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998
Conscious Rabbits Become Tolerant to Multiple
Episodes of Ischemic Preconditioning
Michael V. Cohen, Xi-Ming Yang, James M. Downey
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Abstract Although ischemic preconditioning protects myocardium from infarction in isolated hearts and in anesthetized
open-chest animals, its effects have not been examined in
unanesthetized animals. Furthermore, it is unknown whether
animals become tolerant to multiple episodes of ischemic
preconditioning. Rabbits were chronically instrumented with a
balloon occluder around a major branch of the left coronary
artery for reversible coronary occlusion, a left atrial catheter
for radioactive microsphere injections, ECG electrodes for
monitoring of myocardial ischemia, and, in some cases, a
carotid artery catheter for pressure measurements and timed
withdrawal of reference arterial blood samples. Eight control
rabbits underwent a 30-minute coronary occlusion and then
180 minutes of reperfusion. Five of the eight rabbits developed
ventricular tachycardia or fibrillation during ischemia, and
infarct size averaged 37.7+2.6% of the risk area. Eight rabbits
experienced a 5-minute coronary occlusion and 10 minutes of
reperfusion before the 30-minute occlusion. In these preconditioned animals, potentially fatal arrhythmias during ischemia
were significantly reduced (one of eight, P<.05), and infarct
size was much smaller (5.6+1.1%, P<.0001). The difference
could not be explained by hemodynamics or collateral blood
flow, which were nearly identical in the two groups. But when
the 30-minute coronary occlusion was preceded by 40 to 65
five-minute occlusions during a 3- to 4-day period in seven
animals, protection was markedly attenuated. Potentially lethal arrhythmias were very common, and infarct size averaged
26.5 ±2.9%, substantially larger than in rabbits with oily one
preconditioning occlusion (P<.0001). Finally, if an interval of
2.5 to 3 days of no coronary occlusions was interposed between
this period of multiple occlusions and the terminal preconditioning protocol of 5-minute occlusion and 10-minute reperfusion preceding the long 30-minute occlusion and 180-minute
reperfusion, protection was again evident, and infarct size in
seven rabbits averaged 10.9±1.5% of the risk zone (P<.0001
versus control and P<.001 versus multiple occlusions). It can
be concluded that ischemic preconditioning significantly diminishes the incidence of ischemia-induced ventricular arrhythmias and the extent of infarction after a 30-minute
coronary occlusion in unanesthetized rabbits. Unfortunately,
this protection wanes after multiple 5-minute coronary occlusions have occurred but does reappear after an ischemia-free
period. It is currently being hoped that ischemic preconditioning will have clinical importance, but tolerance may limit its
utility in patients with recurrent angina pectoris. (Circ Res.
1994;74:998-1004.)
Key Words * coronary occlusion * myocardial infarction
* preconditioning * tolerance * ventricular fibrillation
Ischemic preconditioning first demonstrated by
Murry et al' is the phenomenon whereby a brief
episode of myocardial ischemia paradoxically diminishes the amount of necrosis occurring from a subsequent ischemic insult. We have found that myocardial
protection conferred by preconditioning in rabbits is
mediated by release of endogenous adenosine,23 which
in turn occupies adenosine receptors,24 stimulates a
pertussis toxin-sensitive G protein,5 and activates protein
kinase C.6 The ability of ischemic preconditioning to
salvage ischemic myocardium has been demonstrated in
multiple experimental animal models including rabbit,57
dog,1,8'9 pig,10 and rat." However, all known studies have
used open-chest anesthetized animals or isolated heart
preparations. In the past, the physiological significance of
data gathered in anesthetized animal models has been
questioned because of the confounding effects of anesthetic agents on the autonomic nervous system, coronary
dynamics, and responses to vasoactive drugs.'2 Accordingly, it was felt necessary to determine whether the
protection of ischemic preconditioning could be documented in a conscious unsedated animal. Furthermore,
we have recently found that rabbits become tolerant to a
prolonged infusion of an A,-adenosine agonist and that
tolerance is accompanied by a loss of protection from
preconditioning."3 The present study was therefore undertaken to test whether a similar tolerance might develop in rabbits exposed to multiple brief ischemic episodes, each releasing adenosine, as might occur in a
patient with unstable angina pectoris. In the present
study, a recently developed chronically instrumented
rabbit model'4 allowed us to demonstrate that ischemic
preconditioning does result in protection of the ischemic
heart in unanesthetized animals but that tolerance to this
salutary effect soon develops when the hearts are exposed to multiple ischemic episodes.
Received May 17, 1993; accepted January 10, 1994.
From the Division of Cardiology and Departments of Medicine
and Physiology, University of South Alabama College of Medicine, Mobile.
Correspondence to Michael V. Cohen, MD, Department of
Physiology, MSB 3050, University of South Alabama, College of
Medicine, Mobile, AL 36688.
