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37 Open-chest Models of Acute Myocardial Ischemia and Reperfusion Kai Zacharowski, Thomas Hohlfeld and Ulrich K.M. Decking Introduction In acute myocardial ischemia and reperfusion, open chest models allow a) the investigation of cardiac physiology, b) the elucidation of biochemical, functional and morphological changes and c) the evaluation of therapeutic interventions. In chronic studies, open-chest surgery is carried out for instrumentation of animals and as a final step at the end of the study to perform invasive measurements and obtain myocardial tissue. The present overview aims to discuss surgical procedures and steps of open-chest preparations. A selection of techniques to measure parameters of cardiovascular function in open-chest preparations is also discussed. Description of Methods and Practical Approach Experimental Set-Up and Procedures To study ischemia and reperfusion in open-chest models, the laboratory needs to be appropriately equipped for surgery, including an adjustable operating table and coldlight lamps for adequate lighting of the operating area. A complete set of preferably sterile surgical instruments; threads, needles, swabs etc. must be prepared and set out before the start of the experiment. To minimize blood loss during surgery, bleeding can be stopped by careful cauterization or ligature around the relevant vessel. Once the chest is opened, thorax retractors are fitted to keep it open during the experiment. For open-chest surgery in experimental animals, anesthesia, ventilation, thoracotomy and intrathoracic surgical manipulation are steps of primary importance. These will be discussed in the following. Anesthesia and Mechanical Ventilation For ethical and experimental reasons, open-chest models can only be performed under deep anesthesia. A wide range of anesthetics has been used with success in open-chest models. However, anesthetics can cause unwanted side effects, such as hemodynamic depression, and can compromise the outcome of the experiment. Therefore, the phar- 1.3 1 38 Models of Cardiovascular Disease Table 1 Selection of drugs commonly used in open-chest studies and their potential to interfere with experimental models of ischemia and reperfusion Dr u g Mechanism of action Practical importance sedation azaperone and other neuroleptics inhibition of α-adrenergic effects of endo- and exogenous catecholamines hypotension, loss of baroreceptor reflex anesthesia barbiturates depression of vasomotor control and myocardial contractility hypotension, loss of baroreceptor reflex opioids (morphine and congeners) histamine liberation, preconditioning of myocardium against ischemia via activation of myocardial δ-opioid receptors hypotension, decrease of ischemic myocardial injury halothane decreased control of body temperature, cardiodepression preconditioning against ischemia malignant hyperthermia in some breeds of pigs alteration of myocardial function reduction of experimental infarct size isoflurane arterial dilation, preconditioning (like halothane), vasodilation systemic hypotension and impairment of coronary autoregulation, reduction of experimental infarct size, hypotension sevoflurane preconditioning (like halothane) minor vasodilatation minor cardiodepression reduction of experimental infarct size propofol protection against ischemia reduction of experimental infarct size pancuronium ganglionic and peripheral anticholinergic action hypotension, tachycardia succinylcholine slight sy mpathetic stimulation increase of heart rate heparins platelet activation or inhibition anti-inflammatory effects surgical bleeding, alteration of thrombogenesis and microcirculation, reduction of ischemic injury In-Vivo Techniques Indication muscle relaxation anti-coagulation Open Chest Models of Acute Myocardial Ischemia and Reperfusion 39 macological properties of an anesthetic and the species in which it will be used should be carefully considered. Examples are summarized in Table 1 and information about specific anesthetics is given as described below. Surgical opening of the thorax immediately causes the lungs to collapse; this can be prevented by mechanical ventilation following intubation of the trachea. The ventilator should be set to apply a positive end-expiratory pressure of 1–6 mmHg, to prevent airway collapse and atelectasis. Single-sided opening of the pleura may lead to respiratory problems by asymmetric ventilation. The most useful indicator for the appropriate control of tidal volume and respiratory rate is the end-expiratory CO2. Arterial pH, pO2 and pCO2 can additionally be determined at regular intervals. Depending on the duration of the experiment, prewetting and -warming of the inhalation gasses are useful to prevent airway dehydration. Special precautions should also be taken to prevent hypothermia, which may ensue due to loss of endogenous temperature control and excessive heat loss via ventilation and dissipation from the open chest. Surgical Preparation Open-chest models require invasive surgery and therefore some experience in basic surgical techniques is essential. Common steps in open-chest preparations are thoracotomy (usually left-side or midsternal), incision of the pericardium and the applica- Figure 1 Schematic example of an open chest preparation for induction of myocardial ischemia and reperfusion in a larger animal, such as a dog or a pig. Generally, many physiological parameters are assessed in parallel. More detailed information about the depicted experimental techniques and measurements is given in the text. Abbreviations: CO cardiac output, ECG electrocardiogram, LAD left anterior descending coronary artery, LV left ventricle 1.3 40 Models of Cardiovascular Disease In-Vivo Techniques 1 Figure 2 Open chest preparation of a rat, achieved by mid-sternal thoracotomy (Zacharowski et al. 1999). The chest is kept open by the branches of a retractor. The LAD is represented by a dotted line (inset). A hairline suture around the proximal LAD allows experimental coronary occlusion, resulting in myocardial ischemia and infarction tions of various catheters for blood collection and drug administration. In addition, probes are applied or inserted for data acquisition. Additional surgery is required to perform experimental interventions such as the positioning of coronary occluders or the connection of extracorporal perfusion systems (Fig. 1). Many steps of the surgical procedure