Determination of infarct size in isolated perfused rat hearts subjected

Practical course: Basic biochemical methods and
ischemic heart models
Determination of infarct size in isolated perfused
rat hearts subjected to ischemia/reperfusion
A practical manual
Prepared by
Csaba Csonka MD PhD
Tamas Csont MD PhD
2011
Supported by: HURO/0901/069/2.3.1
HU-RO-DOCS
1
Content:
Ex vivo organ perfusion
3
Langendorff rat heart
3
Species
6
The perfusion system
7
The perfusion fluid
8
Oxygenization
10
Thermostation
10
Getting started
11
Anesthesia and anticoagulation
12
Cutting out the heart
14
Cannulation
14
Induction of ischemia followed by reperfusion
16
End points
18
Detection of infarct size
19
Slicing the hearts and TTC incubation
19
Evaluation of the scanned pictures
20
Cleaning
21
References
22
2
Ex vivo organ perfusion
In science, the term ex vivo (Latin: "out of the living") refers to
experimentations or measurements done in or on tissue/organ in an
artificial environment outside the organism. Ex vivo conditions allow
experimentation under more controlled conditions than is possible in in
vivo experiments (in the intact organism), at the expense of altering
the "natural" environment. The term ex vivo is not synonymous to the
term in vitro ("within the glass").
Langendorff rat heart
Coronary artery disease and its most severe manifestation, ischemiareperfusion-induced myocardial infarction, continue to be the leading
cause of mortality in the “western” type societies. Therefore,
attenuation of ischemia/reperfusion injury is of great importance.
There is a conflict between the quantity and quality of data that can be
acquired from an experimental model versus its clinical relevance.
However, a primary advantage of using ex vivo tissues is the ability to
perform tests or measurements that would otherwise not be possible
or ethical in living subjects.
At a practical level, the isolated heart, especially from small mammals,
provides a highly reproducible preparation which can be studied
quickly and in large numbers at relatively low cost.
The isolated perfused mammalian heart preparation was established
by the German physiologist Oskar Langendorff in 1895 as a tool for
studying heart biology. Since then it has been one of the most widely
accepted technique in modern cardiovascular and pharmacological
research to study physiological, pharmacological, biochemical,
molecular biological aspects of basic and pre-clinical research using
animals in spite of a few shortcomings.
These measurements can be made in the absence of the confounding
effects of other organs, the systemic circulation, and a host of
peripheral complications such as circulating neural and hormonal
factors.
3
This characteristic may be considered as an investigational advantage
in that it allows the dissection of peripheral from cardiac responses or
a disadvantage in that it makes the preparation one step further
removed from the in vivo state
It must be recognized that, as an ex vivo preparation, the isolated
heart is a constantly deteriorating preparation but nonetheless it is
capable of study for several hours
Figure 1. The original setup by O. Langendorff
The main principle of the Langendorff technique is to maintain cardiac
activity by perfusing the heart via the coronary arteries using an aortic
cannula inserted into the ascending aorta. Perfusion solution is
delivered to the heart in a retrograde manner via this cannula. This
retrograde perfusion forces the aortic valve closed and shunts the
entire perfusate flow via the coronary ostia into to the coronary
arteries. After passing through the coronary circulation the perfusate
drains into the right atrium via the coronary sinus and finally enters the
right atrium via the coronary sinus and is driven out via the right
ventricle and the pulmonary artery. drops out from the heart.
4
The Langendorff technique is well suited to many different models of
cardiac disease and dysfunction. Some of the more widely used
models include:
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anoxia or hypoxia at various degrees of oxygen deprivation
ischemia (blockade of circulation; total or low flow)
ischemia (global or regional)
ischemia followed by reperfusion
ischemic pre- and ischemic postconditioning
drug response
toxicology (e.g., screening for novel test compounds)
donor-heart transplantation storage techniques
stem-cell therapy for myocardial diseases
Types of the Langendorff perfusion:
(i) Constant Flow Mode - perfusate is pushed through the heart at a
constant rate and the perfusion pressure is measured to give a
representation of the coronary resistance
(ii) Constant Pressure Mode - the desired pressure is maintained and
the resulting fluctuations in the rate of coronary flow are measured.
Both type of Langendorff perfusion can be performed in recirculating
(recycling of perfusate) and non-recirculating modes.