Materials and Methods
Animal Surgery
All procedures were approved by the institutional animal
care and use committee. New Zealand White rabbits weighing
between 2 and 3 kg were anesthetized with intravenous sodium
pentobarbital (35 mg/kg), intubated with an endotracheal
tube, and mechanically ventilated with a positive pressure
respirator (MD Industries, Mobile, Ala) and 100% oxygen.
After intravenous administration of 100 mg Kefzol, a left
thoracotomy was performed in the third intercostal space. The
technique for placement of the pneumatic occluder around the
coronary artery has previously been described.'4 Briefly, a
balloon occluder fashioned from 18-gauge Tygon tubing was
wedged onto the blunt end of a curved taper needle, which was
passed beneath a prominent branch of the left coronary artery.
Cohen et al Ischemic Preconditioning in Conscious Rabbits
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The balloon occluder was pulled through the myocardium, and
the end was doubled over the artery and anchored to the
myocardium with 4-0 suture. Proper functioning of the occluder was confirmed by noting cyanosis of the distal myocardium and cessation of effective contraction after inflation of
the balloon and hyperemia and resumption of contraction
after deflation.
An 18-gauge Tygon catheter was inserted into the left atrium
through the appendage and secured. The chest wound was closed
in layers, and air was aspirated from the thorax. Before approximation of the skin layer, ECG wires were anchored in the
subcutaneous tissue with one lead at each end of the incision.
The left atrial catheter and balloon occluder were brought
through the chest wall, and they, along with the ECG leads, were
tunneled subcutaneously to exit between the scapulas. In a subset
of rabbits, the carotid artery was exposed through an incision in
the left neck. The vessel was ligated distally, and an 18-gauge
Tygon catheter was inserted into the vessel and advanced retrogradely ~2 to 4 cm. The free end of the catheter was tunneled
subcutaneously to exit between the scapulas near the other
catheters and leads, and the neck incision was closed. Three
additional rabbits were prepared expressly for quantification of
coronary and collateral blood flows. In these animals, balloon
occluders, left atrial catheters, and ECG leads were implanted as
already described. In lieu of a carotid catheter for withdrawal of
arterial blood, a PE-60 catheter was inserted directly into the
descending thoracic aorta. The proximal end of the catheter was
brought through the chest wall and, along with the other devices,
was tunneled subcutaneously to exit between the scapulas. Left
atrial, carotid, and aortic catheters were filled with a dilute
solution of heparin. After surgery and again 2 days later, rabbits
were given intramuscular injections of 600 000 U benzathine
penicillin.
Protocol
Single-Occlusion Groups
Five to 7 days after surgery, when rabbits appeared to be
fully recovered, they were brought to the laboratory and
randomly divided into control and experimental groups based
on the day of the week. In any given week, equal numbers of
rabbits were selected for each group. The ECG leads were
attached to an ECG amplifier. In those rabbits with a carotid
artery catheter, the latter was connected to a disposable
transducer (Cobe, Lakewood, Colo). All signa were recorded
on a multichannel oscillograph.
A 5-minute period of regional ischemia was induced in the
preconditioned group by manually inflating the balloon occluder and clamping the tubing to prevent deflation. During
balloon inflation and myocardial ischemia, there were never
any observed behavioral changes in the rabbits that would
suggest perception of a noxious stimulus. Consequently, sedation or analgesia was not felt to be required. The ECG and
carotid artery pressure, when available, were recorded immediately before inflation and every 30 seconds for 2 minutes and
then again at 5 minutes. Appearance of ECG ST segment
elevations in addition to changes in the QRS complex confirmed the onset of myocardial ischemia. After 5 minutes, the
clamp was released, and the balloon was deflated by the
aspiration of air. The ECG and carotid pressure were recorded
every 30 seconds for 2 minutes and then at 5 and 10 minutes
after reperfusion. Normalization of the ECG documented
successful reperfusion of the coronary artery. At this point,
the balloon occluder was reinflated, and the tubing was
clamped for 30 minutes. Recordings were made every 30
seconds for 2 minutes, at 5 minutes, and every 5 minutes
thereafter.
A single measurement of regional myocardial blood flow was
made in these groups. Five minutes after the onset of the
30-minute occlusion, 0.5 to 1x106 microspheres (NEN, Boston, Mass) labeled with either 'Sc, 85Sr, or 141Ce, suspended in
saline, and mechanically agitated and ultrasonicated for 10 to
15 minutes were injected into the left atrium during 5 seconds,
999
followed by a saline flush. For those animals with a carotid
artery catheter, a reference arterial sample was collected by
withdrawing arterial blood at a rate of 1.5 mL/min with a
constant withdrawal pump (Harvard Apparatus) beginning a
few seconds before injection of the microspheres and ending 1
minute after completion of the injection. After 30 minutes of
coronary occlusion, the clamp was removed, and the balloon
was deflated to reperfuse the heart.