must be adapted to the species and the particular aims of the experiment. For example, median thoracotomy may be preferable when access to the anterior wall of the ventricles is intended (e. g., occlusion of the left anterior descending coronary artery, LAD), while left lateral thoracotomy may be preferred for access to the left lateral and posterior left ventricular wall, including the circumflex coronary artery. After the completion of surgery, a stabilization period is required before experimental interventions or data acquisition are initiated. Figure 1 summarizes a selection of well-established experimental techniques commonly used in open-chest preparations of larger animal species, such as dogs and pigs. A typical small animal preparation is shown in Fig. 2. Open Chest Models of Acute Myocardial Ischemia and Reperfusion 41 Experimental Manipulation of Coronary Perfusion A significant number of experiments involving open-chest preparations aim to measure and manipulate coronary blood flow. Obviously, these experiments are of considerable value for the investigation of the pathophysiology of myocardial ischemia and help to develop new or improved therapeutic strategies. Controlled Perfusion In larger animal species (dog, pig), the main branches of the common left coronary artery can be cannulated and supplied by a coronary bypass, withdrawing blood at a predefined rate from another arterial vessel (e. g. carotid or femoral artery). This allows the perfusion of a segment of the left ventricle at a defined flow rate or perfusion pressure (see Fig. 1). This technique has greatly contributed to an improved understanding of coronary autoregulation. Moreover, the tight control of the flow rate enables the intracoronary infusion of drugs or substrates at well-defined arterial concentrations. This may be important for drugs with a small therapeutic range. The approach can also be employed to selectively modify vascular tone or myocardial contractility in the area supplied by a bypass or to provide a metabolic substrate at a given concentration to this region (Decking 2001). Subtotal Ischemia When studying the effects of flow reduction on myocardial contractility and metabolism, the bypass technique described above enables reduction in coronary flow in a step-wise manner in order to investigate functional and metabolic adaptation processes, such as perfusion-contraction-matching (Ross 1991) and myocardial hibernation (Heusch 1998). In these studies, flow was classically reduced by 50% of baseline, and the effects on contractile function and myocardial energetics were analyzed. Using a similar approach, the sensitivity of cytosolic adenosine to an imbalance in the oxygen-supply to -demand ratio was demonstrated (Deussen 1998). Open-chest models with reduced coronary flow and controlled reperfusion have also been extensively used to define and study the phenomena of stunning and preconditioning, including pharmacotherapeutic strategies to improve myocardial function and biochemical outcome after subtotal ischemia. Intracoronary Thrombosis Open-chest preparations are useful to simulate myocardial ischemia in conjunction with its pathophysiological “trigger”, coronary thrombosis. Several experimental techniques are available to induce coronary arterial thrombi. One of the more popular models, originally introduced by Folts (1991), combines an acute injury of the coronary artery (transient clamping at a defined force) with a critical stenosis of the injured segment (plastic cylinder around the injured region). Stenosis and vessel wall injury will cause a mural thrombus to develop within minutes to hours, depending on the extent of arterial injury. This results in a gradual decline in coronary flow. Blood 1.3 In-Vivo Techniques 1 42 Models of Cardiovascular Disease flow often suddenly recovers due to the mobilization of the thrombus into the distal circulation, causing recurrent (“cyclic”) coronary flow variations. Intravenous administration of adrenaline can be used to exacerbate thrombosis. Other strategies aim to injure the endothelium by intracoronary injections of concentrated saline, alcohol or hot solutions with consequent formation of an intracoronary thrombus. Arterial thrombosis can also be induced by the administration of an electrical current (about 150 µA) to the endothelial surface of an artery (Romson et al. 1980). This is done by inserting an electrode (anode) into the artery. The electrical current results in local platelet adhesion and thrombus development. The electrical cathode may be applied to the adventitia of the vessel. Open-chest models of coronary thrombosis have contributed to the development and investigation of anticoagulants, inhibitors of platelet function and thrombolytics, of which many have become indispensable agents for antithrombotic treatment in clinical medicine. Coronary Occlusion The surgical occlusion of a coronary artery (see Figs. 1 and 2) with consequent myocardial infarction has been performed in many species during the past 50 years, including dogs, pigs, cats and rabbits. Since the use of small rodents is less expensive and keeps drug consumption low, models of coronary occlusion have also been developed for smaller species, including rats and mice. Genetically manipulated mice offer new possibilities of characterizing the pathomechanisms of ischemia-reperfusion injury. It must be realized, however, that the experimental occlusion of a coronary artery does not ideally simulate the natural course of myocardial infarction, which often involves subtotal thrombotic occlusion and intermittent phases of spontaneous thrombus dislocation or thrombolysis. Nevertheless, this technique can provide valuable insight into the functional, morphological, biochemical and molecular events that lead to the development of ischemic myocardial injury. Many techniques have been used to occlude a coronary artery. A simple suture around the vessel can only be recommended for continuous ischemia without reperfusion. If reperfusion is intended, a surgical “snare” should be applied, consisting of a thread sling placed around the artery with both ends pulled through a piece of soft silicon tubing. The snare is tightened for induction of ischemia. Care must be taken not to injure the artery by pulling the occluded segment into the tubing. Soft arterial clamps and hydraulic occluders are also available and may be less traumatic. Assessment of Myocardial Integrity and