Mouse Models for Myocardial Ischaemia/Reperfusion

The past few years have witnessed a remarkable advance in our understanding of the pathophysiology of coronary heart disease. Myocardial ischaemia usually occurs on the basis of coronary atherosclerosis. Although the functional consequences of depriving the myocardium of its blood supply have been appreciated for many years, the coronary heart disease is still the leading cause of morbidity and mortality in the western world. This has focused the attention of physicians on restoring blood flow to the ischaemic region in order to prevent tissue necrosis and regain organ function. Reperfusion of ischaemic tissues is often associated with microvascular dysfunction that is manifested as impaired endothelial-dependent dilatation in arterioles and leukocyte plugging in capillaries. The availability of a broad variety of knockout mice provides important clues about the progression of the ischaemia/reperfusion (I/R) injury. Therefore mouse models for I/R are of great importance for the development of new therapeutic strategies for humans. Kurzfassung: Mausmodelle in der myokardialen Ischamie/Reperfusion. Die letzten Jahre wurden durch bemerkenswerte Fortschritte im Verstandnis der Pathophysiologie der koronaren Herzkrankheit gepragt. Die myokardiale Ischamie ist hauptsachlich durch die Atherosklerose der Koronargefase bedingt. Obwohl die funktionellen Folgen von verminderter Blutversorgung am Herzmuskel hinlanglich bekannt sind, ist die koronare Herzkrankheit immer noch die haufigste Todesursache in der westlichen Welt. DesMyocardial Ischaemia and Reperfusion Transient myocardial ischaemia is defined as a state of myocardial impairment due to an imbalance between the level of coronary perfusion and myocardial energy demand. Clinical hallmark is the characteristic chest pain known as angina pectoris. However, episodes of silent myocardial ischaemia also occur, particularly in individuals with diabetes mellitus and cardiac transplants. Pathophysiologic manifestations of transient ischaemia include electromechanical uncoupling, impaired ventricular function, which may be manifest as hypokinesis or akinesis, and altered electrical activity, which may be relevant by an acute injury pattern on the electrocardiogram, arrhythmus and/or conduction disturbances. If coronary occlusion is removed within approximately 20 minutes after onset, tissue viability is preserved, and its only transient damage often results in the phenomenon of stunning, which exhibits temporary contractile failure of the myocardium, but it is not associated with development of necrosis. Acute myocardial infarction is the irreversible injury and subsequent necrosis in a wavefront from subendocardium to subepicardium due to severe and prolonged reduction in coronary perfusion. The major determinants of myocardial infarct size are duration and severity of ischaemia, size of the myocardial area at risk, and magnitude of collateral blood flow available shortly after coronary occlusion. Infarct size also can be influenced by heart rate, wall tension and myocardial contractility. Systemic alterations in the adrenergic nervous system and local alterations in the adrenergic receptor-adenylate cyclase system also are operative in influencing the progression of myocardial ischaemic injury. Myocardial reperfusion is the restoration of coronary blood flow, which either occurs spontaneously or is therapeutically induced, after a period of coronary occlusion. The effects of reperfusion include not only reversible, functional changes, but also irreversible injury. A wide spectrum of deleterious effects results from reperfusion per se when it is superimposed on already ischaemically altered myocardium. Reperfusion also has the potential to salvage ischaemic myocardium. The net effect depends upon the severity and duration of the ischaemic insult before reperfusion [1]. Myocardial ischaemia/reperfusion induces a profound inflammatory condition with activation of multiple cell types, including leucocytes and endothelial cells (Fig. 1). Reduced NO bioavailability signals important patho-physiological halb richten sich die therapeutischen Bemuhungen auf die Wiederherstellung der Perfusion in den ischamischen Arealen, um die Entstehung einer Gewebsnekrose zu vermeiden und um die Funktion des Organs wiederherzustellen. Die Reperfusion von ischamischem Gewebe geht allerdings oft mit mikrovaskularer Dysfunktion einher, die sich durch Beeintrachtigung der endothelabhangigen Vasodilatation in Arteriolen und Ansammlung von Leukozyten in den Kapillaren manifestiert. Die Verfugbarkeit einer grosen Anzahl an Knockout-Mausen ermoglicht wichtige Erkenntnisse uber das Zustandekommen der Myokardschadigung