gersch, Sophia Tincey, Lloyd H. Michael, Mark L. Entman, Nikolaos G. Frangogiannis,
Tópico(s)Congenital heart defects research
ResumoLarge animal models have provided much of the descriptive data regarding the cellular and molecular events in myocardial infarction and repair. The availability of genetically altered mice may provide a valuable tool for specific cellular and molecular dissection of these processes. In this report we compare closed chest models of canine and mouse infarction/reperfusion qualitatively and quantitatively for temporal, cellular, and spatial differences. Much like the canine model, reperfused mouse hearts are associated with marked induction of endothelial adhesion molecules, cytokines, and chemokines. Reperfused mouse infarcts show accelerated replacement of cardiomyocytes by granulation tissue leading to a thin mature scar at 14 days, when the canine infarction is still cellular and evolving. Infarcted mouse hearts demonstrate a robust but transient postreperfusion inflammatory reaction, associated with a rapid up-regulation of interleukin-10 and transforming growth factor-β. Unlike canine infarcts, infarcted mouse hearts show only transient macrophage infiltration and no significant mast cell accumulation. In correlation, the growth factor for macrophages, M-CSF, shows modest and transient up-regulation in the early days of reperfusion; and the obligate growth factor for mast cells, stem cell factor, SCF, is not induced. In summary, the postinfarction inflammatory response and resultant repair in the mouse heart shares many common characteristics with large mammalian species, but has distinct temporal and qualitative features. These important species-specific differences should be considered when interpreting findings derived from studies using genetically altered mice. Large animal models have provided much of the descriptive data regarding the cellular and molecular events in myocardial infarction and repair. The availability of genetically altered mice may provide a valuable tool for specific cellular and molecular dissection of these processes. In this report we compare closed chest models of canine and mouse infarction/reperfusion qualitatively and quantitatively for temporal, cellular, and spatial differences. Much like the canine model, reperfused mouse hearts are associated with marked induction of endothelial adhesion molecules, cytokines, and chemokines. Reperfused mouse infarcts show accelerated replacement of cardiomyocytes by granulation tissue leading to a thin mature scar at 14 days, when the canine infarction is still cellular and evolving. Infarcted mouse hearts demonstrate a robust but transient postreperfusion inflammatory reaction, associated with a rapid up-regulation of interleukin-10 and transforming growth factor-β. Unlike canine infarcts, infarcted mouse hearts show only transient macrophage infiltration and no significant mast cell accumulation. In correlation, the growth factor for macrophages, M-CSF, shows modest and transient up-regulation in the early days of reperfusion; and the obligate growth factor for mast cells, stem cell factor, SCF, is not induced. In summary, the postinfarction inflammatory response and resultant repair in the mouse heart shares many common characteristics with large mammalian species, but has distinct temporal and qualitative features. These important species-specific differences should be considered when interpreting findings derived from studies using genetically altered mice. For almost a century, experimental models of myocardial infarction have contributed to our understanding of the pathobiology of myocardial infarction. Large animal models have been extensively used to study the mechanisms involved in myocardial injury and repair1Jugdutt BI Hutchins GM Bulkley BH Becker LC Myocardial infarction in the conscious dog: three-dimensional mapping of infarct, collateral flow and region at risk.Circulation. 1979; 60: 1141-1150Crossref PubMed Scopus (149) Google Scholar, 2Michael LH Lewis RM Brandon TA Entman ML Cardiac lymph flow in conscious dogs.Am J Physiol. 