Another method where the perfusion of the coronary arteries is
maintained by the own work of the heart: the working heart
preparation.
As shown in Figure 3, this is a more complex preparation where both
the left atrium and ventricle are cannulated. The perfusion fluid enters
to the left atrium from the left atrial reservoir through the left atrial
cannula. From the left atrium, and than from the left ventricle the own
work of the heart ejects the perfusion fluid through the aorta against a
certain resistance ultimately into the artificial lung. This resistance
(called afterload) renders coronary circulation possible. Coronary flow
is collected after dropping out from the heart and pumped to the lung.
After oxygenation and filtration all the perfusion fluid arrived back to
the left atrial reservoir and the cycle starts again.
5
Species
Hearts from any mammalian species including also humans
(together with non-mammalian hearts such as those from reptiles or
birds) may be ex vivo perfused. Isolated perfusions of large animal
hearts such as pigs, or dogs are less frequently used. This is probably
due to the high cost, greater variability, large volumes of perfusion
fluids and large equipment that is required.
Without doubt the most frequently studied and the best
characterized heart is the rat heart. There are numerous reports of
studies with other rodents. The advent of transgenic technology will
undoubtedly result in increasing numbers of studies using mice.
Animal
limitations
Rabbit
Rat
difficulties with anesthesia
very short action potential duration (not perfect for
arrhythmia studies)
miniaturization (eg. intraventricular pressure recordings
are much more difficult), very high heart rate
totally collateralized, non-similarly to human
Mouse
Guinea pig
Figure 2. Main species used in our lab
6
The perfusion system
O2
O2
Langendorff
chamber
elastic
chamber
lung
3-way tap
KH
perfusion pressure
aortic
flowmeter
left atrial
reservoire
filter
KH
pump
heart
chamber
Figure 3. Outline of our Langendorff and Neely perfusion systems
Units:
Storage
Neely chamber
Langendorff chamber
Elastic chamber and bubble trap
Left atrial reservoir
Heart suspending chamber
Connectors
rigid connectors
elastic connectors
cannulae
Basic requirements against components
Fix volume, fix shape
Heat-, acid-, lye-resistant; do not absorb drugs
Transparent
Easy to clean, cheap, replaceable
7
The perfusion fluid
As a perfusion fluid, crystalloid-based perfusion buffers (eg Ringerlactate, Krebs-Henseleit buffer, Tyrode’s solution, etc.) are preferred,
rather than blood.
Its main functions:
 nutrients for energy production (eg glucose, pyruvate, free fatty
acids, lactate, proteins)
 insure Ca2+ for contractions
 maintain pH 7.4±0.05 as buffer system (eg CO2/HCO3-, PO43-)
 sustain extracellular necessary ionmilieu (K+, Na+, Mg2+, Cl-)
 maintain osmolarity (290-300 mOsm)
 oxygen supply (see later)
 washout of metabolites
Krebs Henseleit solution:
In our system the Krebs-Henseleit solution content in mmol/L: NaCl
118.6, KCl 4.3, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, glucose 11.1
and CaCl2 1.5, pH 7.3–7.5, gassed with carbogen [95% O2, 5% CO2])
at a constant perfusion pressure of 100 H2Ocm at 37 °C.
Components for 5L Krebs-Henseleit solution:
NaCl
NaHCO3
KCl
MgSO4×7H2O
KH2PO4
Glucose×1H2O
CaCl2×2H2O
Weight (g)
34.66
10.5
1.6
0.72
0.817
11.0
1.1
MW
58.44
84.01
74.56
120.37
136.09
198.17
147.02
final cc (mmol/L)
118.6
25.0
4.3
1.2
1.2
11.1
1.5
Procedure
1. Measure out 4L of distilled water. Water temperature should be
room temperature.
2. While gently stirring the water, add the powdered components
except for CaCl2. Stir until dissolved. Do not heat.
3. Rinse original package with a small amount of water to remove
all traces of powder. Add to solution in step 2.
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4. Solution should be gassed for 5 min with 95% O2 and 5% CO2
carbogen.
5. Separately, measure out 1L of distilled water. While gently
stirring the water, add CaCl2×2H2O. Stir until dissolved.
