Ultrastructural characterization of the border zone surrounding early experimental myocardial infarcts in dogs.

The existence of a border zone composed of reversibly injured myocardium surrounding an evolving infarct has been the subject of controversy. In experiments designed to search for such a border zone by electron microscopy, 12 mongrel dogs underwent permanent ligation of the left anterior descending coronary artery (LAD). Two to 6.5 (average = 4.2) hours later, the hearts were excised, the area at risk (myocardium perfused by the LAD) was outlined by injection of fluorescent microspheres, and the myocardial infarct was demonstrated by the nitro blue tetrazolium (NBT) gross histochemical method. Myocardial samples for electron-microscopic study were obtained from the periphery of the infarct (tissues unstained by NBT) and serially from the immediately adjacent myocardium, which was stained deep blue by NBT. Grossly, the infarcts always involved the subendocardial myocardium, extended for a variable distance in the epicardial direction, and closely approximated the lateral margins of the area at risk. When examined by electron microscopy, the infarct periphery showed evidence of irreversible damage, thus confirming the ability of NBT to detect early myocardial necrosis. Multiple samples of the NBT-stained myocardium immediately adjacent to the infarct showed varying degrees of reversible ischemia, thus demonstrating, at the ultrastructural level, the existence of a border zone of intermediate myocardial injury. This border zone was substantial (3--4 mm in width) along the subepicardial aspect of the infarct and very thin (1--2 mm) laterally. In conclusion, a significant border zone was demonstrable by electron microscopy in the subepicardial myocardium of 8 out or 12 canine hearts with recent coronary artery occlusion. In the remaining 4 hearts, the infarcts had already reached the epicardium at the time of study, and only a thin lateral border zone was present.     

Autofluorescence in the identification of myocardial infarcts

A method is described for demonstrating myocardial necrosis by changes in the autofluorescence of routine hematoxylin and eosin or hematoxylin and eosin-phloxine stained sections using a standard microscope with an epifluorescence attachment. Regions of necrosis fluoresce with a brilliant yellow color clearly distinguishable from the dull olive-green to red-brown fluorescence of the neighboring viable cardiac muscle.     

Continuum of the thickness of surviving myocardial wall with single myocardial infarcts

The pathologic correlate of the clinical terms subendocardial and transmural applied to myocardial infarcts is uncertain. To examine this question, we reviewed the morphology of 204 hearts with single myocardial infarcts studied at autopsy after coronary arteriography and fixation in distention. The thickness of surviving myocardium with the infarct (S) and the thickness of the adjacent noninfarcted myocardium (A) were measured microscopically and expressed as a ratio. The S:A ratios ranged from 0.00 to 0.83. Necrosis of the entire wall (S:A ratio = 0.00) was seen in 37 (18%) cases. Distribution of the remaining cases by 10% intervals of wall necrosis (ie, from 0.00 less than S:A ratio less than or equal to 0.10 through 0.90 less than S:A ratio less than or equal to 1.00) were present in 27, 33, 31, 24, 25, 12, 8, 6, 1, and 0 cases, respectively. A lower S:A ratio was correlated with infarct size, infarct expansion, infarct rupture, proximal location of the coronary artery lesion causing the infarct, recency of the infarct, and degree of endocardial mural thrombus. A higher S:A ratio was correlated with the degree of left ventricular hypertrophy. No correlation was observed between S:A ratio and several measures of coronary artery disease. Multivariate regression analysis showed that infarct expansion, infarct age, and rupture were distinct predictors of infarct thickness. The study shows that thickness of myocardium surviving with an infarct forms a continuum; there is no evidence of separate populations that would correspond to infarcts of transmural or subendocardial extent.     