Reperfusion proceeded for 180 minutes. Recordings were
made every 30 seconds for 2 minutes, at 5, 10, and 15 minutes,
and then again at 180 minutes. The ECG was continuously
displayed on an oscilloscope. If arrhythmias were observed,
additional recordings were made. No antiarrhythmic agents
were administered to the rabbits at any time. If ventricular
fibrillation occurred, external defibrillation was attempted at
an energy level of 150 W. seconds. If the animal fibrillated
during a coronary occlusion, resuscitation was tried without
deflating the balloon. Control rabbits underwent the same
protocol except that the initial 5-minute coronary occlusion
was omitted. In control hearts, collateral flow was also measured 5 minutes after the onset of the 30-minute occlusion.
Each of the three rabbits with catheters in the descending
thoracic aorta also underwent a 30-minute coronary occlusion
and 3-hour reperfusion. Coronary and collateral blood flows
were measured 5 minutes after onset of the coronary occlusion. In these animals, 2x106 microspheres labeled with 'Sc
were injected into the left atrium while arterial blood was
withdrawn from the aortic catheter at a rate of 2.7 mL/min.
Multiple-Occlusion Groups
To determine whether protection was maintained after
multiple episodes of ischemic preconditioning, animals were
prepared as above, but the coronary artery was occluded for a
5-minute interval every half hour for 8 hours each day.
Balloon inflation and deflation were effected with compressed
air and a vacuum pump, respectively. A previously described
timing circuit14 connected to a solenoid valve controlled the
duration of inflation and deflation. If ventricular fibrillation
occurred, attempts were always made to defibrillate the animal
with external DC countershocks. After --3 to 4 days of
repetitive occlusions, most rabbits underwent a final 30minute coronary occlusion. The 30-minute ischemic insult was
produced at the end of a day when the rabbit had already
undergone at least 12 five-minute occlusions. After the last
5-minute occlusion, a 10-minute period of reperfusion was
allowed, as in the preconditioned rabbits described above.
Then the balloon occluder was manually inflated for 30
minutes. The heart was reperfused for 180 minutes, and then
the animal was killed so that risk zone and infarct sizes could
be determined as described below.
To determine whether the repeated 5-minute occlusions
themselves might have induced a change in collateral flow,
microspheres with one radioactive label were injected 2 minutes after the onset of the second 5-minute occlusion. Microspheres with a second label were injected 2 minutes after the
onset of the final 30-minute coronary occlusion.
In four rabbits, the final 30-minute coronary occlusion was
omitted to test whether the 5-minute occlusions might be
causing infarction. After reperfusion for 180 minutes following
the final 5-minute occlusion, the animals were killed, and the
hearts were removed for analysis. In these rabbits, the second
injection of radioactive microspheres was made 2 minutes after
the onset of the final 5-minute occlusion.
Finally, in a group of seven rabbits having successfully
undergone multiple occlusions over 3 to 4 days, occlusions
were suspended for the next 2.5 to 3 days. Thereafter, a single
5-minute occlusion followed by a 10-minute reperfusion period was produced, followed by the prolonged 30-minute
coronary occlusion and subsequent 3-hour reperfusion. Hearts
were then excised and evaluated as indicated below.
Myocardial Sample Preparation
After completion of the 180-minute period of reperfusion,
all experimental and control rabbits were reanesthetized with
1000
Circulation Research Vol 74, No 5 May 1994
TABLE 1. Hemodynamics in Control and lschemically Preconditioned Conscious Rabbits Before, During, and After
Coronary Occlusion
5-Minute
15-Minute
10-Minute
5-Minute
304Minute
Occlusion
Occlusion
Occlusion
Baseline
Occlusion
Reperfusion
HR,
BP,
HR,
BP,
HR,
BP,
HR,
BP,
HR,
BP,
HR,
BP,
mm Hg
bpm mm Hg
bpm mm Hg
bpm mm Hg
bpm mm Hg
bpm mm Hg
Group
bpm
...
...
...
...
280±+11 63±5 274±+13 68±3 277±12 63±5
Control 241 +9*
70+2t
260±+13
63±3
249±+16
66+5
262±+12
69±5 254±+10 69±4 271±15 68±3
240±14* 67±+lt
PC
HR indicates heart rate; bpm, beats per minute; BP, mean blood pressure; and PC, ischemic preconditioning. Values are mean±SEM.
*Observations in entire group of seven control and seven PC rabbits.
tObservations in subset of three control and three PC rabbits.
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sodium pentobarbital (35 mg/kg IV), intubated through a
tracheotomy, and mechanically ventilated with 100% oxygen.
