Combined enzyme histochemical and ultrastructural study on cryostat sections of pig heart to detect early reperfusion damage after ischaemia

In cardiac surgery, recognition of peroperative myocardial infarction may improve patient selection for prolonged circulatory support. The value of enzyme histochemistry to discriminate between reversible and irreversible myocardial damage at short periods of reperfusion was studied in an in vivo model of regional ischaemia in pig hearts. The left anterior descending coronary artery (LADCA) was clamped for 45 min followed by 2 h reperfusion (group 1, n=3). Post-mortem heart tissue showed markedly decreased activities of lactate dehydrogenase (LDH) and beta-hydroxybutyrate dehydrogenase (BDH) as demonstrated in cryostat sections, accompanied by massive leakage of LDH in the venous effluent. The depleted areas showed irreversible cell damage based on the presence of flocculent densities in mitochondria. In group 2 (n=6), LADCA flow was reduced to 40 per cent of the base-line value followed by 2 h reperfusion. Heart tissue showed normal LDH and BDH activities, except for some cells surrounding blood vessels, which activity was minimally decreased. Flocculent densities in mitochondria were never observed. We conclude that enzyme histochemistry of LDH and BDH activity on cryostat sections is a useful tool for detecting irreversible myocardial cell damage as early as 2 h reperfusion after ischaemia of the pig heart. The technique has potential applications in the detection of peroperative infarction in human biopsies.     

REDUCTION OF RAT MYOCARDIAL ISCHAEMIA/REPERFUSION INJURY BY A SYNTHETIC SELECTIN OLIGOPEPTIDE

Neutrophils infiltrate into myocardial tissue subjected to ischaemia followed by reperfusion and play a major role in myocardial reperfusion injury. The infiltration of neutrophils begins within 2 h after reperfusion, indicating the engagement of rapidly inducible adhesion molecules, such as P-selectin, on vascular endothelial cells of myocardial tissue. To investigate the essential role of P-selectin in myocardial reperfusion injury, this study examined the expression of P-selectin in rat hearts subjected to 30 min of ischaemia followed by reperfusion. The induction of P-selectin was also evaluated on the surface of cultured rat vascular endothelial cells subjected to 60 min of hypoxia, followed by reoxygenation in vitro . Finally, the effects of in vitro administration of a synthetic selectin oligopeptide on myocardial necrosis were analysed. Reperfusion of ischaemic myocardial tissue resulted in enhanced expression of P-selectin on the luminal surface of vascular endothelium and surface expression of P-selectin was induced on cultured vascular endothelial cells by hypoxia/reoxygenation in vitro . The in vitro administration of a synthetic selectin oligopeptide significantly reduced the area of myocardial infarction produced by 30 min of ischaemia, followed by 48 h of reperfusion. These data offer therapeutic possibilities for acute myocardial infarction.     

Remote vs. local ischaemic preconditioning in the rat heart: infarct limitation,suppression of ischaemic arrhythmia and the role of reactive oxygen species

The unmet clinical need for myocardial salvage during ischaemia–reperfusion injury requires the development of new techniques for myocardial protection. In this study the protective effect of different local ischaemic preconditioning (LIPC) and remote ischaemic preconditioning (RIPC) protocols was compared in the rat model of myocardial ischaemia–reperfusion, using infarct size and ischaemic tachyarrhythmias as end‐points. In addition, the hypothesis that there is involvement of reactive oxygen species (ROS) in the protective signalling by RIPC was tested, again in comparison with LIPC. The animals were subjected to 30‐min coronary occlusion and 90‐min reperfusion. RIPC protocol included either transient infrarenal aortic occlusion (for 5, 15 and 30 min followed by 15‐min reperfusion) or 15‐min mesenteric artery occlusion with 15‐min reperfusion. Ventricular tachyarrhythmias during test ischaemia were quantified according to Lambeth Conventions. It was found that the infarct‐limiting effect of RIPC critically depends on the duration of a single episode of remote ischaemia, which fails to protect the heart from infarction when it is too short or, instead, too prolonged. It was also shown that RIPC is ineffective in reducing the incidence and severity of ischaemia‐induced ventricular tachyarrhythmias. According to our data, the infarct‐limiting effect of LIPC could be partially eliminated by the administration of ROS scavenger N‐2‐mercaptopropionylglycine (90 mg/kg), whereas the same effect of RIPC seems to be independent of ROS signalling.     