Function Open-chest preparations with access to the intrathoracic cavity allow for an almost unlimited evaluation of alterations in myocardial tissue integrity and function. Blood and tissue samples can be analyzed for biochemical and morphological parameters. Open Chest Models of Acute Myocardial Ischemia and Reperfusion 43 Plasma Markers of Myocardial Injury Acute models of myocardial ischemia and reperfusion often require the quantitative determination of infarct size. This may prove to be difficult, because some myocytes are clearly viable or necrotic, while others are still in the process of recovery or transition from viable to necrotic or apoptotic cell death. Therefore, terminal infarct size should ideally be determined after stabilization for several days of survival. However, many studies have obtained a reasonable differentiation of necrotic from viable tissue after much shorter periods, but it must be kept in mind that long-term processes such as ischemia-induced inflammation, thrombosis and apoptosis may not be adequately reflected in these studies. It depends on the experimental aims whether or not shortor long-term effects on myocardial viability are of primary interest. Cytosolic or myofibril-bound markers released into plasma from the ischemicreperfused myocardium are commonly used to estimate infarct size. These include lactate dehydrogenase (LDH), creatine kinase (CK), myoglobin, cardiospecific CK (CK-MB) and cardiac troponins. Commercial enzymatic assays are available for routine measurements. During reperfusion after severe ischemia, the concentration (or activity) of these markers rapidly rises in peripheral blood. Depending on collateral coronary blood flow during ischemia, this rise may already become evident during ischemia. Morphological Infarct Size A widely accepted technique for measuring infarct size is histochemistry. Many studies have determined the loss of myocardial dehydrogenase activity by tetrazolium dyes in order to detect severe and probably irreversible myocardial injury. In the presence of intact dehydrogenase enzyme systems (viable myocardium), tetrazolium salts form blue or red formazan pigments, whilst areas of necrosis lack dehydrogenase activity and therefore fail to stain. Since the dehydrogenase activity slowly disappears when myocardial tissue becomes necrotic, relevant results can only be expected after several hours of reperfusion (Fishbein et al. 1981). Tetrazolium staining is inexpensive and requires the reperfused ischemic tissue to be incubated in aqueous solutions of triphenyl or nitroblue tetrazolium. Ex vivo perfusion with tetrazolium salts is also possible. Vital (stained) and necrotic tissues (unstained) are easily distinguished (Fig. 3). In order to quantitate infarct volume, the heart must be cut into slices, which are analyzed by computer-assisted planimetry to provide a representative measure over the entire heart. As important additional information, many studies have also determined the amount of tissue, which is subjected to ischemia and reperfusion and within which necrosis is expected. This “area at risk” is identified ex vivo by re-occlusion of the coronary artery and subsequent perfusion with Evans Blue or other dyes, which results in staining of the perfused myocardium and leaves the formerly ischemic area unstained (see Fig. 3). Infarct size can be expressed as a fraction/percentage of the entire heart, of the left ventricle or of the area at risk. 1.3 In-Vivo Techniques 1 44 Models of Cardiovascular Disease Figure 3 Top: transversal slice of a pig heart after 1 h of LAD occlusion, followed by 3 h of reperfusion. At the end of reperfusion, the coronary artery has been re-occluded and saline with Evans Blue dye perfused into the coronary ostia. Nonischemic myocardium is stained dark blue within the posterior (post) and lateral wall of the left ventricle (LV). The anterior (ant) septum and a small part of the anterior left ventricular wall, located distal to the occluded coronary artery (area at risk, Ri), remained unstained. The risk area amounted to about 30% of the left ventricle. Additional staining of vital myocardium with triphenyl tetrazolium delineated the necrotic tissue (Ne), which was approximately 50% of the area at risk in this experiment. Bottom: determination of the area at risk and infarct size in a rat heart by a modified technique (Zacharowski et al. 1999b). Rats were subjected to 25 min of LAD occlusion and 2 h of reperfusion. After re-occlusion of the LAD, Evans blue dye was injected, staining perfused tissue blue (non-perfused area stains red; left panel). Subsequent incubation of the heart slices with nitro-blue tetrazolium stained vital tissue (normally perfused plus area at risk) dark-blue. Necrotic myocardium is not stained (right panel). For coloured version see appendix There are additional techniques to measure irreversible myocardial injury. For example, myocardial cell integrity has been determined by perfusion with horseradish peroxidase, followed by in vitro detection of intracellular peroxidase after transition of the enzyme through the injured plasma membrane into the intracellular space. This procedure appears to identify infarcted myocardium after rather short periods of reperfusion (Farb et al. 1993). As compared with histochemical staining, light and electron microscopy do not usually quantify myocardial infarct size. Nevertheless, histology provides invaluable morphological information about ischemia-induced myocardial injury. Myocardial Contractile Function As in closed-chest models, global measures of myocardial contractile function can be assessed by catheter-based manometers. Open-chest preparations allow direct insertion of miniature pressure tip catheters into the atria and ventricles for the sensitive Open Chest Models of Acute Myocardial Ischemia and Reperfusion 45 detection of pressures and dP/dt. Myocardial contractile function can nowadays be assessed by imaging techniques such as ultrasound and magnetic resonance. These techniques usually achieve better signal-to-noise ratios and thus a higher spatial resolution in the open-chest animal. The measurement of regional ventricular dynamics is usually performed by sonomicrometry, which has developed as a common standard to measure ventricular segment length, wall thickness or vessel diameters. The devices commercially available allow a high resolution of time (milliseconds) and dimensions (micrometers). Traditionally, a