The heart was exposed through a left thoracotomy, and the site
where the balloon occluder encircled the coronary artery was
uncovered. The balloon occluder was removed and replaced
with a silk suture. The heart was excised and hung by the aortic
root from a Langendorff apparatus. The silk suture was tied
tightly, thus reoccluding the coronary artery branch. The
aortic root was perfused with saline for 1 minute, and then 2
mL of a 1% solution of 1 to 10 ,gm ZnCd sulfide fluorescent
microspheres (Duke Scientific Corp, Palo Alto, Calif) was
added to the perfusate. Hence, the tissue supplied by the
occluded branch (risk region) would be nonfluorescent.
The heart was frozen and then cut into 2-mm slices from
apex to base perpendicular to the long axis of the heart. The
slices were incubated at 37°C in a 1% solution of triphenyltetrazolium chloride (TTC) to identify infarcted myocardium.
TTC stains normal myocardium red; necrotic tissue appears
pale or gray. The slices were illuminated with UV light, and
the fluorescent and nonfluorescent areas were traced on
plastic overlays. On the same overlays, the areas of infarction
illuminated with white light were traced. Risk and infarct areas
were quantified by planimetry with the aid of a digitizer (SAC,
Norwalk, Conn) interfaced with a computer. The nonfluorescent tissue was carefully separated from the fluorescing myocardium. Both fluorescent and nonfluorescent pieces were cut
into wedges, weighed, and put into glass tubes, and the
radioactivity of these tissues as well as the arterial blood
reference samples from those rabbits with carotid and aortic
catheters was counted with a gamma spectrometer (LKBWallac 1282 Compugamma, Turku, Finland).
Data Analysis
Collateral flow was defined as flow to the nonfluorescent
myocardium, whereas flow to fluorescent tissue was assumed
to be normal. Raw radioactive counts per minute were
summed for all fluorescent and nonfluorescent pieces and
normalized for the respective collective tissue mass. To avoid
contamination of ischemic myocardium by inadvertent inclusion of some tissue with normal flow, nonfluorescent risk zone
pieces bordering normally perfused fluorescent myocardium
were omitted. Collateral flow was expressed as a percentage of
the normal flow to the same heart. In those animals with
reference arterial samples, absolute flows were calculated by
multiplying the raw counts per minute of the tissue sample by
the withdrawal rate of the pump and then dividing by the
counts per minute of the reference blood sample. Flows were
normalized for mass. The volume of ischemic or risk zone
tissue was calculated by multiplying planimetered areas by
slice thickness. Infarct size was calculated as a percentage of
the risk zone.
Statistics
Data are presented as mean+SEM. Statistical significance
of comparisons between groups was assessed by ANOVA with
replication, Student's unpaired t test, and the X 2test.15 A value
of P<.05 was considered to be significant.
Results
Sixteen (eight preconditioned and eight control) rabbits were prepared for the first protocol evaluating the
effects of a single 5-minute preconditioning stimulus. Of
the control group, four rabbits developed ventricular
fibrillation after 5, 6.5, 8, and 24 minutes of coronary
occlusion. The rabbit fibrillating after only 5 minutes
could not be resuscitated, whereas the other three were
successfully defibrillated. A fifth rabbit developed nonsustained ventricular tachycardia. Of the eight preconditioned animals, only one had a ventricular arrhythmia.
This animal could not be resuscitated when ventricular
fibrillation occurred after 5 minutes of the intended
30-minute coronary occlusion. Thus five of eight control
and one of eight preconditioned rabbits developed
ventricular arrhythmias during ischemia. This difference
is statistically significant (P<.05). No reperfusion arrhythmias were detected.
Three control and three preconditioned rabbits were
instrumented with carotid artery catheters. Although
attempts were made to chronically cannulate the artery
in other rabbits, inability to pass the catheter into the
aortic arch and subsequent thrombosis of the proximal
carotid artery resulted in poor pressure recordings and
suboptimal blood sampling. Therefore, to simplify surgery this part of the procedure was eliminated, accounting for availability of pressure and absolute flow data in
only a subset of animals.
As detailed in Table 1, there were no differences in
either heart rate (seven control and seven preconditioned
rabbits) or blood pressure (three control and three preconditioned rabbits) between the two groups throughout
the observation period. In the control group, there was a
significant trend for heart rate to increase after occlusion
(P<.025), but there were no differences between any two
time points. Collateral flow in the control rabbits averaged
6.4±1.4% of normal myocardial flow, no different than
6.5 + 1.1% in the preconditioned animals. In animals with
flow quantification, normal myocardial flow averaged
5.1+0.6 mL. minm . g`l. Collateral flow was 0.4±0.2
mL min' g`.