6. Mix solutions in a 5-L dish.
7. While stirring, adjust the pH of the medium to 7.4 using of 1N
HCl
8. Filter immediately using vacuum pump and membrane with a
porosity of 5 microns.
Storage and Stability
Store Krebs-Henseleit solution 2-8 °C, use it before 3 days.
Deterioration of the liquid medium may be recognized by any or all of
the following: (i) pH change, (ii) precipitate or particulates, (iii) cloudy
appearance (iv) color change.
9
Oxygenization
Carbogen (mixture of 95% O2 and 5% CO2) is used to oxygenize
Krebs-Henseleit solution via a sintered glass gas distributor connected
to the high-pressure gas cylinder using a gas reductor. Gassing with
CO2 is necessary to maintain pH, therefore, use of pure O2 deteriorate
quickly cardiac function.
O2 solubility in Krebs-Henseleit solution is approximately 17 mL/L
using carbogen at 37 °C.
In case of heart of a 300-g rat, CF is approximately 15-20 mL/min
Oxygen consumption in different tissues:
Cardiac State
MVO2(ml O2 min-1 100g-1)
Arrested heart
2
Resting heart rate 8
Heavy exercise
70
Brain
3
Kidney
5
Skin
0.2
Resting muscle
1
Contracting muscle 50
Thermostation
In order to maintain experimental environment closest to the
physiological milieu, maintenance of an accurate temperature setup is
one of the most critical factor during the experiments. Therefore, all
containers of the perfusion system has a water-jacket connected to a
large capacity thermostat pump.
Procedure:
1. Open carbogen gas flow by adjusting the gas redactor to get
approximately 200 mL/min gas flow
2. Switch on the thermostat and check temperature before onset
of experiments
10
Getting started
1.
Pay attention to the good fit of all connections and close taps
well.
2.
Open carbogen, adjust gas flow to ~200 mL/min. Check
pressure on the manometer of the gas cylinder. Do not forget to open
the tap connected to Langendorff system.
3.
Fill up the perfusion system with Krebs-Henseleit solution using
a syringe of 50 ml in a reverse manner (from the bottom) to prevent
accumulation of air below the glass frit filter. Open the 3-way tap so
that it remains closed to the direction of the cannula. Then fit the
syringe into the tap and squish the Krebs-Henseleit solution as quickly
as you can do it. Level of Krebs-Henseleit solution should be over the
level of glass frit in the Langendorff reservoir. Air bubbles are not
allowed to remain under the glass frit because they jeopardize the
function of isolated hearts due to the possibility of aeroembolism. If
perfusion fluid has flown out for any reason dropping should be
stopped as soon as possible.
4.
Then fill up the Langendorff reservoir from the top with KrebsHenseleit solution. The pressure of fluid column (perfusion pressure)
should be 100 H2Ocm. It means that the vertical distance between the
end of the cannula and the liquid level of the perfusion fluid in the
Langendorff reservoir should be exactly 100 cm. Check distance with
tape measure.
5.
Prepare the tools:
2 pcs scissors
3 pcs forceps (one pair are anatomic)
1 pc bulldog clamp
1 pc glass baker for the isolated heart (fill the baker with KrebsHenseleit solution and store it on 4 ˚C or on ice)
 aluminium foil
 permanent marker for writing the animal code, date, and study
name on the aluminium foil.
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Anesthesia and anticoagulation
Isolation of the heart requires the donor animal to be rendered
unconscious prior to excision. Anesthesia can be induced by
inhalation of agents such as isoflurane or injection (intravenously or
intraperitoneally) with agents such as pentobarbitone. For intravenous
injection, femoral vein is the preferred route the vein is accessed by a
small skin incision. An alternative to anesthesia is cervical dislocation,
however it is allowed only for newborn rats. Ether is hazardous as it is
irritant to the animal. Therefore, it is not allowed to use according to
the latest regulations.
Whatever the choice of procedure (and this may be influenced
by local animal welfare regulations), every effort should be made to
minimize stress prior to and during anesthesia. Therefore, keep the
animal in a quiet environment and minimize handling.
In order to prevent the formation of thrombi in the excised heart,
it is strongly recommended to administer anticoagulant, namely
heparin before isolation. Heparin is given parenterally because it is not
absorbed from the gut, due to its high negative charge and large size.
Heparin is preferably injected intravenously to femoral vein which is
accessed by a small skin incision.