Impaired sarcolemmal membrane permeability in reperfused ischemic myocardium

Summary Sarcolemmal membrane permeability to intravenously injected horseradish peroxidase HRP (MW=40,000) was examined in 8 Wistar rats which had temporary ischaemia produced by left coronary artery ligation. HRP reaction product was identified following 6 min of circulation time by light and electron microscopy. Controls included 4 uninjected animals with coronary ligation, 2 uninjected animals without myocardial ischaemia and 2 injected non operated rats.In normal myocardium, the tracer permeated endothelial plasmalemmal vesicles, intercellular spaces and intracellular vesicles of the T-tubule system, but never permeated the cytoplasm of myocardium cells.As early as 15 min after coronary artery ligation followed by 6 min of reperfusion with circulation of the tracer, HRP product could be seen in the cytoplasm of muscle cells randomly distributed in the subendocardial area. The quantity of permeated cells increased when the ischaemic myocardium is reperfused during 10 min before injecting the tracer.These data indicate that sarcolemmal membrane alteration is an early event in myocardial ischaemic injury and precede the irreversible cellular degenerative changes.This work was supported by research grant from INSERM (ATP 78-95)     

Effects of acute vagal nerve stimulation on the early passive electrical changes induced by myocardial ischaemia in dogs: heart rate-mediated attenuation

Parasympathetic activity during acute coronary artery occlusion (CAO) can protect against ischaemia-induced malignant arrhythmias; nonetheless, the mechanism mediating this protection remains unclear. During CAO, myocardial electrotonic uncoupling is associated with autonomically mediated immediate (i.e. type 1A) arrhythmias and can modulate pro-arrhythmic dispersion of repolarization. Therefore, the effects of acutely enhanced or decreased cardiac parasympathetic activity on early electrotonic coupling during CAO, as measured by myocardial electrical impedance (MEI), were investigated. Anaesthetized dogs were instrumented for MEI measurements, and left circumflex coronary arterial occlusions were performed in intact (CTRL) and vagotomized (VAG) animals. The CAO was followed by either vagotomy (CTRL) or vagal nerve stimulation (VNS, 10 Hz, 10 V) in the VAG dogs. Vagal nerve stimulation was studied in two additional sets of animals. In one set heart rate (HR) was maintained by pacing (220 beats min(-1)), while in the other set bilateral stellectomy preceded CAO. The MEI increased after CAO in all animals. A larger MEI increase was observed in vagotomized animals (+85 +/- 9 Omega, from 611 +/- 24 Omega, n = 16) when compared with intact control dogs (+43 +/- 5 Omega, from 620 +/- 20 Omega, n = 7). Acute vagotomy during ischaemia abruptly increased HR (from 155 +/- 11 to 193 +/- 15 beats min(-1)) and MEI (+12 +/- 1.1 Omega, from 663 +/- 18 Omega). In contrast, VNS during ischaemia (n = 11) abruptly reduced HR (from 206 +/- 6 to 73 +/- 9 beats min(-1)) and MEI (-16 +/- 2 Omega, from 700 +/- 44 Omega). These effects of VNS were eliminated by pacing but not by bilateral stellectomy. Vagal nerve stimulation during CAO also attenuated ECG-derived indices of ischaemia (e.g. ST segment, 0.22 +/- 0.03 versus 0.15 +/- 0.03 mV) and of rate-corrected repolarization dispersion [terminal portion of T wave (TPEc), 84.5 +/- 4.2 versus 65.8 +/- 5.9 ms; QTc, 340 +/- 8 versus 254 +/- 16 ms]. Vagal nerve stimulation during myocardial ischaemia exerts negative chronotropic effects, limiting early ischaemic electrotonic uncoupling and dispersion of repolarization, possibly via a decreased myocardial metabolic demand.     