1979; 237: H311-317PubMed Google Scholar and have significantly contributed to our understanding of the pathological process of myocardial infarction. However, large animal studies have significant limitations in investigating the functional role of specific genes in myocardial ischemia. Recent advances in transgenic and gene targeting approaches have allowed sophisticated manipulations of genes whose functions may be important in injury and repair following myocardial infarction.3Chien KR Genes and physiology: molecular physiology in genetically engineered animals.J Clin Invest. 1996; 97: 901-909Crossref PubMed Scopus (81) Google Scholar Because of technical and economic considerations, these experiments are largely confined to the mouse.4James JF Hewett TE Robbins J Cardiac physiology in transgenic mice.Circ Res. 1998; 82: 407-415Crossref PubMed Scopus (154) Google Scholar, 5Franz WM Mueller OJ Hartong R Frey N Katus HA Transgenic animal models: new avenues in cardiovascular physiology.J Mol Med. 1997; 75: 115-129Crossref PubMed Scopus (25) Google Scholar To capitalize on these advances in gene targeting technology murine models of experimental myocardial infarction have been developed6Michael LH Entman ML Hartley CJ Youker KA Zhu J Hall SR Hawkins HK Berens K Ballantyne CM Myocardial ischemia and reperfusion: a murine model.Am J Physiol. 1995; 269: H2147-2154PubMed Google Scholar, 7Nossuli TO Lakshminarayanan V Baumgarten G Taffet GE Ballantyne CM Michael LH Entman ML A chronic mouse model of myocardial ischemia-reperfusion: essential in cytokine studies.Am J Physiol Heart Circ Physiol. 2000; 278: H1049-1055PubMed Google Scholar and have been extensively used to dissect the mechanisms involved in ischemic myocardial injury.8Heymans S Luttun A Nuyens D Theilmeier G Creemers E Moons L Dyspersin GD Cleutjens JP Shipley M Angellilo A Levi M Nube O Baker A Keshet E Lupu F Herbert JM Smits JF Shapiro SD Baes M Borgers M Collen D Daemen MJ Carmeliet P Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.Nat Med. 1999; 5: 1135-1142Crossref PubMed Scopus (702) Google Scholar, 9Orlic D Kajstura J Chimenti S Jakoniuk I Anderson SM Li B Pickel J McKay R Nadal-Ginard B Bodine DM Leri A Anversa P Bone marrow cells regenerate infarcted myocardium.Nature. 2001; 410: 701-705Crossref PubMed Scopus (4652) Google Scholar, 10Ducharme A Frantz S Aikawa M Rabkin E Lindsey M Rohde LE Schoen FJ Kelly RA Werb Z Libby P Lee RT Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction.J Clin Invest. 2000; 106: 55-62Crossref PubMed Scopus (684) Google Scholar However, extrapolation of the findings derived from murine experiments to the human pathobiology requires similar disease mechanisms in both species. Despite the widespread use of murine models of myocardial infarction, detailed studies of the cellular and molecular events associated with repair of the mouse infarction are lacking. This study examines the pathological features of myocardial infarction in mice, and compares the sequence of cellular and molecular events noted following murine coronary occlusion and reperfusion with the characteristics of reperfused infarcts in an established canine model. Both canine and murine infarcts exhibit a robust inflammatory response associated with induction of chemokines, cytokines and adhesion molecules. In comparison with higher mammals, mice show a more rapid and transient response and do not demonstrate up-regulation of growth factors supporting sustained survival of inflammatory leukocytes. Male and female wild-type C57BL/6 mice (Harlan Sprague-Dawley, Houston, TX), 8 to 12 weeks of age (18.0–22.0 g body weight) were anesthetized by an intraperitoneal injection of sodium pentobarbital (60 μg/g). A closed-chest mouse model of myocardial ischemia-reperfusion was used as previously described,7Nossuli TO Lakshminarayanan V Baumgarten G Taffet GE Ballantyne CM Michael LH Entman ML A chronic mouse model of myocardial ischemia-reperfusion: essential in cytokine studies.Am J Physiol Heart Circ Physiol. 