pair of piezoelectric crystals (emitter and receiver) is applied to the tissue of interest (often the ventricular wall) and the transit times of repetitive ultrasound pulses (1 MHz or higher) are recorded. These are automatically converted into distances, assuming a constant speed of ultrasound propagation (1540 m/s) in biological tissue. Single crystal systems, which measure pulse reflection from interfaces between different tissues (e. g., blood/myocardium), are also available. Two- and three-dimensional arrangements of ultrasound crystals are used to estimate ventricular volumes. Sonomicrometry can provide very valuable information about regional myocardial function in open-chest preparations (see below). Electrophysiological Measurements The conventional ECG (chest wall) is often difficult to interpret in open-chest preparations due to the altered electrical environment of the heart. Therefore, ECG recordings are frequently derived from uni- or bi-polar electrodes attached to the epicardium. Open-chest preparations also provide an opportunity to simultaneously record many ECG signals from the epicardial surface with high geometrical resolution, in order to map the propagation of action potentials across the surface of the heart under normal and pathological conditions. The origin and spreading of arrhythmias can also be investigated, giving valuable information about the effects of antiarrhythmic drugs. A large number of channels (> 1000) on an epicardial surface of about 50 cm2 can be achieved in larger species (pigs or dogs), with a resolution of about 2 mm2. In addition to ECG recordings, it is possible to derive cardiac monophasic action potentials (MAP) from almost any ventricular location (Franz 1999). The MAP signal is thought to represent a local injury potential, which is created between the normal tissue beneath a reference electrode and the locally injured (depolarized) area beneath another electrode. The depolarization can be generated by exerting a gentle mechanical pressure on the tissue under one of the electrodes. Different designs apply KCl solutions locally. More recently, catheter-based MAP electrodes have become available for closed-chest experiments and human use. A general problem is that a relative rather than the absolute action potential voltage is recorded. There is also a limitation in time, because stable signals are obtained only over minutes to hours. Nevertheless, MAP measurements can provide information about depolarization, refractory period and after-depolarizations. They are well suited for the in vivo evaluation of antiarrhythmic drugs. 1.3 1 46 Models of Cardiovascular Disease Techniques to Measure Coronary Blood Flow and Perfusion In-Vivo Techniques Coronary blood flow is of great importance for numerous types of studies and mandatory to calculate myocardial consumption of substrates or oxygen and the release of metabolites. The open-chest preparation provides excellent means in this context, because arterial and coronary venous blood from the coronary sinus or an epicardial coronary vein are easily accessible for the transcardiac measurement of metabolites and elaborate techniques are available to determine coronary perfusion with high precision. In the open-chest animal, coronary flow and myocardial perfusion are conventionally studied using 1) flow probes or 2) microspheres. Flow Probes Until 20 years ago, electromagnetic flow probes were the state-of-the-art for determinations of vascular volume flow (ml/min). The current standard equipment is ultrasound transit time flow probes. These probes cover vessel diameters from about 0.5 to 16 mm. Perivascular flow probes measure local volume flow with high temporal resolution and precision. Since a perivascular probe has to surround the interrogated vessel, the relevant arterial vessel has to be carefully dissected, which can injure smaller side branches and impair local sympathetic control. Due to size constraints, flow probes in mice and rats have only been employed in the assessment of cardiac output (ascending aorta) or carotid or femoral artery flow. In larger animals (e. g. dogs and pigs), coronary flow can also be assessed at the level of the left anterior descending or left circumflex artery. Many of the flow probes available are not only suitable for measurement in open-chest models, but can be chronically implanted for monitoring of cardiac output or coronary artery flow. Microspheres Without quantitative knowledge of the region supplied by a given vessel, the vascular volume flow, e. g. of a coronary artery, is not a direct measure of local myocardial perfusion, which is generally given in ml/min/g. In the open-chest animal this is most frequently assessed using microspheres of 10 to 15 µm in diameter. In the context of ischemia and reperfusion, microsphere flow measurements are almost mandatory to define the extent of residual collateral flow during coronary artery occlusion or stenosis in the area at risk and also to determine local flow in the border zone. Microspheres (e. g. 0.2 million/kg) are usually injected during a period of 5–30 s into the left atrium. Following natural mixing in the atrium and ventricle they are homogeneously distributed in the cardiac output and are distributed to all arterially supplied organs in proportion to their respective share of cardiac output. Since the microspheres are clearly greater than the internal diameter of any capillary (5–7 µm), they are almost completely extracted during the first pass of organ perfusion, most probably in pre-capillary arterioles and capillaries. The number of microspheres per organ is therefore a relative measure of arterial blood supply. To obtain an absolute measure of flow, a virtual reference organ is frequently employed by withdrawing blood from a central artery (e. g., aorta) at a given volume flow (e. g., 10 ml/min) for Open Chest Models of Acute Myocardial Ischemia and Reperfusion 47 Figure 4 Fluorescence emission spectra of 7 differently labeled fluorescent microspheres (Molecular Probes), dissolved in 2-ethoxyethylacetate. Excitation wavelengths in nm are given in brackets. For coloured version see appendix 2 to 3 min into a syringe during and after microsphere application. Thus, if 10,000 microspheres are deposited in the reference organ (10 ml/min), 1000 microspheres in a given organ would represent a blood supply of 1 ml/min. The technique required to measure microsphere deposition depends on