Plasma nitrotyrosine in reversible myocardial ischaemia

BACKGROUND: Nitric oxide (NO) plays a vital role in vascular homeostasis and in the pathophysiology of coronary heart disease. Its metabolites, nitrite and nitrate, have vasculoprotective properties, whereas peroxynitrite, an oxidant metabolite of NO, is cytotoxic and can aggravate myocardial damage during ischaemic reperfusion injury. Peroxynitrite nitrates free and protein bound tyrosine residues to produce nitrotyrosine. The measurement of nitrotyrosine provides an indirect estimation of plasma peroxynitrite concentrations.AIMS: To measure plasma nitrotyrosine concentrations to see whether peroxynitrite could contribute to myocardial dysfunction during myocardial ischaemia induced by an exercise tolerance test (ETT). MATERIALS/METHODS: Plasma free nitrotyrosine concentrations were compared before and after exercise in 29 subjects with a positive ETT and 34 subjects with a negative ETT. RESULTS: Plasma nitrotyrosine concentrations were similar in patients with exercise induced myocardial ischaemia and controls. CONCLUSION: Peroxynitrite does not contribute to the myocardial dysfunction in reversible myocardial ischaemia.     

Remote ischaemic pre- and delayed postconditioning - similar degree of cardioprotection but distinct mechanisms

Myocardial ischaemia-reperfusion injury can be significantly reduced by an episode(s) of ischaemia-reperfusion applied prior to or during myocardial ischaemia (MI) to peripheral tissue located at a distance from the heart; this phenomenon is called remote ischaemic conditioning (RIc). Here, we compared the efficacy of RIc in protecting the heart when the RIc stimulus is applied prior to, during and at different time points after MI. A rat model of myocardial ischaemia-reperfusion injury involved 30 min of left coronary artery occlusion followed by 120 min of reperfusion. Remote ischaemic conditioning was induced by 15 min occlusion of femoral arteries and conferred a similar degree of cardioprotection when applied 25 min prior to MI, 10 or 25 min after the onset of MI, or starting 10 min after the onset of reperfusion. These RIc stimuli reduced infarct size by 54, 56, 56 and 48% (all P < 0.001), respectively. Remote ischaemic conditioning applied 30 min into the reperfusion period was ineffective. Activation of sensory nerves by application of capsaicin was effective in establishing cardioprotection only when elicited prior to MI. Vagotomy or denervation of the peripheral ischaemic tissue both completely abolished cardioprotection induced by RIc applied prior to MI. Cardioprotection conferred by delayed remote postconditioning was not affected by either vagotomy or peripheral denervation. These results indicate that RIc confers potent cardioprotection even if applied with a significant delay after the onset of myocardial reperfusion. Cardioprotection by remote preconditioning is critically dependent on afferent innervation of the remote organ and intact parasympathetic activity, while delayed remote postconditioning appears to rely on a different signalling pathway(s).     

Intracoronary delivery of DNAzymes targeting human EGR-1 reduces infarct size following myocardial ischaemia reperfusion

Despite improvements in treatment, myocardial infarction (MI) remains an important cause of morbidity and mortality. Inflammation arising from ischaemic and reperfusion injury is a key mechanism which underpins myocardial damage and impairment of cardiac function. Early growth response-1 (Egr-1) is an early immediate gene and a master regulator that has been implicated in the pathogenesis of ischaemia-reperfusion (IR) injury. This study sought to examine the effect of selective inhibition of Egr-1 using catalytic deoxyribonucleic acid molecules (DNAzymes, DZs) delivered via the clinically relevant coronary route in a large animal model of myocardial IR. It was hypothesized that Egr-1 inhibition with intracoronary DZ would reduce infarction size by modulating its downstream effector molecules. Egr-1 DZs inhibited the adherence of THP-1 monocytes to IL-1β-activated endothelial cells in vitro and retained its catalytic activity up to 225 min after in vivo administration. In a porcine model of myocardial IR (45 min ischaemia/3 h reperfusion), DZ was taken up in the cytoplasm and nuclei of cardiomyocytes and endothelial cells in the myocardium after intracoronary delivery. Egr-1 DZs reduced infarct size and improved cardiac functional recovery following intracoronary delivery at the initiation of IR in this large animal model of MI. This was associated with inhibition of pro-inflammatory Egr-1 and ICAM-1 expression, and the reduced expression of TNF-α, PAI-1, TF, and myocardial MPO activity in tissue derived from the border zone of the infarct. Taken together, these data suggest that strategies targeting Egr-1 via the intracoronary route after IR injury in pigs have potential therapeutic implications in human MI.     