心肌缺血的膜损伤及蝮蛇抗栓酶修饰效应的实验研究

本实验以异丙肾上腺素性心肌缺血(ISP-MI)为模型,对心肌膜脂、膜酶及自由基系统三个方面进行了研究,旨在探讨蝮蛇抗栓酶(Svate)对膜损伤的修饰作用。结果发现,大鼠皮下注射异丙肾(ISP,85mg/kg)24h后,心肌膜脂、膜酶及自由基代谢发生明显紊乱,标志着膜损伤的存在。舌下静脉注射Svate(0.25U/kg)治疗组,与单纯缺血组比较,心肌线粒体Ch/PL比降低,膜脂流动性明显恢复;心肌膜脂过氧化产物丙二醛(MDA)下降,谷胱甘肽过氧化物酶(GSH-Px)活力增加,Na~+-K~+-ATPase活力明显升高及Ca~(2+)-Mg~(2+)-ATPase活力恢复正常。以上结果说明,Svate对缺血心肌膜损伤有一定的修饰效应。     

Absolute morphometric study of myocardial hypertrophy in experimental hypertension. I. Determination of myocyte size.

A method for determining the mean absolute volume of a specific population of cells within a tissue is described and applied to the measurement of endocardial and epicardial myocytes in the left ventricle of normal and hypertensive rats. The technique, based on nuclear counts per unit area in tissue slices of different known thicknesses, measures the mean cell volume per nucleus independent of previously unknown nuclear dimensions and systematic counting errors. Duplicate determinations, demonstrating reproducibility, were made in mutually perpendicular longitudinal and transverse sections of the myocardium. Combining these light microscopic measurements with electron microscopic data enabled the evaluation of the mean diameter and length of the cylindrical myocyte nuclei showing those in the epicardial cells to be significantly longer than the nuclei in endocardial cells. It was estimated that 2 to 4 per cent of ventricular myocytes are binucleate. After 1 to 4 weeks of hypertension, induced by constriction of the left renal artery, endocardial myocytes were enlarged 21 per cent, from 10,370 +/- 410 to 12,520 +/- 490 cu. micrometer., while epicardial myocytes showed a 37 per cent hypertrophy, from 12,600 +/- 1,600 to 17,300 +/- 1,100 cu. micrometer. The availability of a reliable determination of cell volume will make possible the interpretation of much biochemical, functional, and morphometric data at the whole cell level.     

The MRL mouse repairs both cryogenic and ischemic myocardial infarcts with scar.

OBJECTIVE: The MRL mouse strain shows extraordinary wound healing capacities. Some years ago, Leferovich et al. (Proc Natl Acad Sci U S A 2001;98:9830-35) have reported the absence of scar formation after cryogenically-induced right ventricular myocardial infarcts in adult MRL mice. An independent group (Oh et al., Cardiovasc Pathol 2004;13:203-6) found that MRL mice repair left ventricular ischemic infarcts after coronary artery ligation with regular scar formation. Given the divergent outcomes in infarct healing in MRL mice reported by those two studies, we have investigated whether MRL mice heal myocardial infarcts without scar both in the cryoinjury and in the coronary ligation model. METHODS AND RESULTS: Four different protocols of cryogenically induced right and left ventricular injury, as well as permanent ligation of the left anterior descending coronary artery, were tested in adult MRL and control C57Bl/6 mice. At 60 days after experimental infarction, MRL mice showed pronounced scarring of the affected right and left ventricular areas, with no significant differences in infarct size and thickness between MRL and C57Bl/6 mice using any of the five experimental protocols. Analysis of cell proliferation by 5-bromo-2'-deoxyuridine (BrdU) incorporation into the DNA did not show any difference between the two strains of mice after infarction. Histological analysis of infarct areas using picrosirius red staining did not show differences in extent of collagen and distribution between the two mouse strains. CONCLUSIONS: MRL mice heal myocardial infarcts with scar formation in response to ischemic as well as to cryogenic injuries.     