their labeling. Commercially available radioactive microspheres are labeled with 46Sc, 85Sr, 113 Sn, 153Gd (all γ-radiation), display a uniform activity per microsphere (e. g. 1 Bq/ sphere) and can be detected in tissue or tissue samples using a γ-counter without additional preparation. The measurement of fluorescent (Fig. 4) and colored (absorbing) spheres, which are available from different manufacturers, requires tissue degradation in KOH (4 mol/l, 50 °C, 4 h, > 5 ml KOH/g tissue) and the separation of the microspheres from the tissue digest by 8 µm polyethylene filters. Thereafter, filters and fluorescent microspheres are quantitatively transferred into cups and the microspheres are dissolved by 2-ethoxyethylacetate, enabling the detection of fluorescent emission intensities at defined excitation wavelengths. Similar protocols apply for colored microspheres. The choice of microspheres depends on the available detection equipment and the number of measurements required. Radioactive microspheres require little manipulation but are more expensive, expose the laboratory to radiation and require the disposal of radioactive waste. For colored microspheres, only 3 different colors can be reliably quantified in one sample, which limits the number of time points that can be measured in one experiment. Since in the microsphere technique local blood perfusion is inferred from the deposition of discrete particles, there are two caveats of practical importance: 1.3 In-Vivo Techniques 1 48 Models of Cardiovascular Disease 1. The stochastic nature of the particle distribution pattern in tissue requires a given number of counted spheres for a desired precision of measurement. It has been calculated that about 400 spheres are required to estimate local deposition density with 5% precision (Buckberg et al. 1971). This already limits on theoretical grounds the spatial resolution of flow measurements attainable using microspheres. 2. In the arterial vascular tree, the deposition of microspheres may not be identical to plasma or erythrocyte flow. Indeed, Bassingthwaighte et al. compared microsphere deposition with the uptake of methylimipramine, which is almost completely extracted during its first capillary passage, and observed a small bias of microspheres towards areas of higher flow. Most probably, the bias will increase at higher spatial resolution, where flow at small arterial bifurcations may control the direction of deposition of an individual microsphere. In general, however, microsphere deposition correlates well with local plasma flow (reviewed by Prinzen and Bassingthwaighte 2000). Microsphere studies revealed that the distribution of myocardial blood flow is not entirely homogenous. There is a transmural gradient, with higher subendocardial and lower subepicardial values, the ratio being about 1.2–1.4/1. Moreover, microsphere measurements at high spatial resolution revealed in several species a substantial spatial variability within each myocardial layer. For example, at a resolution of 0.3 g, about 6% of samples receive less than 50% and another 6% more than 150% of the average flow, which cannot be explained by the transmural gradient. This spatial heterogeneity is temporally stable for weeks (Decking et al. 2002) and correlates with indices of substrate uptake, energy turnover and local protein expression (reviewed by Deussen 1998 and Decking 2002). Hence, when applying microspheres for flow determinations, the microsphere density in small myocardial samples may not be a valid measure of the average myocardial blood flow, e. g. in the LV free wall or a distinct myocardial region. However, providing the number of counted spheres exceeds 400 and the tissue size is greater than 1 g per sample, the flow value determined may be representative of a larger area of myocardium under similar experimental conditions. Methods to determine myocardial perfusion are not limited to microsphere deposition. Uptake of radioactive molecular markers would be an alternative, but most (e.g. K+ or Rb+) suffer from incomplete first pass extraction or rapid wash-in and wash-out kinetics (e.g. 3H2O). Local perfusion measures using positron emission (PET), magnetic resonance imaging (MRI) or echocardiography are also available. Examples Experimental Preparation Anesthesia and Mechanical Ventilation Many anesthetics with different pharmacological properties have been used in openchest studies. Examples are barbiturates, propofol, opioids and the volatile anesthetics halothane, isoflurane and sevoflurane. Chloralose, while obsolete in clinical Open Chest Models of Acute Myocardial Ischemia and Reperfusion 49 medicine, has occasionally been preferred for cardiovascular studies because the vasomotor tone is less affected than by many other anesthetics. The anesthetic is of great importance for the desired experiments and can be a source of severe problems. As in clinical anesthesia, experimental protocols often use a combination of different anesthetics, allowing for an acceptable narcotic and anesthetic effect with a minimum of circulatory depression. Numerous anesthetics have been used with good success in open-chest experiments. For acute studies with open-chest rats, for example, the authors routinely use barbiturates, such as thiopentone sodium (120 mg/kg, i.p.) or pentobarbitone (60 mg/ kg i.p.), followed by supplementary small doses, as required. Artificial ventilation is mandatory during anesthesia. Thiopentone has a longer anesthetic effect than pentobarbital but tends to reduce blood pressure. For chronic experiments with intended survival, midazolame (5 mg/kg, i.p.) with the neuroleptanalgesic combination of fentanyl (0.1 mg/kg, i.m.) plus fluanisone (3 mg/kg, i.m.) is a better alternative, because spontaneous respiration recovers more rapidly at the end of the experiment. For open-chest studies with larger animals, such as swine, the authors start anesthesia by an i.m. injection of azaperone (4 mg/kg), followed by i.m. ketamine (10 mg/ kg). Atropine (0.02 mg/kg) may be helpful to prevent excessive tracheobronchial mucus production. After tracheal intubation and start of artificial ventilation, pancuronium bromide (0.1 mg/kg) is given i.v. for skeletal muscle relaxation. Thereafter, fentanyl (0.01–0.02 mg/kg) is injected i.v. to provide sufficient analgesia, later re-administration being required for longer surgical procedures. Anesthesia is maintained by isoflurane (1–1.5%). Food should be withheld