2000; 278: H1049-1055PubMed Google Scholar to avoid the confounding effects of surgical trauma and inflammation, which may influence the baseline levels of chemokines and cytokines. Briefly, after thoracotomy, the pericardium was dissected and an 8–0 Prolene suture (Ethicon, Sommerville, NJ) with the U-shaped tapered needle was passed under the left anterior descending (LAD) coronary artery. The needle was then cut from the suture, and the two ends of the 8–0 suture were then threaded through a 0.5-mm piece of PE-10 tubing (Becton Dickinson, Sparks, MD), forming a loose snare around the LAD. The PE-10 tubing was previously soaked for 24 hours in 100% ethanol. Each end of the suture was then threaded through the end of a size 3 Kalt suture needle (Fine Science Tools, Foster City, CA), and exteriorized through each side of the chest wall. The chest was closed with 3 interrupted stitches using 6–0 Prolene, carefully avoiding pneumothorax, and the animal was removed from the respirator. The ends of the exteriorized 8–0 suture were tucked under the skin, which was then also closed with 6–0 Prolene. The endotracheal tube was withdrawn, and the animal was kept warm with a heat lamp and allowed to breathe 100% oxygen via nasal cone until full recovery of consciousness. Seven to ten days postinstrumentation, the animals were anesthetized with 1.5% MAC isoflurane in 1 l/min oxygen flow under spontaneous breathing. The extremities were taped to a lead II electrocardiogram (ECG) board to measure S-T elevations during the I/R protocol. The skin above the chest wall was then re-opened. The 8–0 suture, which had been previously exteriorized outside the chest wall and placed under the skin, was cleared of all debris from the skin and chest, and carefully taped to heavy metal picks. Occlusion of the LAD was accomplished by gently pulling the heavy metal picks apart until an S-T elevation appeared on the ECG. The ECG was constantly monitored throughout the entire ischemic interval to ensure persistent ischemia. After 1 hour of coronary occlusion, reperfusion was accomplished by pushing the metal picks toward the animal, cutting the suture close to the chest wall, and removing it completely. Subsequently, the skin was closed with a 6–0 Prolene and the animals were kept in the vivarium until the end of the reperfusion interval. At the end of the experiment, mice were anesthetized with sodium pentobarbital overdose the chest was opened and the heart was immediately excised, fixed in Z-fix (Anatech Ltd., Battle Creek, MI) and embedded in paraffin for histological studies, or snap frozen and stored at −80°C for RNA isolation. Sham animals were prepared identically without undergoing myocardial infarction protocols. Animals used for histology underwent 6-hour, 24-hour, 72-hour, 7-day, and 14-day reperfusion protocols (8 animals per group). To identify mast cells in the murine heart, additional animals underwent 3, 5, 7 and 14 days of reperfusion (5 animals per group) and were fixed in Carnoy's to ensure detection of the formalin sensitive mast cell population. Mice used for RNA extraction underwent 3 hours, 6 hours, 24 hours, 72 hours, and 7 days of reperfusion (8 animals per group). An established protocol of canine circumflex coronary occlusion/reperfusion was used.11Frangogiannis NG Lindsey ML Michael LH Youker KA Bressler RB Mendoza LH Spengler RN Smith CW Entman ML Resident cardiac mast cells degranulate and release preformed TNF-α, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion.Circulation. 1998; 98: 699-710Crossref PubMed Scopus (404) Google Scholar, 12Frangogiannis NG Mendoza LH Lindsey ML Ballantyne CM Michael LH Smith CW Entman ML IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury.J Immunol. 2000; 165: 2798-2808PubMed Google Scholar Healthy dogs were instrumented with a hydraulic occluder and underwent 1 hour of coronary occlusion, followed by 24 hours, 72 hours, 5 days, and 7 days of reperfusion (5 animals per group). After the reperfusion periods, hearts were stopped by the rapid intravenous infusion of 30 mEq of KCl and removed from the chest for sectioning from apex to base into four transverse rings ∼1 cm in thickness.Tissue samples were isolated from infarcted or normally perfused myocardium based on visual inspection. Myocardial segments were fixed in B*513Beckstead JH A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.J Histochem Cytochem. 