Cardiovascular haemodynamics and ventriculo-arterial coupling in an acute pig model of coronary ischaemia–reperfusion

Although reperfusion after coronary occlusion is mandatory for myocardial salvage, reperfusion may trigger a cascade of harmful events (reperfusion injury) adding to myocardial injury. We investigated effects of reperfusion on left ventricular (LV) haemodynamics and ventriculo-arterial (VA) coupling in pigs following acute myocardial ischaemia induced by coronary artery occlusion. Experiments were performed in six animals, with measurements of cardiac and arterial function at baseline, after 60 min of ischaemia (T60) and after 2 (T180) and 4 h of reperfusion (T300). Ventriculo-arterial coupling was assessed using the ventriculo-arterial elastance ratio of paper, as well as using a 'stiffness coupling' and 'temporal coupling' index. Reperfusion following ischaemia (T180 versus T60) induced a progressive decline in cardiovascular function, evidenced by a decrease in mean arterial blood pressure, cardiac output and ejection fraction which was not restored at T300. Although reperfusion also induced an increase in slope of the end-systolic pressure-volume relationship (ESPVR), the ESPVR curve shifted to the right, associated with a depression of contractile function. Histology demonstrated irreversible myocardial damage at T300. The ventriculo-arterial elastance ratio and the 'stiffness coupling' index were unaffected throughout the protocol, but the 'temporal coupling' parameter indicated a relative shift between heart period and the time constant of the arterial system. It is unlikely that these alterations are attributable to ischaemic injury alone. The combination of both the stiffness and temporal coupling index may provide more information when studying ventriculo-arterial coupling than the more commonly used ventricular end-systolic stiffness/effection arterial elastance (E(es)/E(a)) ratio.     

Cardiomyocyte differentiation of mouse and human embryonic stem cells*

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function. Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post‐infarction cardiac failure. We are searching for methods for inducing pluripotent stem cells to differentiate into transplantable cardiomyocytes. We have already shown that an endoderm‐like cell line induced the differentiation of embryonal carcinoma cells into immature cardiomyoctyes. Preliminary results show that human and mouse embryonic stem cells respond in a similar manner. This study presents initial characterization of these cardiomyocytes and the mouse myocardial infarction model in which we will test their ability to restore cardiac function.     

Myocardial potassium balance associated with regional ischaemia in the pig: effects of beta-adrenoceptor blockade, duration of ischaemia and preceding ischaemic periods.

The effects of beta-adrenoceptor blockade, duration of ischaemia and preceding ischaemic periods on ischaemia-induced changes in myocardial K+ balance were studied in 12 open-chest pigs. Coronary venous blood was directed through a shunt from the coronary sinus to the right atrium. Continuous recordings of arterial, shunt blood [K+] and shunt flow enabled us to compute myocardial K+ balance during and after consecutive 2-, 2-, 5-, 10- and 2-min periods of regional ischaemia separated by 30 min of reperfusion. beta-adrenoceptor blockade (propranolol 1 mg kg-1 i.v.) given between the first and second ischaemic period did not alter the effects of 2 min ischaemia on myocardial K+ balance. Total K+ losses induced by 2, 5 and 10 min of ischaemia were 67.1 (40.6-93.3), 106.7 (69.4-176.8) and 192.2 (117.7-332.6) mumol 100 g-1, respectively. Thus, the plateau observed in extracellular [K+] between 2 and 10 min of regional ischaemia could, at least partly, be explained by continuous drainage of K+ from ischaemic myocardium into the surrounding normally perfused tissue. The total K+ loss induced by the second and last 2-min ischaemic period were 67.1 (40.6-93.3) and 35.6 (23.1-53.6) mumol 100 g-1 (P less than 0.001), respectively. This reduction shows that ion homeostasis during ischaemia was greatly changed in myocardium which had been 'preconditioned' and 'stunned' by 5 plus 10 min of ischaemia. Total amount, maximal rate and duration of post-ischaemic K+ reuptake increased with the duration of the preceding ischaemia. Moreover, K+ re-uptake after 2 min of ischaemia and the number of sarcolemmal Na/K pumps ([3H]ouabain binding), were normal in stunned myocardium. From these observations we conclude that progressive stimulation of the Na/K-pump occurred when ischaemia was prolonged from 2 to 10 min, and that Na/K-pump function was preserved in stunned myocardium.     