for 12 h before anesthesia with free access to water. In chronic experiments, the administration of prolonged acting opioids (e.g. piritramide) may be required to prevent pain during the recovery from surgery. Additional information about anesthesia is also given below. Surgical Preparation The surgical procedure, as described above, must remember that the intrathoracic anatomy may differ between species. For example, the left coronary artery is predominant in rats without a true equivalent of a circumflex artery. Most of the blood of the left coronary artery is carried by the left descending branch, which runs in an almost straight line from its origin towards the apex of the heart and supplies the left ventricular wall by almost horizontal, lateral branches. Species differences in coronary anatomy also include the degree of collateral blood supply during ischemia, which is high in dogs and much lower in pigs (Hearse 2000). Manipulation of Coronary Perfusion The duration of ischemia required to achieve a certain degree of injury is determined by many experimental variables. Examples are species differences in collateral supply, anesthetics, hemodynamic parameters and body temperature. Pilot experiments are usually needed for adjustment of the experimental conditions to achieve the desired degree of experimental ischemic injury. Fig. 5 shows an example of a series of experi- 1.3 In-Vivo Techniques 1 50 Models of Cardiovascular Disease Figure 5 Effects of varying lengths of regional myocardial ischemia (LAD occlusion for 0–60 min) followed by 2 h of reperfusion in anesthetized rats. Each group n = 5–11. Top: Infarct size expressed as percentage of the area at risk (AR), which amounted to approximately 50% of the left ventricle in these experiments. Bottom: cardiac troponin T release expressed as plasma concentrations. *, p < 0.05 vs. control experiments without ischemia. ND not done ments that have been performed to characterize the time-dependent increase of infarction. There is a steep interrelation between the duration of ischemia and injury, as measured by the release of cardiac troponin T into the plasma or morphological infarct size. Moreover, the experiments show that parameters of ischemic injury respond clearly with different temporal kinetics (see below for further discussion). Therefore, assessment of more than one parameter of ischemic myocardial injury is generally re-commended, if this is an important endpoint in a particular study. Many studies have suggested that the re-introduction of oxygen or the occurrence of inflammation during reperfusion may aggravate ischemic myocardial injury (“reperfusion injury”). The amount of reperfusion-induced injury also depends on the particular experimental conditions and is probably most prominent within the first minutes of reperfusion. An aggravation of myocardial injury during the first minutes of reperfusion is, however, difficult to detect because the methodology to identify infarcted myocardium (e. g., tetrazolium staining) requires a minimum duration of reperfusion (hours). Nevertheless, the viability of ischemic myocardium before re-perfusion is of little practical interest, because reperfusion is a conditio sine qua non for tissue survival. Longer times of reperfusion may be less critical. In a systematic evaluation in openchest rats, an increase of the reperfusion period from 2 to 8 h did not result to a major increase in infarct size (K. Z., unpublished). Open Chest Models of Acute Myocardial Ischemia and Reperfusion 51 Assessment of Myocardial Integrity and Function Measurement of Myocardial Infarct Size Regional ischemia induces myocardial infarction, the extent of which correlates to the duration of ischemia. For example, coronary artery occlusion in the rat causes necrosis after several minutes of ischemia, with a continuous increase until approximately 45 min. At this time, about 75% of the area at risk has become necrotic. Longer periods of ischemia often do not lead to a further increase in infarct size, because marginal regions of the area at risk may be supplied by diffusional oxygen or collateral blood flow from the surrounding normally perfused tissue, the lumen or the epicardial surface. Figure 5 shows representative examples from open-chest rats. There is also a positive correlation between plasma markers of ischemic injury and soluble markers of ischemia, such as cardiac troponin T (see Fig. 5). Nevertheless, the time courses of troponin T in plasma and morphological infarct size are not identical. Obviously, the kinetics of troponin release following impairment of myocardial cell membrane permeability on the one hand, and the release and degradation of myocardial dehydrogenases (used for tetrazolium staining) on the other hand are only loosely related to one another. Similar considerations may apply to reperfusion. In open-chest rats, for example, morphological infarct size remains almost unchanged between 2 and 8 h of reperfusion, while there is a remarkable (more than 50%) decline in the plasma concentration of cardiac troponin T within the same time (K.Z., unpublished). The clearance or redistribution of troponin is apparently high enough to cause a decrease in plasma troponin within only a few hours. Sonomicrometry Measurements of regional contractile function with sonomicrometric crystals usually require larger animal species, mostly pigs or dogs. Within the ventricular wall, distances between two crystals may be measured circumferentially in a short-axis plane, longitudinally (base to apex) or in a diagonal orientation. Which one gives the best results depends on the local fiber orientation. In addition, endo- and epicardial placement of crystals enables the measurement of wall thickness, which is preferred by many investigators because it is independent of the local fiber direction. Epicardial single-crystal devices, which determine wall thickness from the endocardial echo signal, are particularly convenient. Sonomicrometric measurements are very useful in evaluating regional myocardial contractile function during ischemia and reperfusion, whereas global parameters, such as intraventricular pressures or cardiac output, may not adequately reflect myocardial function within an ischemic area. This is demonstrated in Fig. 6, where LAD occlusion in an open-chest pig causes only moderate changes of left ventricular pressure and aortic flow. In contrast, the sonomicrometric registration of left ventricular