Table 2 presents the infarct and risk zone data. In
control rabbits, infarct size averaged 37.7+2.6% of the
risk zone. In animals preconditioned with 5 minutes of
ischemia before the prolonged 30-minute coronary occlusion, infarcts were significantly smaller (5.6+1.1 % of
the risk zone, P<.0001). This difference is perhaps
better appreciated in Fig 1. It can be seen that there was
no overlap between the two groups. The difference
between the two groups is virtually identical if only
animals not developing ventricular fibrillation are
considered.
-
Cohen
et
al Ischemic Preconditioning in Conscious Rabbits
1001
the 30-minute occlusion at the end of the protocol. In
TABLE 1.
each of these instances, ventricular fibrillation
Continued
15-Minute
5-Minute
Reperfusion
278 +15
67+5
bpm
272±+14
BP,
mm Hg
69+5
254 ±17
65±3
266±15
69±3
HR,
bpm
BP,
mm
Hg
HR,
successfully converted to a sinus mechanism with DC
countershocks. If the arrhythmia occurred toward the
180-Minute
Reperfusion
Reperfusion
HR,
bpm
BP,
mm
Hg
end of the 5-minute occlusion, the balloon was deflated
before electrical defibrillation. However, the balloon
was never
deflated if ventricular fibrillation occurred
272±11
62±4
during the 30-minute coronary occlusion. There was no
273±12
63±3
65developing ventricular fibrillation (0.88+0.04 cm') and
those without any arrhythmias (0.84±+0.03 cm3).
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As presented in Table 2, there was no significant
difference in the size of risk zones between the two
groups, although there was a tendency for the risk zone
to be smaller in the experimental rabbits. To calculate
risk zone-to-heart weight ratios, risk zone volumes
were converted to mass by multiplying by 1.05, the
specific gravity of myocardium. The ratios were not
different in the two groups. Absolute infarct size was
significantly smaller (P<.0001) in preconditioned rabbits. Because of the possible influence of the size of the
risk zone on the extent of infarction, infarct volume was
plotted against risk zone volume for control and preconditioned rabbits in Fig 2. The relation between risk
region and infarct size was strikingly
the single preconditione
was
different between
difference
in
size
of the
risk zone
in the 7 survivors
Between 40 and 65 (average 58+1) 5-minute coronary occlusions were performed over 3 to 4 days in 18
rabbits. To document any possible change in coronary
collateral flow, radioactive microspheres were injected
during both the second and final balloon occlusion. In
these animals, initial collateral flow averaged 3.8±0.5%
of normal tissue flow, which was unchanged (4.2±0.8%)
at the end of the protocol. In 4 control rabbits with only
multiple 5-minute occlusions, no infarction was detectable with TTC staining (Fig 1). In 7 animals that
additionally underwent a final 30-minute balloon inflation 10 minutes after the last
3- to 4-day
averaged
5-minute occlusion of the
multiple-occlusion protocol, infarct size
26.5±2.9% of the risk
zone.
As
seen
in
Fig
1,
infarcts
significantly larger than those obandthecontrolhearthese
thecontrolhearts,aserved in rabbits preconditioned with a single 5-minute
preofonditionedtand
theicsingl
protection.
ndicating profound
determine whether coronary collateral flow might
percentage of
be influencing infarct size, infarction as
risk area was plotted against collateral flow as a perTo
a
centage of flow to normally perfused tissue (Fig 3). For
control animals with only a 30-minute occlusion, there
was a linear relation between the two variables (r= .88).
However, the range of collateral flows
the two animals with collateral flows
was
small. In only
>t10%
was there a
possible effect oninfarct size. Of note, the animalswith
ischemic preconditioning displayed virtually identical
collateral flows, but in this group there was no relation
between infarct size and flow. For the same collateral
flow, infarct sizewas obviouslymuch smaller in precon-
ditioned
than control
rabbits.
To evaluate the effect of multiple ischeinic episodes
on arrhythmias and infarction, the coronary artery of 21
rabbits was subjected to repeated 5-minute periods of
occlusion. Intractable ventricular fibrillation appearing
after occlusions 5 and 15 of the first day and occlusion
12 of the second day resulted in the exclusion of 3
rabbits. For the remaining 18 animals completing the
protocol, ventricular fibrillation also occurred in 7 of
them. Interestingly, ventricular fibrillation occurred after occlusion 11 of the first day in 1 rabbit, after
occlusion 13 of both the first and second days in 2
rabbits, and after occlusions 13, 14, and 15 of the second
day in 3 additional rabbits, respectively. Additionally, 3
of these rabbits as well as 1 other without a prior
arrhythmia also developed ventricular fibrillation during
were
ischemic episode (P<.0001). Infarcts in this group approached the size seen in the nonpreconditioned control hearts, although they were still significantly smaller
(P<.025). Although 6 of the 7 rabbits in this group had
infarcts comparable in size to those in the control group,
1 rabbit had a very small infarct (8.8% of the risk zone).