Multifunctional roles of macrophages in the development and progression of atherosclerosis in humans and experimental animals

In atherosclerosis, macrophages are important for intracellular lipid accumulation and foam cell formation. Monocytes respond to chemotactic factors, cytokines, and macrophage growth factors produced by vascular endothelial cells, smooth muscle cells, and infiltrated cells, by migrating from peripheral blood into the arterial intima and differentiating into macrophages in atherosclerotic lesions. Although various chemotactic factors are known to induce monocyte migration, monocyte chemoattractant protein-1 is the most important and powerful inducer of migration into atherosclerotic lesions. Macrophage colony-stimulating factor is crucial for monocyte/macrophage differentiation and proliferation, and for the survival of macrophages in these lesions. A minor population of macrophages can proliferate in the atherosclerotic lesions themselves, particularly in the early stage. The macrophages express a variety of receptors, particularly scavenger receptors, and take up modified lipoproteins, including oxidized low-density lipoprotein, beta-very-low-density lipoprotein, and/or enzymatically degraded low-density lipoprotein. These cells accumulate cholesterol esters in the cytoplasm, which leads to foam cell formation in lesion development. Among various scavenger receptors, class A type I and type II macrophage scavenger receptors (MSR-A I,II) play the most important role in the uptake of oxidized low-density lipoprotein by macrophages. In addition, macrophages and macrophage-derived foam cells produce ceroid and advanced glycation end-products (AGEs) and accumulate these substances in their cytoplasm. Extracellularly generated AGEs are taken up by macrophages via receptors for AGEs, including MSR-AI,II. Most foam cells die in loco because of apoptosis, and some foam cells escape from the lesions into peripheral blood. Macrophages also play multifaceted roles in inducing plaque rupture, blood coagulation, and fibrinolysis via the production of various enzymes, activators, inhibitors, and bioactive mediators. During the development of atherosclerosis, macrophages interact with vascular endothelial cells, medial smooth muscle cells, and infiltrated inflammatory cells, particularly T cells and dendritic cells. This review, based on data accumulated in studies of atherosclerosis in humans and experimental animals, focuses on the multifunctional roles of macrophages in the pathogenesis and progression of atherosclerosis.     

Cardioprotection against experimental myocardial ischemic injury using cornin

Phosphorylated-cyclic adenosine monophosphate response element-binding protein (Phospho-CREB) has an important role in the pathogenesis of myocardial ischemia. We isolated the iridoid glycoside cornin from the fruit of Verbena officinalis L, investigated its effects against myocardial ischemia and reperfusion (I/R) injury in vivo, and elucidated its potential mechanism in vitro. Effects of cornin on cell viability, as well as expression of phospho-CREB and phospho-Akt in hypoxic H9c2 cells in vitro, and myocardial I/R injury in vivo, were investigated. Cornin attenuated hypoxia-induced cytotoxicity significantly in H9c2 cells in a concentration-dependent manner. Treatment of H9c2 cells with cornin (10 µM) blocked the reduction of expression of phospho-CREB and phospho-Akt in a hypoxic condition. Treatment of rats with cornin (30 mg/kg, iv) protected them from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics, and reduction of severity of myocardial damage. Cornin treatment also attenuated the reduction of expression of phospho-CREB and phospho-Akt in ischemic myocardial tissue. These data suggest that cornin exerts protective effects due to an increase in expression of phospho-CREB and phospho-Akt.     

The combination of L-arginine and ischaemic post-conditioning at the onset of reperfusion limits myocardial injury in the pig

Aim: To investigate whether ischaemic post‐conditioning (IPoC) combined with i.v. infusion of the nitric oxide (NO) substrate l ‐arginine at the onset of reperfusion exerts cardioprotective effect that is superior to either treatment given separately. Methods: Twenty‐six anesthetized pigs were subjected to coronary artery (left anterior descending artery, LAD) ligation for 40 min followed by 4 h reperfusion. The pigs were randomized into five different groups receiving either i.v. vehicle, i.v. l ‐arginine, IPoC 4 × 60 s together with i.v. vehicle or IPoC together with i.v. l ‐arginine and a group with IPoC 8 × 30 s. All infusions were started 10 min before reperfusion. Results: The infarct size of the vehicle group was 82 ± 4% of the area at risk. l ‐Arginine alone (79 ± 8%), IPoC 4 × 60 s vehicle (86 ± 3%) or IPoC 8 × 30 s vehicle (94 ± 7%) did not affect infarct size. l ‐Arginine together with IPoC significantly reduced infarct size to 59 ± 4% (P < 0.01). Except for higher LAD flow during early reperfusion in the IPoC l ‐arginine group, haemodynamic parameters did not differ between the four main groups. Heart rate and rate pressure product were lower during ischaemia and reperfusion in the IPoC 8 × 30 s vehicle group. In comparison with the vehicle group, there were no changes in the expression of Akt, phosphorylated Akt Ser⁴⁷³, inducible NO synthase, endothelial NO synthase (eNOS) or phosphorylated eNOS Ser¹¹⁷⁷ in the ischaemic/reperfused myocardium. Conclusion: l ‐Arginine given systemically at the onset of reperfusion protects the pig heart against ischaemia and reperfusion injury only when combined with IPoC. These results indicate that the combination of the two treatment strategies exerts cardioprotection.     