Heparin activates lipases, therefore, in studies studying lipid or
fatty acid metabolism it is advisable to choose another anticoagulant.
Coagulation tests are much shorter in rats than in humans, therefore
without heparin the ex vivo model should be prepared extremely fast.
The conventional dose for heparine is 500 IU/kg.
Coagulation tests:
Blood clotting time (s)
Bleeding time (s)
Rat
125±4
88±4
Human
508±5
270±3
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Procedure
1.
2.
3.
4.
Measure animal weight
Calculate necessary amount of pentobarbital (Euthasol)
Inject requested volume intraperitoneally
During time when anesthetic takes effect, prepare heparine
(final concentration is 500 U/mL)
5. When animal is unconscious, Inject requested volume
intravenously to femoral vein (500 U/kg) accessed by a small
skin incision
6. Wait 1 min
7. Go to next step
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Cutting out the heart
Once the animal is anesthetized the heart can be excised.
Generally, the diaphragm is accessed by a transabdominal incision
and cut carefully to expose the thoracic cavity. The thorax is opened
by a bilateral incision along the lower margin of the last to first ribs, the
thoracic cage is then reflected over the animals head, exposing the
heart. We then cradle the heart between their fingers (it is essential to
do this gently to avoid contusion injury) and then lift the heart slightly
before incising the aorta, vena cava and pulmonary vessels.
Immediately after excision, hearts are immersed in cold perfusion
solution (4 °C to limit any ischemic injury during the period between
excision and the restoration of vascular perfusion).
Cannulation
For adequate perfusion of the heart a suitably sized cannula is
required for insertion into the aorta.
The external diameter is typically similar to, or slightly larger
than, that of the aorta (about 3mm for a heart from a 250g rat).
Several small circumferential grooves is usually machined into the
distal end of the cannula to prevent the aorta from slipping off (fig 4).
It is advisable to have the perfusion fluid gently dripping from
the aortic cannula prior to cannulation since this helps minimize the
chance of air emboli at the time the heart is attached to the cannula.
Before cannulation cut the aorta transversally just before the
origin of the innominate artery to form a intact regular aortic ostia for
cannulation.
Hearts should be held gently between the tips of blunt-ended
fine curved forceps, taking care to avoid stretching or ripping of the
aortic wall. The aorta is then gently eased over the end of the cannula,
taking care not to insert the cannula too far (5-7 mm) into the aorta
since this would occlude the coronary ostia or damage the aortic
valve. The aorta is then clamped to the cannula with a small blunt
artery clip (bulldog clamp), whilst a ligature is rapidly tied around the
aorta, locking into the grooves; the artery clip can then be removed.
Full flow of perfusate should be initiated as soon as the heart is
mounted on the cannula.
Once the heart is securely attached to the cannula any surplus
tissue (such as bits of thymus, fat or lungs) can be trimmed away.
14
Drainage of coronary perfusate from the right side of the heart via the
pulmonary artery should be unimpeded, however, in the course of
cannulation it is possible to accidentally ligate the pulmonary artery.
Thus, to facilitate adequate drainage it is advisable to make a small
incision in the base of the pulmonary artery using small pointed
scissors. Coronary flow passed through the coronary vasculature can
either be discarded or collected for analysis; if recirculating perfusion
is required the coronary effluent can be returned (preferably via a 5µm
filter) to the perfusion fluid reservoir for reoxygenation.
Once cannulation is completed and coronary perfusion initiated,
contractile function and regular heart rhythm will return within a few
seconds but it may be 10 minutes or more before maximum function is
established.
Once preparation of the heart has been completed, the
temperature regulated heart chamber is placed around the heart.
Procedure
1. Cannulation of the heart according to the instructions above
Figure 4: The aortic cannula with a 3-way cock above the heart chamber
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Induction of ischemia followed by reperfusion
The induction of regional ischemia is performed by the ligation
of the left anterior descending coronary artery usually after a 10-15
min long normoxic (equilibration) perfusion period. To stabilize the
heart, hold it gently with your finger and place a small suture (3-0 nonabsorbable suture silk, Mersilk®) from the mid level of the left atrium to
the mid level of the pulmonary tract. The suture must be deep enough,
which you could carry out easily with a “half moon”-shaped suture.
The ligature ends are passed through a short plastic tube to form a
snare.