A quantitative histochemical study of 5'-nucleotidase activity in rat liver after ischaemia

The lead salt method of Wachstein and Meisel15 has been applied using incubation media containing polyvinyl alcohol for the localization and quantification of 5'-nucleotidase (E.C.3.1.3.5) activity in cryostat sections from rat liver after ischaemia in vitro and ischaemia in vivo followed by different periods of re-perfusion. 5'-Nucleotidase activity at the bile canaliculi, especially in the pericentral areas, had already decreased after 60 min of ischaemia in vitro, although the total activity as measured densitometrically was not changed. After 120-240 min of ischaemia, a significant decrease of the total 5'-nucleotidase activity was found. At that stage, signs of irreversible cell damage were recognized. Short periods of re-perfusion (1 h) after ischaemia in vivo induced a decreased bile canalicular 5'-nucleotidase activity throughout the entire liver, but a restoration after longer periods of re-perfusion was observed (5, 24, and 48 h). Necrotic areas recognized by a decreased lactate dehydrogenase activity after all periods of re-perfusion showed decreased total 5'-nucleotidase activities. A correlation was observed between the decrease in bile canalicular 5'-nucleotidase activity and the disappearance of microvilli of the bile canaliculi. It is concluded that a decrease in the bile canalicular 5'-nucleotidase activity can be used as a very sensitive marker for ischaemic liver cell damage. Assessment of the irreversibility of the cell injury has to be determined using additional parameters such as a decreased lactate dehydrogenase activity.     

An experimental evaluation of staining techniques for the detection of early ischaemic injury to the myocardium.

A series of experimental infarcts of the posterior papillary muscle of the canine heart was used to assess the value of 6 special stains in the histological detection of early myocardial infarction. The infarcts were of 5-720 min duration and were compared not only with normal control myocardium but also with normal myocardium autolyzed for similar periods of time. All tissue was stained with H & E, PAS, PAS-diastase, PTAH, Masson's trichrome, Connor's modification of the acid fuchsin method, Puchtler's PAS-navy blue, and Lie's haematoxylin-basic fuchsin-picric acid. The zone of severely altered myofibres which separated normal from ischaemic tissue in infarcts aged 2 or more hours was demonstrated by all but PAS. Normal, border, and ischaemic zones of heart muscle were clearly differentiated only by PTAH and PAS-navy blue. PAS distinguished normal from glycogen-depleted ischaemic myocardium after only 40-60 min, but this change was also seen in autolyzed tissue.     

The use of antithrombotic drugs in artery disease

Evaluating the use of antithrombotic drugs in artery disease has been a long and difficult process, which is far from complete. The aims of treatment have ranged from the primary prevention of myocardial infarction or stroke, through the restoration of blood flow to ischaemic organs in order to salvage threatened tissue, to the prevention of recurrent vascular occlusion. Drugs studied in depth by clinical trial include the oral anticoagulants, antiplatelet drugs (especially aspirin), and thrombolytic agents. Their results are considered under the headings of coronary artery disease, cerebral ischaemia, and peripheral vascular disease. Aspirin, with or without dipyridamole, prevents progression of unstable angina to myocardial infarction or death, probably reduces long-term mortality after myocardial infarction, and prevents aortocoronary bypass graft occlusion. It decreases the risks of stroke or death in patients with transient cerebral ischaemia, diminishes cardiovascular morbidity after a thrombotic stroke, and may improve the outcome after some kinds of surgery for peripheral vascular disease. The benefits of oral anticoagulant treatment to prevent artery occlusion remain poorly defined. Oral anticoagulants prevent systemic embolism in many groups of high-risk patients, and probably reduce the risk of recurrence after embolism has occurred. Whether their long-term use to prevent reinfarction in patients with a previous myocardial infarct can be justified remains uncertain. They are of little or no proven value in patients with transient cerebral ischaemia or thrombotic stroke. On the other hand, there is increasing support for early thrombolytic treatment after myocardial infarction, especially since two multicentre trials have now shown reduced mortality in patients treated with intracoronary streptokinase within 4-6 hours of infarction and a further large multicentre study also demonstrated reduced mortality in patients treated with early intravenous streptokinase. In addition, the local infusion of streptokinase leads to recanalization in a high proportion of patients with a recent peripheral artery occlusion who are poor candidates for surgery.     