Yet the risk zone in this animal was large (1.04 cm3),
and the rabbit fibrillated twice during the repetitive
5-minute occlusion protocol and again during the final
30-minute coronary occlusion. Therefore, significant
myocardial ischemia was evident during the coronary
occlusions. Except for this 1 rabbit, animals with multiple 5-minute preconditioning cycles were nearly indistinguishable from control nonpreconditioned rabbits
(Fig 2).
Finally, seven rabbits with multiple episodes of myocardial ischemia allowed to rest in their cages for 2.5 to
3 days without any coronary occlusions had a single
5-minute ischemic preconditioning stimulus before the
standard 30-minute occlusion and reperfusion. In these
animals, infarct size averaged 10.9±1.5% of the risk
zone (Fig 1), significantly smaller than that in rabbits
with no ischemic preconditioning (P<.0001) as well as
in those with multiple episodes of ischemic preconditioning without a prolonged ischemia-free interval
(P<.001). There was no difference in infarct size between this group and that with only a single ischemic
preconditioning period.
In the rabbits studied above, collateral blood flow to
ischemic myocardium consistently averaged 4% to 6.5%
TABLE 2. Risk Zones, Infarct Sizes, and Collateral Blood Flows in Control and lschemically Preconditioned Rabbits
Body Weight, Heart Weight, Risk Zone, Risk Zone/Heart Infarct Size, Infarct/Risk Zone, Collateral/Normal
%
BF, %
cm3
cm3
Weight, %
kg
Group
9
3.4+0.8
37.7±2.6
0.31
+0.03
10.8+0.9
0.83±0.06
7.8+0.2
2.2+0.1
Control
4.0±1.3
5.6±1.1*
0.04+0.01*
9.1+0.7
0.73±0.06
8.1±0.2
2.1+0.0
PC
are
mean+SEM.
Values
ischemic
preconditioning.
BF indicates blood flow; PC,
*P<.00011 vs control group.
1002
Circulation Research Vol 74, No S May 1994
(.-
hU
P<0.001 v 30' 0CCL aud
REPETITIVE PC + 90' OCCL
** P<0.025 vs 30' OCCL
*
50
a
V
40 F
v
30 -
0
cn
ER
o
r=
8
o
0
20
Or
0
10 _
8
1
U
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
PC
REPETVE PC ~~~REpETITiVE
3 DAyS
PC
30' OCCL SINGLE PC
_
MU~
S[N0Lic PC
NG
30' OCCL
30' OCCI
PC
REP
v
30' OCCL
FIG 1. Infarct size plotted as a percentage of volume of risk zone for control rabbits with a 30-minute coronary occlusion (OCCL), rabbits
with ischemic preconditioning (PC) from a single 5-minute coronary occlusion before the 30-minute occlusion, rabbits with only multiple
5-minute occlusions, rabbits with repetitive 5-minute coronary occlusions preceding the 30-minute occlusion, and finally rabbits with a
single 5-minute ischemic preconditioning period after repetitive occlusions and an ischemia-free interval. Open circles represent
individual data points; filled circles represent means. Vertical bars depict SEM. One cycle of ischemic PC significantly decreased infarct
size after a 30-minute coronary occlusion (P< .0001). This protection was markedly attenuated if animals had multiple 5-minute coronary
occlusions before the 30-minute occlusion. Infarct size in this latter group was still less than that observed in control rabbits (P<.025)
but greater than that in rabbits with only one preconditioning stimulus (P<.0001). However, protection from a 5-minute coronary
occlusion was restored in rabbits with multiple occlusions and an interposed ischemia-free period (P<.0001 vs control rabbits and
P<.001 vs rabbits with multiple occlusions).
of simultaneously measured flow to normal cardiac
tissue. In control animals with only a single 30-minute
coronary occlusion and those rabbits with multiple
5-minute coronary occlusions before the 30-minute
coronary occlusion, the large infarcts (average, 27% and
0.5
r--
0.4
* 30' OCCL
O SINGLE PC + 30' OCCL
A REPETIIEPC + 30' OCCL
A REPETTVE PC + 3 DAYS REST
+ SINGLE PC + 30' OCCL
38% of the risk zone, respectively) attest to the inadequacy of the flow. However, in those animals iwith
carotid catheters in which flow was quantified, flows to
normal (5.1 mL * minm' g-') as well as ischemic (0.4
mL* minm g-') tissues were surprisingly high. Because
flow quantification is very dependent on the adequacy
of the arterial withdrawal sample and because it could
not be determined whether transient neck flexion or
*
60
A.0
0.3
Q
Iin
A-
* 30' OCC.