durch Ischamie und Reperfusion. Deshalb sind Mausmodelle von grosem Interesse fur die Entwicklung neuer therapeutischer Strategien beim Menschen. J Kardiol 2006; 13: 239–44. Received and accepted: February 28th, 2006. From the Department of Cardiology, University Hospital of Internal Medicine, Innsbruck, Austria Correspondence to: Bernhard Metzler, MD, M. Sc. , Department of Cardiology, University Hospital of Internal Medicine, A-6020 Innsbruck, Anichstrase 35; E-Mail: bernhard.metzler@uibk.ac. at Figure 1: Scheme of involvement of the adhesion molecules in ischaemia/reperfusion. Under normal conditions, there are virtually no interactions between the endothelium and neutrophils. After coronary ischaemia/reperfusion, the blood vessel’s ability to form NO is severely reduced. With decreased supplies of NO, the coronary vessel may constrict. Additional adhesion molecules were expressed on the endothelium and polymorphonuclear leukocytes (PMNs) begin with rolling as the first step in PMN sequestration. As the PMN slows, tighter interactions between CD18 (on PMN) and ICAM-1 (on endothelium) cause the second phase of the PMN-EC interaction called sticking. The final step, the diapedesis, involves the coordination of many factors. For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH. 240 J KARDIOL 2006; 13 (7–8) Ischaemia/Reperfusion Models changes, such as leucocyte adherence and transmigration of mononuclear cells [2]. At least three major pathways may contribute to lethal reperfusion injury. The first mechanism involves massive calcium overload via Na/H and Na/Ca exchange during reperfusion, which leads to mitochondrial calcification and contraction band necrosis. A second mechanism leads to intracellular accumulation of osmotic active catabolites during ischaemia that may induce massive cell swelling and sarcolemmal rupture when reperfusion provides exposure of the injured cells to abundant extracellular fluid. A third way involves the consequences of a burst of oxygenbased free radical generation. Thus, myocardial reperfusion can cause accelerated progression of cell death in myocytes with prior severe ischaemic damage coupled with salvage of myocytes with less severe ischaemic injury. Some of the known risk factors for cardiovascular disease (hypercholesterolaemia, diabetes and hypertension) appear to exaggerate many of the microvascular alterations elicited by ischaemia and reperfusion. Although much experimental evidence exists in support of the reperfusion component of injury, ischaemia without following reperfusion will cause the destruction of most of the ischaemic myocardium. This leads to an obvious paradox: the need for re-establishing blood flow at the expense of a profound inflammatory response [3]. Mouse Types Used in Cardiovascular Research Ischaemia/reperfusion research with animal models has being carried out for a considerable time. Knowledge of the pathogenesis and therapeutic intervention of disease conditions and the use of animal models in the research have evolved almost simultaneously. Animal models are designed to be preliminary tools for better understanding of the pathogenesis and improvement in diagnosis, prevention and therapy of heart ischaemic diseases in humans. The evaluation of a risk factor as a single independent variable, with almost complete exclusion of other factors, can be performed in animals free of intercurrent diseases or abnormalities and with well known genetic characteristics. Furthermore, experiments using animals are the only way to develop and test new diagnostic, preventive and therapeutic procedures for both ethical and practical reasons. The investigator can choose the species, time and method, as well as obtain tissue, serum samples and other materials needed for measurements under optimal conditions, selective circumstances that are difficult, if not impossible, in studies with human subjects. Attracted by its well-defined genetic map, a number of investigators have begun to use the mouse as an experimental system for research on cardiovascular diseases. Hundreds of inbred lines have been established and both congenic and recombinant strains are available to facilitate genetic experimentation. Recently, genetic models have been developed in which genes are either overexpressed, deleted or mutated. Such mouse models have considerable advantages, because they overcome the need to administer factors or their inhibitors, which can be problematic and often difficult to quantify. They also seem to tolerate prolonged monoclonal antibody treatments better. Beside the use of conventional wild type mice the availability of a broad variety of genetically altered mice in the last years lead to a big rise in interest for these strains.