1994; 42: 1127-1134Crossref PubMed Scopus (225) Google Scholar or Carnoy's fixative for histological analysis. Samples from mouse myocardium were fixed in zinc-formalin (Z-fix; Anatech, Battle Creek, MI), and embedded in paraffin. Sections were cut at 3 μm and stained immunohistochemically with the following antibodies: monoclonal anti-α smooth muscle actin (α-SMA) antibody (Sigma, St. Louis, MO), rat anti-mouse neutrophil antibody (Serotec, Oxford, UK), rat anti-mouse macrophage antibody F4/80 (Research Diagnostics Inc, Flanders, NJ),14Gersch C Dewald O Zoerlein M Michael LH Entman ML Frangogiannis NG Mast cells and macrophages in normal C57/BL/6 mice.Histochem Cell Biol. 2002; 118: 41-49PubMed Google Scholar and rat anti-mouse CD31 antibody (PharMingen, San Diego, CA).15Ismail JA Poppa V Kemper LE Scatena M Giachelli CM Coffin JD Murry CE Immunohistologic labeling of murine endothelium.Cardiovasc Pathol. 2003; 12: 82-90Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Staining was performed using a peroxidase-based technique with the Vectastain ELITE rat kit (Vector Laboratories, Burlingame, CA) and developed with diaminobenzidine (DAB)+nickel (Vector Laboratories). The Mouse on Mouse (MOM) kit (Vector Laboratories) was used for α-SMA immunohistochemistry. For CD31 staining, sections were pretreated with trypsin and staining was performed using the tyramide signal amplification (TSA) kit (Perkin Elmer, Boston, MA) as previously described.16Enholm B Karpanen T Jeltsch M Kubo H Stenback F Prevo R Jackson DG Yla-Herttuala S Alitalo K Adenoviral expression of vascular endothelial growth factor-C induces lymphangiogenesis in the skin.Circ Res. 2001; 88: 623-629Crossref PubMed Scopus (190) Google Scholar Slides were counterstained with eosin and examined in a Zeiss microscope equipped with a Leaf Lumina digital camera. To identify mast cells additional mouse hearts were fixed in Carnoy's and the sections were stained with toluidine blue as previously described.14Gersch C Dewald O Zoerlein M Michael LH Entman ML Frangogiannis NG Mast cells and macrophages in normal C57/BL/6 mice.Histochem Cell Biol. 2002; 118: 41-49PubMed Google Scholar, 17Frangogiannis NG Burns AR Michael LH Entman ML Histochemical and morphological characteristics of canine cardiac mast cells.Histochem J. 1999; 31: 221-229Crossref PubMed Scopus (56) Google Scholar Canine samples were fixed in B*5 fixative and sections were stained with the following antibodies: monoclonal anti-α smooth muscle actin antibody (Sigma),18Frangogiannis NG Michael LH Entman ML Myofibroblasts in reperfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb).Cardiovasc Res. 2000; 48: 89-100Crossref PubMed Scopus (170) Google Scholar monoclonal anti-canine neutrophil antibody SG8H619Hawkins HK Entman ML Zhu JY Youker KA Berens K Dore M Smith CW Acute inflammatory reaction after myocardial ischemic injury and reperfusion: development and use of a neutrophil-specific antibody.Am J Pathol. 1996; 148: 1957-1969PubMed Google Scholar (a gift from Dr. C. W. Smith, Baylor College of Medicine), and mouse anti-human CD31 antibody (Dako, Carpinteria, CA).20Ren G Michael LH Entman ML Frangogiannis NG Morphological characteristics of the microvasculature in healing myocardial infarcts.J Histochem Cytochem. 2002; 50: 71-79Crossref PubMed Scopus (137) Google Scholar To identify macrophages, we used immunohistochemical staining with the mouse anti-human macrophage antibody PM-2K (Biogenesis, Kingston, NH), which cross-reacts with canine species21Zeng L Takeya M Ling X Nagasaki A Takahashi K Interspecies reactivities of anti-human macrophage monoclonal antibodies to various animal species.J Histochem Cytochem. 1996; 44: 845-853Crossref PubMed Scopus (32) Google Scholar and detects infarct macrophages as we have previously demonstrated.22Frangogiannis NG Mendoza LH Ren G Akrivakis S Jackson PL Michael LH Smith CW Entman ML MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype.Am J Physiol Heart Circ Physiol. 