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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-
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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
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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
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In-Vivo Techniques
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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.
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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
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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.
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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.
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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
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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.
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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
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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:
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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
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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-
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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).
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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
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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)
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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
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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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