Fibronectin and fibrinogen/fibrin at the focus of an experimental myocardial infarct

The content of fibrinogen/fibrin, plasma and cellular fibronectin, the 1st type collagen, laminin and skeletal muscle myosin in the zone of experimental myocardial infarction was studied by the immunofluorescent method. The infiltration of the necrotized cardiomyocytes with fibrinogen/fibrin and plasma fibronectin was observed 3 hours and later after coronary artery ligation. Fibrinogen/fibrin and plasma fibronectin form a "primary matrix" of the granulation tissue in which the fibers of the 1st type collagen are being formed. Cellular fibronectin starts to be synthesized 3 days after the infarct development and its content in the extracellular matrix (ECM) of the granulation tissue increases 7-15 days after the infarction. The amount of the fibronectin in ECM of the scar tissue decreases 30 days after the infarct. Fibrinogen/fibrin is always found in the granulation tissue replacing the myocardial infarction but its content in ECM decreases during the scar formation.     

塞来昔布对心肌梗死后心肌细胞凋亡的影响

 目的：研究选择性环氧化酶-2抑制剂塞来昔布(celecoxib,Cele)对心肌梗死后心肌细胞抗氧化损伤能力、Bcl-2、Bax蛋白表达及细胞凋亡的影响。方法：选择新西兰兔24只，随机分为3组（每组8只）：假手术组（sham组）、心肌梗死组（MI组）、塞来昔布组(Cele组，10 mg·kg^-1·d^-1,灌胃)。6周后取梗死灶边缘缺血心肌制作组织匀浆进行NO浓度(以NO^-₂/ NO^-₃间接表示)、结构型一氧化氮合酶(cNOS)和诱导型一氧化氮合酶(iNOS)活性及总抗氧化能力（T-AOC）测定，并做病理观察，免疫组化观察各组Bcl-2、Bax蛋白表达，TUNEL法比较其细胞凋亡程度。结果：MI组心肌间质水肿，较多心肌纤维化与炎性细胞浸润,心肌胞浆空泡样变，心肌肌原纤维间隙增宽，排列紊乱，可见局灶性肌丝溶解，肌浆网明显扩张。而Cele组病理改变较轻。Cele组心肌组织匀浆中NO^-₂/ NO^-₃浓度、iNOS活性低于MI组，cNOS活性及T-AOC高于MI组(P<0.05)； Cele组Bcl-2表达阳性率高于MI组而bax表达率低于MI组（P<0.01）；细胞凋亡程度低于MI组（P<0.01）。结论：塞来昔布可增强心肌梗死后心肌细胞抗氧化损伤能力并降低细胞凋亡率。     

RNA kinetics at the wound margins.

The in vitro incorporation rate of 3H cytidine was determined by autoradiography in vital specimens taken from cut wounds in the outer ear of rats and evaluated by means of a Quantimet 920. Seven rats in each group were allowed to survive for the same periods of time between 0 and 60 hours. The following findings were observed: after 10 hours survival time the cytidine incorporation rate in the basal cells increased significantly, while remaining unchanged in all other skin layers. The relationship between the higher RNA synthesis rate and the increase in DNA synthesis--particularly in the basal cell layer--is discussed.