2003; 285: H483-492Crossref PubMed Scopus (91) Google Scholar Staining of canine tissue was performed using a peroxidase-based technique with the Vectastain ELITE mouse kit (Vector Laboratories) and developed with DAB+nickel (Vector Laboratories) Canine cardiac mast cells were identified using toluidine blue staining as previously described.17Frangogiannis NG Burns AR Michael LH Entman ML Histochemical and morphological characteristics of canine cardiac mast cells.Histochem J. 1999; 31: 221-229Crossref PubMed Scopus (56) Google Scholar Quantitative analysis of mast cell density was performed by counting the number of toluidine blue positive mast cells in control and infarct areas. Macrophage quantitation was performed by counting the number of F4/80 positive cells in mice and PM-2K positive cells in dogs. Neutrophil quantitation was performed by counting the number of neutrophils identified in canine and murine hearts using the specific anti-neutrophil antibodies. Mast cell, neutrophil and macrophage density was expressed as cells/mm2. Microvascular density was assessed by counting the number of CD31-positive microvascular profiles in infarcted and noninfarcted myocardium. α-SMA stained area was quantitated as a percentage of the total area of infarcted or control myocardium using Image Pro software (Media Cybernetics Inc., Silver Spring, MD). All solutions for RNA analysis were treated with 0.1% diethylpyrocarbonate and sterilized or prepared in diethylpyrocarbonate-treated water. Glassware was baked at 240°C for 5 hours to remove trace RNases. Total RNA was isolated from whole mouse heart according to the acid-guanidium-thiocyanate-phenol-chloroform. Briefly, whole hearts were homogenized in RNA STAT-60 solution (Tel-Test, Friendswood, TX). For RNA extraction, 0.2 volumes of R-chloroform were then added per volume of homogenate. This mixture was incubated on ice for 15 minutes and then spun at 12,000 × g for 15 minutes at 4°C. The supernatant was transferred to another tube, and an equal volume of isopropanol was added for RNA precipitation overnight at 4°C. The tubes were then spun at 12,000 × g for 15 minutes at 4°C, and the supernatant was decanted. The pellet was washed twice with 75% ethanol, briefly dried, and dissolved in 0.1% diethylpyrocarbonate-treated water. Quantification and purity of RNA was assessed by A260/A280 UV absorption, and RNA samples with ratios above 1.9 were used for further analysis. The mRNA expression level of the chemokines monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, and interferon-γ inducible protein (IP)-10, the IP-10 receptor, CXCR3, the cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, leukemia inhibitory factor (LIF) and IL-10, the growth factors transforming growth factor (TGF)-β1, 2, and 3, stem cell factor (SCF), granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-colony stimulating factor (M-CSF), the adhesion molecules intercellular adhesion molecule (ICAM)-1, CD31/platelet endothelial cell adhesion molecule (PECAM)-1 and E-selectin, the matricellular protein osteopontin (OPN)-1, and the cytokine receptors IL-10 receptor (IL-10R), and GM-CSF receptor was determined using a ribonuclease protection assay (RiboQuant; PharMingen) according to the manufacturer's protocol. Phosphorimaging of the gels was performed (Storm 860; Molecular Dynamics, Sunnyvale, CA) and signals were quantified using Image QuaNT software and normalized to the ribosomal protein L32 mRNA. All data are presented as means ± SEM. Comparison between the groups was done using analysis of variance with a Student's–Newman-Keuls corrected post hoc analysis. Differences with P < 0.05 were considered significant. Reperfused myocardial infarction in both mice and dogs is associated with rapid infiltration of the injured myocardium with inflammatory cells, accompanied by clearance of necrotic cardiomyocytes. Extensive leukocyte extravasation is noted in both murine and canine infarcts after 24 hours of