wall thickness within the ischemic area shows a dramatic decline in the systolic increase in wall thickness (contraction), which is already completely lost 2 min after coronary occlusion. At this time, the ischemic ventricular wall is passively stretched 1.3 52 Models of Cardiovascular Disease In-Vivo Techniques 1 Figure 6 Registrations of left ventricular pressure, aortic flow, wall thickness (sonomicrometry) and epicardial ECG in an anesthetized open-chest pig immediately before (control) and different times after experimental occlusion of the LAD. Myocardial ischemia, which comprised about 20% of the left ventricle in this experiment, resulted in only minor changes of global ventricular function (left ventricular pressure, aortic flow), while regional function measured by sonomicrometry (wall thickness) reveals the deterioration of left ventricular systolic contraction with progressive thinning during systole. Regional contractile function in an area remote from ischemia was preserved, except for a decrease in end-diastolic wall thickness (increasing end-diastolic volume). The epicardial ECG shows the characteristic signs of acute myocardial ischemia (increase of T, loss of R wave) by the intraventricular pressure, as shown by a systolic decrease in wall thickness (bulging). There is also a decrease in the end-diastolic wall thickness of the non-ischemic ventricular wall, which results from an increase in the end-diastolic left ventricular volume due to a moderate degree of ventricular failure. Microspheres Microsphere measurements are, as outlined above, generally stochastic and a sufficient amount of microspheres needs to be trapped within a given volume of myocardial tissue. As long as the number of spheres in the sample of interest exceeds 400, the precision of the measurement in the sample of interest will be > 95%, which conventionally requires (at 200.000 microspheres/kg) a sample size of 0.5–1 g wet weight. Nevertheless, due to the physiological phenomenon of spatial perfusion heterogeneity, the perfusion of an individual 1 g sample may still not be representative of average myocardial blood flow. Transmural analysis of > 10% of the total LV tissue will 3 3 r² = 0.84 r² = 0.57 2 2 MBF in the open-chest animal after pericardiotomy (ml min–1 g–1) 1.3 53 Open Chest Models of Acute Myocardial Ischemia and Reperfusion 1 1 Heart # 1 0 0 1 3 2 Heart # 2 0 3 0 1 2 3 3 r² = 0.75 r² = 0.57 2 2 1 1 Heart # 4 Heart # 3 0 0 0 1 2 3 0 1 2 3 3 2 r² = 0.78 r² = 0.21 2 1 1 Heart # 6 Heart # 5 0 0 0 1 2 0 1 2 3 Myocardial blood flow, awake animal (ml min–1 g–1) Figure 7 Myocardial blood flow (MBF) following midazolam-piritramide anesthesia, open-chest surgery and pericardiotomy as compared to blood flow under resting conditions (awake) in chronically instrumented beagle dogs. Each data point represents the local perfusion of an individual left ventricular tissue sample (300 mg) determined in the anesthetized and conscious animal, respectively In-Vivo Techniques 1 54 Models of Cardiovascular Disease be necessary to obtain a valid measurement of LV perfusion even under physiological conditions. Special attention is required when perfusion is to be measured in models of local ischemia and reperfusion, where the extent of spatial heterogeneity is substantially increased. Figure 7 shows examples of LV free wall flow measurements by microspheres. Microspheres were given first in conscious, chronically instrumented beagle dogs, and thereafter in the anesthetized animal following open-chest surgery and pericardiotomy. Following myocardial excision, the local perfusion of individual 300 mg myocardial samples was determined. The perfusion of each individual sample under anesthesia is related to the basal perfusion in the conscious animal. Two important features become apparent. First, local myocardial blood flow already varies substantially in the conscious animal despite the absence of any coronary stenosis, reflecting spatial heterogeneity of flow. Secondly, in most hearts, areas receiving little flow under basal conditions displayed low flow during anesthesia, and high flow areas remained on average high flow areas during anesthesia. However, on average anesthesia resulted in a decrease in local perfusion since the slope of a linear regression was < 1 in each of the experiments. The lower average blood flow most probably reflects the lower energy demand due to a reduced global workload. While in most hearts flow under anesthesia correlated closely to that under basal conditions, in some hearts flow under anesthesia was almost independent of basal perfusion (see heart #6 in Fig. 7), reflecting a redistribution of perfusion. These factors have to be taken into account in the interpretation of myocardial blood flow in open-chest models. Troubleshooting Anesthesia and Mechanical Ventilation Anesthesia in general and the pharmacological properties of anesthetics specifically exert a profound influence on cardiovascular control. One problem frequently encountered is the potential depression of blood pressure and myocardial contractility. Anesthetics may directly reduce myocardial contractility and alter the autonomic tone with profound effects on myocardial oxygen consumption in normal and underperfused tissue. Hence, the anesthetic agents must be carefully selected according to potential interference with the experiment (see Table 1). The dosing strategy should also consider the pharmacokinetic properties of anesthetics. Depending on the drug, administration by continuous infusion is better than a bolus injection, particularly if constant hemodynamic conditions are required. Nevertheless, the pharmacological properties of some compounds are complex. For example, with pentobarbitone the dosing regimen is critical, because redistribution within the body during early anesthesia may favor underdosing, while longer administration (hours) may lead to accumulation and overdosage. If anesthetics are not given with a loading dose, a duration of at least 4 times the compound’s terminal halflife will be required to achieve a pharmacokinetic steady state. Open Chest Models of Acute Myocardial Ischemia and Reperfusion 55 Volatile anesthetics are frequently used in open-chest preparations. However, there is some indirect evidence that halogenated compounds (e. g., halothane, isoflurane), even when administered for a short period (minutes), may alter myocardial tolerance to ischemia, potentially by acting