Cholinergic agonists may produce preservation of myocardial ischaemia/reperfusion injury

The best treatment for myocardial infarction is to restore blood flow in the ischaemic region, though it will bring new myocardial damage known as myocardial ischaemia/reperfusion (I/R) injury. Both the ischaemia preconditioning and the ischaemia postcondioning have been shown to reduce the myocardial I/R injury, but their deficits restrict wide clinical availability. It has been demonstrated that inflammation plays a critical role in the I/R injury process. Also plasma levels of cytokines and inflammation response can be regulated by specifically augmenting cholinergic signaling via the efferent vagus nerve and α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR). Because cholinergic modalities, acting through vagus nerve- and/or α7nAChR-mediated mechanism, have been confirmed to suppress excessive inflammation during the I/R injury in kidney, liver, lung and intestine, therefore, we hypothesize that cholinergic agonists may also provide a protection for the myocardial I/R injury.     

Evaluation of the membrane attack complex of complement for the detection of a recent myocardial infarction in man

The diagnosis of an acute myocardial infarction (MI) can be cumbersome for pathologists. Even with a positive nitroblue tetrazolium (NBT) reaction, haematoxylin and eosin (H&E) evaluation of the myocardial tissue can remain inconclusive. Early signs presumed diagnostic for myocardial infarction, such as hypereosinophilia, waviness, and contraction band necrosis, have to be considered non-specific and are probably reversible signs of ischaemia. Several studies implicate the complement system, and especially complement factor C9, as part of the membrane attack factor (MAC), in cardiomyocyte damage during MI. In a post-mortem study on well-documented cardiological autopsies, we evaluated the use of complement factor C9 immunostaining as a marker for the detection of very recent MI. Forty-three tissue samples from 40 patients were obtained from the left ventricular free wall only, a region that can be specifically attributed to one corresponding coronary artery. As some patients presented with MIs of various stages in that perfusion area, in total 57 observations were possible. C9 immunostaining specifically detected irreversibly damaged (=infarcted) cardiomyocytes, as is implied by the lytic activity of C9/MAC binding to cell membranes. Most interesting was the group of clinically suspected, NBT-positive MIs resulting from very recent myocardial ischaemia. In this population, where H&E evaluation by (cardio-) experienced pathologists was not conclusive, C9 immunostaining clearly pointed towards myocardial infarction in 47% of the cases. In conclusion, C9 immunostaining, routinely practicable in the pathology laboratory, has an additional value in discriminating between reversible ischaemia and infarcted cardiomyocytes in very early MIs.     

Estimation of the tissue damage after MI through time-frequency analysis of the electromechanical signals

Abstract

Coronary congestion is a heart disease that puts many lives at risk each year. The task of coronary arteries is to distribute blood to the heart tissue and any blockage in them can cause the tissue to absorb less oxygen and nutrients than needed (ischaemia disease). This imbalance will continue until the first cell is destroyed (myocardial infarction). Simulating the myocardial infarction in the laboratory rats, this study tries to determine the extent of tissue damage through the electrocardiogram (ECG) and atrial blood pressure (ABP) synchronic signals. The signals of 50 wistar rats with a weight range of 200–300?g were recorded at 30?min in the normal case and 30?min in the ischaemia and myocardial infarction (MI) case (the artificial complete blockage was in the left anterior descending coronary artery (LAD)). For a different injury in the rats’ heart, the vasopressin (AVP) with different doses was injected to 40 rats. After that the images of the heart sections and the data were extracted, the 50-dimensional feature vector was generated by using the wavelet packet transform (WPT) on the ECG and ABP signals and also by obtaining the entropy of the wavelet coefficients. The extent of tissue damage on the images of the heart tissue was extracted by using the image processing method. Finally, the amount of the damaged tissue was estimated by four artificial neural networks (ANN) (with different structures) with an averaging criterion. The intelligent machine estimated the ischaemia and normal tissues with the average error of 2.91% for all the AVP doses and control cases.     

Myocardial ischaemia-reperfusion injury in haematopoietic cell-restricted beta1 integrin knockout mice.