0 SINGJI PC + 30'CL
50
0
40
0.2
z
0
0
*L
-
0
A
AA
8
0.1
z
0
0
A
A
30
0
Y
A
20
8
V.O'
0.0
'
0.2
0 00
-
0.4
0.6
0.8
0
'
1.0
1.2
RISK ZONE (cm3)
FIG 2. Relation between infarct and risk zone sizes plotted for
control rabbits with only a 30-minute coronary occlusion (OCCL),
rabbits with a single cycle of ischemic preconditioning (PC)
before the 30-minute coronary occlusion, rabbits with multiple
cycles of ischemic preconditioning before the final prolonged
occlusion, and rabbits with a single cycle of ischemic preconditioning after multiple cycles and then an interposed ischemiafree interval. Although risk zones were comparable in the four
groups, infarcts were considerably smaller in rabbits with isolated single preconditioning ischemic periods and those with
single preconditioning ischemic periods after multiple occlusions and ischemia-free intervals than in the other two groups.
10
0
0
_
0
0
nv 0
2
4
6
8
10
12
14
COLLATE1AL LOW ( % OF NORMA )
FIG 3. Relation between infarct size and coronary collateral flow
plotted for control rabbits with only a 30-minute coronary occlusion (OCCL) and rabbits with a preceding cycle of isehemic
preconditioning (PC). Although the range of colateral flows is
narrow, there is a linear relation between the two variables for the
control rabbits (r=.88) but not for the preconditioned rabbits.
Therefore, collateral flow cannot account for the salvage of
jeopardized myocardium in the latter animals.
Cohen et al Ischemic Preconditioning in Conscious Rabbits
rotation in these conscious animals might have interfered with withdrawal from the carotid catheter, three
animals were prepared with catheters in the descending
thoracic aorta. Arterial withdrawal would therefore not
be influenced by animal movement. To further maximize the accuracy of the flow measurements, the number of microspheres injected was increased, only the
radioactive label with the highest energy was selected,
and a faster arterial withdrawal rate was used. In these
three rabbits, infarction averaged 39.0+1.7% of the risk
zone. Blood flow to normal myocardium was 4.3 ± 0.6
mL* min-1 * g-'; flow to ischemic tissue averaged
0.2±0.1 mL * min'm g'. Again, the ratio of collateral to
normal tissue blood flow was 5.4±1.6%.
Discussion
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Ischemic preconditioning is gaining increasing attention as a potentially useful clinical tool to limit the
extent of myocardial infarction. Paradoxically, those
animals exposed to a brief ischemic episode before a
more prolonged ischemic insult have less infarction than
animals experiencing only the longer ischemic insult
despite the additional ischemia.1-5,7-11 The present study
expands these observations to conscious unanesthetized
animals without the confounding difficulties engendered by the effects of anesthesia on reflex pathways
and the autonomic nervous system. Previous studies by
Vatner12 have clearly demonstrated the potential limitations of an anesthetized animal model in the study of
the cardiovascular system. Virtually all previous studies
of ischemic and pharmacologic preconditioning have
used either Langendorff isolated-heart preparations or
animals with hearts exposed through a thoracotomy.
Although these animal models have provided important
information, it was unclear whether the preparation
itself might have influenced the outcome. The present
experiments document the effectiveness of ischemic
preconditioning in yet another animal model and suggest that ischemic preconditioning is not merely a
function of the method of investigation.
As noted in Table 2, control and experimental groups
had comparable heart weights and volumes of risk
region. Furthermore, heart rates in the two groups were
also comparable throughout the protocol. Because of
attempts to simplify the surgical procedure, indwelling
arterial catheters were introduced into only a subset of
the rabbits studied. As noted in Table 1, blood pressures
were also not different in control and preconditioned
rabbits. Collateral flow averaged 6% of normal flow in
these animals, and these observations are consistent
with previous observations by us14 and others.16 Again,
there was no difference in relative or absolute collateral
flows between the two groups.
Fig 1 demonstrates a striking difference in infarct size
between rabbits with an isolated 30-minute occlusion
and those with one preceding 5-minute period of ischemic preconditioning. To further document a significant
difference in infarct size between the two groups, infarct
size as a function of risk area was plotted for these
rabbits. As depicted in Fig 2, two distinctly different
populations of points are apparent. The difference in
infarct size between the two groups was also not explicable by variations in collateral flow to the ischemic
tissue. As demonstrated in Fig 3, individual collateral
flows were comparable in the two groups, yet infarcts
were obviously much smaller in the preconditioned
1003
rabbits. There was no correlation between infarct size
and collateral flow in the latter. Although a linear
relation was apparent in the control group, only the two
animals with collateral flows exceeding 10% seemed to
have a small benefit. The range of collateral flows is
indeed narrow compared with the much greater flow
variability in dogs,16 and the ultimate effect on infarct
size is small.