Figure 5. The place of the ligature to induce regional ischemia
The major coronary arteries min rats, in contrast to larger
mammalian species do not lie on the surface of the heart, rather are
covered with a layer of myocardium throughout their course.
16
Figure 6. Coronary arteries in rats (panle A: arrow, panel B: “L”) are not in the
surface of the heart
For coronary artery occlusion and reperfusion, the plastic tube
is pressed onto the surface of the heart directly above the coronary
artery and released to induce coronary occlusion and released to
induce reperfusion. There are no exact definitions in the literature
about the pressure to induce coronary occlusion. Extra forces to
certainly occlude coronary occlusion can result in tissue damage.
Therefore, in isolated heart perfusions models the same forces are
provided by a 100 g weight flung over a small pulley.
The occlusion is performed with the help of a plastic bead, and
a plastic tube. The silk must be attached to a 100 g heavy weight
without any retraction to maintain the same occlusion force during
experiments for all og the hearts. After a successful occlusion, a 3040% decrease is detectable in coronary flow.
Induction of ischemic preconditioning
Ischemic preconditioning is usually achieved by 3 intermittent 5
min long coronary occlusion and reperfusion. In contrast with
postconditioning it is performed in regional ischemia. Do not forget to
adjust the length of preischemic perfusion in your ischemia/reperfusion
control group (usually 40 min).
17
End points
Once the heart is cannulated and successfully beating, there are
several parameters that can be measured and recorded from the
Langendorff preparation including:
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infarct size
histology/morphology
myocardial contractile function
ventricular pressure and its derivatives
ventricular volume and its derivatives
flows (aortic, coronary, cardiac output)
pressure-volume work
electrophysiology including arrhythmias
tissue and perfusate samples for
measurements
various
biochemical
Note:
In the Langendorff system, because the cardiovascular system is
no longer a closed loop, the ventricles do not fill with the perfusate and
therefore do not perform pressure-volume work. Left ventricular
pressure can however still be measured with the use of a fluid-filled
balloon-tipped catheter connected to a pressure transducer. Once
inserted, the ventricle can contract isovolumetrically against the
balloon. Although this parameter can not be accreted as real left
ventricular pressure, it is used to describe cardiac performance.
18
Detection of infarct size
Using popular Langendorff perfusion, 2,3,5-triphenyltetrazolium
chloride (TTC, CAS 298-96-4) staining became the gold standard of
infarct size determination. Among the advantages of the TTC staining
the relatively low cost, high throughput, easy performance, reliability
and reproducibility can be mentioned. TTC staining is a well described
method used by uncountable researchers; however, it contained
several non-objective parameters. Therefore, a kind of standardization
of this method is necessary in order to minimize errors of
measurements and thus comparison of different working groups all
over to world is more possible.
TTC is a redox indicator commonly used in biochemical
experiments to differentiate between metabolically active and inactive
tissues. This white crystalline powder forms colorless solution with
water is enzymatically reduced to brick-red precipitations of formazan
dye (TPF, 1,3,5-triphenylformazan, CAS 531-52-2) in living tissues
with intact mitochondrial respiration due to the activity of various
dehydrogenases (i.e. succinate dehydrogenase) in the presence of
electron donor NADH. After TTC staining, necrotic zone remains as
pale areas of the heart.
There is a consensus in the literature that in ex vivo isolated rat
heart experiments after ischemia usually a 120 min of reperfusion is
used to measure infarct size.
To delineate from area at risk at the end of reperfusion coronary
arteries are re-occluded and hearts are perfused with 5 mL 0.1%
Evans blue dissolved in Krebs-Henseleit solution followed by a short
rinsing (2 sec) with Krebs-Henseleit.
Slicing the hearts and TTC incubation
Hearts are preferred to stain after slicing rather than perfusing
the heart with TTC. After delineation remove atria from the ventricles
and slice the heart manually to 5-6 uniform slices (with equal
thickness) perpendicular to the long axis of the left ventricle (LV) by
eye. Use a 24-well microplate to incubate slices at 37°C for 10 min. Fill
wells with 2 mL of 1% weight/volume TTC prepared in 0.1 M
phosphate buffer (dissolve 7.12g Na2HPO4×2H2O (MW 178) in 400
mL and 1.56g NaH2PO4×2H2O (MW 120) in 100 mL distilled water,
mix and set pH from 7.2 to 7.4 with Na2HPO4). Prior to slicing and TTC
staining, hearts are stored overnight at -20°C because myocardial
19
tissue can be cut more easily when it is in a semi-frozen state and
yesterdays’ hearts can be incubated all together using completely the
same incubating conditions. After TTC staining slices are fixed with
10% formalin solution for 12 min, rinsed with PBS, and then scanned
between glass plates. Glass plates preferred than plexiglas because
glass can not be easily scratched which can worsen the quality of the
scanned image.