Evidence indicates that the intercellular adhesion molecule-1 and its counter-receptor beta2 integrin are cardioprotective proteins during myocardial ischaemia-reperfusion, but no data are available concerning the role of blood cell beta1 integrins in this process. We studied the effects of temporary myocardial ischaemia and reperfusion in blood cell-restricted beta1 integrin knockout mice (beta1(-/-)). The left descending coronary artery in conditional beta1(-/-) integrin (beta1(-/-)), beta1 integrin +/+ (beta1(+/+)) and beta1 integrin -/- bone marrow chimeric (beta1(-/-) BM) mice was ligated for 30 min, followed by reperfusion of either 3 h or 3 weeks. Plasma levels of troponin T were evaluated as an index of cardiac cellular damage. The histological evaluation of tissue damage was performed with Haematoxylin and Eosin stained sections. Cell infiltrations in the ischaemic area were investigated by immunofluorescence studies. It was found that plasma troponin T was at a similar level in beta1(-/-), beta1(+/+) and beta1(-/-) BM mice treated with 30 min ischaemia and 3 h reperfusion. Histological analysis showed that ischaemia-reperfusion resulted in marked myocardial injury in all groups of animals, but the damage score of the hearts was not significantly different between beta1(-/-), beta1(+/+) and beta1(-/-) BM mice after 3 h of reperfusion following 30 min of ischaemia (2.8 +/- 0.5, 2.6 +/- 0.5 and 2.8 +/- 0.6, respectively, n.s.). Furthermore, no difference in scar sizes in ischaemia-injured hearts was found 3 weeks after ischaemia. Semi-quantification of cells demonstrated that, compared with beta1(+/+) mice, the number of infiltrating neutrophils was significantly reduced in beta1(-/-) and beta1(-/-) BM mice, whereas MAC-1(CD11b/CD18)-positive cells in the ischaemic regions were similar in myocardial tissues of all groups. We conclude that absence of beta1 integrin expression in haematopoietic cells results in reduced neutrophil infiltration in the ischaemic regions, but does not influence myocardial damage of ischaemic hearts.     

Ischaemic contracture and myocardial perfusion in isolated rat heart

Summary The development of left ventricular contracture and myocardial perfusion defect was studied in isolated rat hearts during global ischaemia of 90 min duration. The left ventricular pressure was measured by a balloon catheter inserted into the ventricle and filled with water. The pressure reached the maximum at 16 min of ischaemia. The left ventricular volume and compliance (passive distensibility) were measured by the same balloon, the former by connecting the balloon to an open catheter and the latter by applying a constant additional volume (0.020 ml) into the balloon. The left ventricular volume and compliance both decreased progressively for 20 min of ischaemia after which they remained low for the rest of the observation period (90 min). The myocardial perfusability was tested by infusing 0.1 per cent sodium fluorescein in isotonic saline into the cannulated aortic root of the isolated heart preparation. The percentage perfused with the fluorescent tracer in horizontal frozen myocardial sections was estimated by point counting from colour photographs taken under ultraviolet light. The proportion of the perfused area decreased gradually from 100% at 0 min of ischaemia to 93, 67, 43 and 37% at 15, 30, 60 and 90 min of ischaemia, respectively. It was concluded that ischaemic contracture of the left ventricle is followed by the development of a myocardial perfusion defect in isolated ischaemic rat heart.     

Reversible myocardial damage in gerbil brain ischaemia and its prevention by beta-adrenergic blockade.

Acute cerebral infarction in gerbils, produced by unilateral carotid ligation, was used as a model to investigate secondary myocardial changes. The extent of the myocardial damage revealed by succinic dehydrogenase (SDH) histochemistry and by release of myocardial creatine phosphokinase (MB-CK) was measured in gerbils sacrificed from 3 to 48 h after either carotid ligation, carotid isolation only or skin incision only. For technical reasons dead animals were excluded from analysis. Of surviving ligated animals 74% developed neurological deficits related to brain ischaemia. A significant weight increase in the ipsilateral hemisphere was found at 6-10 h, and maximal histological damage at 16 h, both partially reversible thereafter. Non-ligated animals did not develop neurological changes, and showed neither brain swelling nor cerebral histopathology. Extensive cardiac damage was shown by the SDH method from 3 h postoperatively, and confirmed by the elevated serum levels of MB-CK in the carotid-ligated group. The SDH changes were identical with those described in the hearts of patients with acute intracranial lesions, and appeared to be reversible. The effect of beta-adrenergic blockade was assessed in this model. Metoprolol tartrate injected intraperitoneally 3 h before and 1 h after carotid ligation (10 mg/kg each dose) significantly decreased the extent of myocardial damage as estimated both with SDH histochemistry and MB-CK serum levels. It had no effect on the ischaemic brain changes. These results strongly support the concept of catecholamine mediation of myocardial injury resulting from acute brain lesions.