reperfusion (Figure 1, A and E). After 72 hours of reperfusion, murine infarcts demonstrate almost complete replacement of injured cardiomyocytes with inflammatory cells (Figure 1F). In contrast, canine infarcts demonstrate a significantly slower progression of myocyte replacement and persistent presence of significant numbers of contracted cardiomyocytes surrounded by an inflammatory infiltrate (Figure 1B). After 7 days of reperfusion, murine infarcts show thinning and decreased cellular content (Figure 1G), whereas canine infarcts are filled with inflammatory cells (Figure 1C). Further loss of cellular content is noted in mouse infarcts after 14 days of reperfusion (Figure 1H), whereas the dog infarct (Figure 1D) is still highly cellular. Reperfused canine and murine infarcts demonstrate rapid infiltration with inflammatory leukocytes, leading to the formation of granulation tissue with a high cellular content. Neutrophils rapidly infiltrate both murine and canine infarcts (Figure 2, Figure 3) peaking after 24 hours of reperfusion, but are rarely found after 7 days of reperfusion (Figure 2, C and F). Reperfused mouse infarcts exhibit a transient increase in macrophage and fibroblast accumulation, followed by decreased cellular content after 7 to 14 days of reperfusion. Macrophage density rapidly increases after 6 hours of reperfusion (data not shown), peaking after 24 hours of reperfusion (macrophage density 1805 ± 185.5 cells/mm2; Figure 4, Figure 5). Subsequently as the scar matures, a significant decrease in macrophage density is noted after 7 days (density 136.9 ± 56.9 cells/mm2, P < 0.01 vs. 24 hours; Figure 4, Figure 5). Myofibroblasts, identified as spindle-shaped α-smooth muscle actin positive cells, accumulate in the border zone of the mouse infarct after 72 hours of reperfusion (Figure 6D). Myofibroblast infiltration in infarcted mice appears to be a transient response: after 7 and 14 days of reperfusion the mature mouse infarct has relatively low myofibroblast content and decreased α-SMA % staining when compared with 3-day infarcts (Figure 6, E–G).Figure 3Quantitative analysis of neutrophil density in reperfused murine (A) and canine infarcts (B). Neutrophil infiltration in both mouse and dog infarcts is robust but transient, decreasing after 3 days of reperfusion. Neutrophil density in infarcts after 7 days of reperfusion is very low. ***, P < 0.001, **, P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Macrophage identification in reperfused canine (A–C) and mouse infarcts (D–F) using the monoclonal antibodies PM-2K (for the dog), and F4/80 (for the mouse). Canine macrophages progressively accumulate in the infarct after 24 hours (A, arrows) to 72 hours (B) of reperfusion, peaking after 7 days of reperfusion (C). In contrast, murine macrophages peak after 24 hours of reperfusion (D), remain abundant after 72 hours (E) and decrease significantly after 7 days of reperfusion (F, arrows). Sections counterstained with eosin (magnification, ×400).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Quantitative analysis of macrophage density in reperfused murine (A, F4/80 staining) and canine infarcts (B, PM2K staining). Macrophage accumulation in reperfused mouse infarcts is robust but transient, decreasing after 7 days of reperfusion. In contrast, macrophage infiltration in dog infarcts is sustained, peaking after 7 days reperfusion. ***, P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6α-smooth muscle actin (α-SMA) staining in reperfused canine (A–C) and murine infarcts (D–F). Dog infarcts show occasional α-SMA positive spindle-shaped cells (arrows) after 72 hours of reperfusion (A). These cells are phenotypically modulated fibroblasts, termed myofibroblasts. Myofibroblast accumulation increases dramatically after 7 days of reperfusion in the canine infarct (B, arrows). After 14 days of reperfusion α-SMA staining is predominantly localized in smooth muscle cells and pericytes coating maturing wound neovessels (C, arrowheads). In contrast, mouse infarcts exhibit a rapid and transient myofibroblast response. After 72 hours of