Additionally, preconditioning appears to attenuate serious and frequently life-threatening ventricular arrhythmias. Whereas five of eight control rabbits developed
either ventricular tachycardia or fibrillation, only one
preconditioned rabbit was similarly affected. Studies in
anesthetized rats have concluded that preconditioning
ischemia can attenuate serious ventricular arrhythmias
during both ischemia and early reperfusion,17-20 although
observations in dogs have yielded less consistent results.12'-23 However, rat and canine myocardium, unlike
rabbit and human cardiac tissue, contains xanthine oxidase, which in these models may be largely responsible for
these arrhythmias by generating free radicals after the
reintroduction of oxygen. The present study may be the
first to clearly demonstrate that ischemic preconditioning
also has a salutary effect on the occurrence of potentially
lethal arrhythmias in xanthine-deficient hearts of conscious rabbits. It is interesting to note that arrhythmias of
this type are rare in the anesthetized rabbit and have been
too few to quantify. Clearly, the unanesthetized rabbit
should provide a useful small-animal xanthine oxidasedeficient model for ischemia-induced arrhythmias.
But perhaps more important than the observation
that the preconditioning phenomenon applies to conscious unanesthetized animals is the realization that the
protection of ischemic preconditioning is markedly attenuated in the face of recurring preconditioning stimuli
but can be reinstated after an ischemia-free interval.
Although it has been reported that the protection after
one episode of ischemic preconditioning in the pig could
not be renewed after several hours during which the
protective effect naturally waned,24 we have found that
in the rabbit full protection can be achieved with a
second episode of ischemic preconditioning 2 hours
after the first.25 Therefore, it appears that many episodes of ischemic preconditioning are required before
tachyphylaxis develops.
Previous work from this laboratory has already demonstrated that acute pretreatment with the A,-adenosine agonist 2-chloro-N6-cyclo-pentyladenosine (CCPA)
can mimic ischemic preconditioning.4 The protection
after adenosine agonist administration was not maintained, however, when CCPA was infused over a 72hour period.13 Chronic exposure to the A1-adenosine
agonist presumably resulted in downregulation of the
myocardial receptor or its signal transduction pathway.
Liang and Donovan26 and Longabaugh et a127 have
already demonstrated downregulation of adenosine receptors and regulatory G proteins, respectively, after
chronic exposure of cells to A,-adenosine agonists. It is
also possible that downregulation may occur at the level
of protein kinase C. These CCPA-tolerant animals also
could not be protected with ischemic preconditioning.
CCPA-induced downregulation of adenosine receptors
or their signaling pathways apparently also made the
heart insensitive to the endogenously released adenosine responsible for triggering the protection after a
5-minute period of ischemia. The present data suggest
1004
Circulation Research Vol 74, No 5 May 1994
that this same type of tolerance can be induced physi-
ologically by repeated bursts of endogenous adenosine
production during multiple 5-minute coronary occlusions. This suggestion is further supported by the additional observation that the protective effect is reinstated
after an ischemia-free interval, during which upregulation would be expected to restore baseline sensitivity of
receptors and/or the signal transduction pathway.
In addition to the larger infarcts observed in these
rabbits with multiple prior 5-minute coronary occlu-
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
sions, ventricular fibrillation was very common. Ten of
21 rabbits developed ventricular fibrillation, and 4 had
multiple episodes. This is in striking contrast to the
apparent diminution in potentially lethal arrhythmias
seen in animals preconditioned with a single 5-minute
ischemic period. Of note, when ventricular fibrillation
occurred, it appeared quite consistently after 11 to 15
ischemia/reperfusion cycles, or 5.5 to 7.5 hours after
initiation of the protocol on a given day. This interval
for day 1 may be too short for significant receptor
downregulation to have occurred since in the study
reported by Tsuchida et al13 protection of ischemic
myocardium and bradycardia (an A,-mediated effect on
atrial tissue) were still apparent after 6 hours of CCPA
infusion. One possible explanation is that suppression
of arrhythmias and protection from infarction may be
controlled by separate and distinct mechanisms. Prior
observations in the rat also suggest that arrhythmias and
infarction may have separate controlling pathways,
since a single 5-minute coronary occlusion attenuated
ventricular arrhythmias during a subsequent coronary
occlusion despite the lack of any salutary effect on
infarct size."
It is not yet known how many repeated ischemic
episodes can be tolerated before the protective effect of
ischemic preconditioning is lost, but the clinical implications are striking. Whereas the individual with only
one episode of angina pectoris may be protected if a
coronary thrombosis occurs shortly thereafter, the patient with unstable angina or one with a history of stable
but frequent angina may lose this protective effect.
Furthermore, such a patient may not be amenable to
protection from an adenosine-based drug either. Additional investigations are needed to determine the precise mechanism of this tolerance.
Acknowledgment
This study was supported in part by National Heart, Lung,
and Blood Institute grant HL-17809.
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M V Cohen, X M Yang and J M Downey
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Circ Res. 1994;74:998-1004
doi: 10.1161/01.RES.74.5.998
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