Evaluation of the scanned pictures
Cumulative planimetry appears to be superior and to more
accurately reflect the degree of tissue damage than manual dissection
of stained from unstained tissue. Moreover, documentation and later
control of the evaluation is also missed without computer based
planimetry. There are several useful softwares.
InfarctSize 2.4™ software is a standard computer program with
the usual picture editing functions specially developed to evaluate 2D
scanned images of heart slices by Pharmahungary Group.
Procedure:
2.
3.
4.
5.
6.
7.
Prepare Evans blue dye
Perfusate the heart with Evans blue to delineate area at risk
Incubate the heart in TTC to detect infarct size
Measure area at risk and infarct size with manual dissection
Express area at risk in % of the total heart mass
Express infarct size in % of the area at risk
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Cleaning
After the experiment has been completed, the equipment
should be thoroughly cleaned. It is important to remember that the
solutions used to provide isolated organ or tissue preparations with
nutrients will also provide an ideal environment for bacteria and fungal
growth.
Regular procedure of glassware maintenance and post experimental
cleanup:
The apparatus is manufactured of heat-resistant borosilicate
glass and can be easily cleaned. When the experiment is finished,
remove heart and let the Krebs-Henseleit fluid run away the system.
Then flush out carefully and accurately all of the components of the
perfusion system (glasswares: reservoirs and chambers, stopcocks,
aerators, cannulae, flowmeters, transducers, connectors and other
associated parts) using 1 liter of deionized water. Repeat this step 4
times. The last cycle should be done with boiling distilled water. After
the final water rinse, disassemble the perfusion system and let all the
cleaning water run away. Aerators should be carefully blown dry using
gas or air. Regularly clean the top and fixing bolts using distilled water
to avoid the formation of saline deposits.
Covering equipment to reduce air borne contamination from
microbes and spores is useful.
Last steps:
 Switch off thermostat
 Close carbogen flow
 Wash up hand tools
 Take care (hazardous) waste material
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References
Csonka C, Kupai K, Kocsis GF, Novák G, Fekete V, Bencsik P, Csont T, Ferdinandy
P. Measurement of myocardial infarct size in preclinical studies. J Pharmacol Toxicol
Methods. 61:163-70. (2010) Review.
DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL on the protection of animals used for scientific purposes
Hearse DJ, Sutherland FJ. Experimental models for the study of cardiovascular
function and disease. Pharmacol Res. 41:597-603. (2000)Review.
http://www.colby.edu/chemistry/CH331/O2%20Solubility.html
http://www.experimetria.com/
http://www.radnoti.com/
http://www.usouthal.edu/ishr/help/hearse/
García-Manzano A, González-Llaven J, Lemini C, Rubio-Póo C. Standardization of
rat blood clotting tests with reagents used for humans. Proc West Pharmacol Soc.
44:153-5. (2001)
Krebs, H. A. and Henseleit, K. Untersuchungen über die Harnstoffbildung im
Tierkörper. Hoppe-Seyler's Zeitschrift für Physiol. Chemie. 210, 33-66. (1932)
Langendorff O. Untersuchungen am uberlebenden Saugethierherzen. Pflugers
Archives fur die Gesamte Physiologie des Menschen and der Tiere 61:291-332.
(1895)
Neely JR, Liebermeister H, Battersby EJ and Morgan HE. Effect of pressure
development on oxygen consumption by isolated rat heart. American Journal of
Physiology 212:H804-H814. (1967)
Sutherland FJ, Hearse DJ. The isolated blood and perfusion fluid perfused heart.
Pharmacol Res. 41:613-27. (2000) Review.
Zimmer HG. The Isolated Perfused Heart and Its Pioneers. News Physiol Sci.
13:203-210. (1998)
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