reperfusion mouse infarcts show extensive myofibroblast infiltration (D, arrows). However, after 7 days of reperfusion myofibroblasts in the infarcted mouse heart significantly decrease and α-SMA staining is predominantly localized in pericyte-coated vessels (E, arrowheads) indicating rapid maturation of the infarct neovasculature. α-SMA immunoreactivity remains localized in vascular structures (arrowheads) after 14 days of reperfusion (F, arrowheads). G: Quantitative analysis of α-SMA staining in healing mouse infarcts. Infarct α-SMA % staining peaks after 3 days of reperfusion (***, P < 0.001 compared with non-infarcted heart) and decreases significantly after 7–14 days of reperfusion (#, P < 0.01 compared with % staining after 3 days of reperfusion).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast, canine infarcts show a progressive increase in macrophage and myofibroblast accumulation, peaking after 7 days of reperfusion. Macrophage density in reperfused canine infarcts is significantly higher than in control myocardial areas after 24 hours of reperfusion and continues to increase for the following 6 days, (Figure 4, A–C, and Figure 5B). In addition, dog infarcts demonstrate a delayed and sustained myofibroblast response, which is first noted after 72 hours of reperfusion and peaks after 7 days (Figure 6, A and B). As we have previously demonstrated α-SMA % staining in the canine infarct peaks after 5 to 14 days of reperfusion, decreasing significantly after 3 to 4 weeks of reperfusion18Frangogiannis NG Michael LH Entman ML Myofibroblasts in reperfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb).Cardiovasc Res. 2000; 48: 89-100Crossref PubMed Scopus (170) Google Scholar (Table 1).Table 1Pathologic Features of Reperfused Myocardial Infarcts: Comparison of a Canine and a Murine ModelFeatureMouseDogGranulation tissue formationRapid formation of granulation tissue; injured cardiomyocytes replaced with granulation tissue after 72 h of reperfusion in animals undergoing 1-h coronary occlusion protocols (Figure 1)Slower formation of granulation tissue; injured cardiomyocytes are replaced with granulation tissue after 120 h of reperfusion in animals undergoing 1-h coronary occlusion protocolsNeutrophil recruitmentRapid and transient neutrophil infiltration (Figure 2, Figure 3)Rapid and transient neutrophil infiltration (Figure 2, Figure 3)Macrophage recruitmentRapid influx of monocytes; macrophage accumulation peaks early and decreases significantly after 7 days of reperfusion (Figure 4, Figure 5)Rapid influx of monocytes; sustained macrophage accumulation peaks after 7 days of reperfusion (Figure 4, Figure 5)Myofibroblast accumulationMaximal myofibroblast accumulation after 72 h of reperfusion; myofibroblast density decreases after 7 days of reperfusion (Figure 6)Low number of myofibroblasts after 72 h of reperfusion (Figure 6); myofibroblast accumulation peaks after 7 days of reperfusion18Frangogiannis NG Michael LH Entman ML Myofibroblasts in reperfused myocardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb).Cardiovasc Res. 2000; 48: 89-100Crossref PubMed Scopus (170) Google ScholarMast cell recruitmentNo increase in mast cell density found in healing mouse infarcts (Figure 7)Significant mast cell accumulation in infarcted dog hearts after 72 h of reperfusion23Frangogiannis NG Perrard JL Mendoza LH Burns AR Lindsey ML Ballantyne CM Michael LH Smith CW Entman ML Stem cell factor induction is associated with mast cell accumulation after canine myocardial ischemia and reperfusion.Circulation. 1998; 98: 687-698Crossref PubMed Scopus (158) Google Scholar; mast cell infiltration persists for at least 2 weeks after coronary occlusionWound angiogenesisLow capillary density in the infarcted area after 7–14 days of reperfusion (<500 microvessels/mm2); formation of large dilated vessels; progressive coating of the neovessels with pericytes; coating and maturation of infarct neovessels complete after 14 days of reperfusion (Figure 8)High capillary density in the infarcted area after 7–14 d
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