via myocardial K+ATP channels (Kwok 2002). Intravenous agents such as pentobarbital, ketamine-xylacine and propofol do not appear to have this property. Nevertheless, ketamine may interfere with ischemic preconditioning (Walsh et al. 1994). Mechanical ventilation can change hemodynamic parameters, in particular by increasing the intrathoracic pressure, which decreases venous blood return into the thorax and reduces ventricular preload and cardiac output. This may be of particular significance if insufficient amounts of fluid are administered and can be prevented by infusing sufficient amounts of fluid. Experimental Preparation Open-chest preparations require major surgical experience. A typical complication during thoracotomy is the injury of a large cranial vein near the cranial thorax aperture with severe venous bleeding and lethal air embolism. In some species (e. g., pigs) the anterior ventricular wall is very close behind the sternum, resulting in risk of injury to the heart during sternotomy. Moreover, the dissection of coronary arteries may injure unrecognized diagonal or marginal branches, particularly when the anatomy is complex. In larger species (dogs, pigs), it is helpful to keep heart rate low (< 100 beats per min) by appropriate anesthesia. Surgical blood loss increases the tendency of open chest preparations to develop hypovolemia and, therefore, must be minimized. Minor unrecognized bleeding may cause significant blood loss into the thoracic cavity. Whenever possible, blunt dissection should be preferred to sharp incision and anticoagulants should not be administered until surgical manipulations are completed. Surgery generally causes an inflammatory response. This may be of particular importance for manipulations at the epicardial surface of the heart, such as are required to apply epicardial electrodes, flow probes, vascular cannulas or sonomicrometric crystals (see Fig. 1). Within only a few hours, an accumulation of neutrophil granulocytes can be observed within the subepicardial layers of the ventricles. It may therefore be difficult to distinguish between an inflammatory response caused by an experimental intervention (e. g., ischemia and reperfusion) and one caused by an artifact of surgery. Tilting of the heart, as sometimes required to expose structures which are difficult to access (lateral segments of coronary arteries, posterolateral biopsies, coronary sinus) may cause deterioration in myocardial function and circulatory stress, which can profoundly alter coronary blood flow and cause an unwanted preconditioning of the heart against ischemia. An open pericardium can also change ventricular geometry, for example diastolic over-distension of the ventricles at higher end-diastolic pressures. An intact or reclosed pericardium is, hence, recommended if increased ventricular filling pressures are expected. 1.3 1 56 Models of Cardiovascular Disease The open chest represents a large wound, making it vulnerable to the development of hypothermia. Mechanical ventilation and infusion of cold solutions contributes to this condition. The continuous monitoring of body temperature and, if necessary, external heating (infrared lamp, heating pads) are required. Hypothermia can critically influence the experimental outcome. For example, a fall in temperature by only 1°C reduces infarct size by 10% (Chien et al. 1994). In-Vivo Techniques Manipulation of Coronary Perfusion Extensive manipulation of a coronary artery, often required in open-chest studies for cannulation or placement of occluders, can induce vasospasm with myocardial ischemia, arrhythmias and infarction. It can be helpful to administer a short-acting local vasorelaxant such as glycerol trinitrate topically to prevent or terminate arterial spasm. Vascular compression must be avoided to prevent injury to the vascular wall with subsequent spasm or intravascular thrombosis. Assessment of Myocardial Integrity and Function Markers of Myocardial Injury Plasma markers of ischemic injury (e.g., troponins) may correlate with morphological infarct size, such as tetrazolium staining (O’Brien et al. 1997), but disparities between cardiac troponins and infarct size have also been reported (Kawakami et al. 1999). These may be explained by delayed washout of the markers from injured myocardium during ischemia, as demonstrated in Fig. 5. In addition, the plasma levels of soluble infarct markers are also influenced by the excretion and distribution kinetics, both of which are poorly defined in experimental animals. The estimation of ischemic injury from plasma markers can also be limited by insufficient myocardial perfusion during reperfusion (“no reflow phenomenon”), potentially preventing marker washout from infarcted tissue. The relation between plasma markers and myocardial injury is also complex in clinical myocardial infarction (Omura et al. 1995). Microspheres All types of microspheres may form aggregates during storage, which can prevent stochastic distribution in the microcirculation. This is particularly important for radioactive microspheres due to their higher specific density. Microsphere aggregation can be prevented by careful sonication (5–10 min) immediately before application. Commercial microsphere preparations may contain low concentrations of detergents to prevent aggregation. Moreover, homogenous distribution within the syringe should always be ensured by rapid shaking or vortexing the syringe before use, because microspheres tend to sediment rapidly. Microspheres may cause hemodynamic changes due to microvascular blockade, limiting the number of spheres that can be injected. In dogs, for example, repeated measurements with left atrial injection of Open Chest Models of Acute Myocardial Ischemia and Reperfusion 57 15 µm spheres, totaling up to 48×106, are unlikely to cause significant changes in systemic hemodynamics or regional myocardial flow. Larger numbers of spheres may impair circulation, which results, for example, in a depression of myocardial function. Long-term studies with an intended survival for days, weeks or months must also take into account the possible release of label from the microspheres. While fluorescent microspheres appear to be physically stable and to continue to reside at the location of their first deposition (Van Oosterhout 1998), radioactive microspheres may clearly leak. Their use cannot be recommended for chronic studies. 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