Download Reduction of Canine Myocardial Infarct Size by a Diffusible Reactive

1652
Reduction of Canine Myocardial Infarct
Size by a Diffusible Reactive Oxygen
Metabolite Scavenger
Efficacy of Dimethylthiourea Given at the
Onset of Reperfusion
Frank P. Carrea, Edward J. Lesnefsky, John E. Repine,
Robert H. Shikes, and Lawrence D. Horwitz
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
A number of scavengers of reactive oxygen metabolites reduce myocardial injury when given
before ischemia and reperfusion, but few, if any, have proven to be effective when given near the
onset of reperfusion. This is particularly true when infarct size is measured after at least 48
hours of reperfusion, when the full extent of myocardial damage has become apparent.
Dimethylthiourea (DMTU) is an extremely diffusible, potent scavenger of hydroxyl radical,
hydrogen peroxide, and hypochlorous acid, with a long half-life of 43 hours. Sixteen chloraloseanesthetized dogs underwent 90 minutes of left anterior descending coronary artery (LAD)
occlusion followed by 48 hours of reperfusion. Collateral flow was measured by radioactive
microspheres. Infarct size and risk area were measured by a postmortem dual-perfusion
technique using triphenyl tetrazolium chloride and Evan's blue dye. In eight dogs, therapy with
DMTU (500 mg/kg i.v.) was given during the last 15 minutes of ischemia and the first 15
minutes of reperfusion. In eight control dogs, the same volume of 0.9% saline was given during
the last 15 minutes of ischemia through the first 15 minutes of reperfusion. Infarct size as a
percent of risk area was reduced in the DMTU-treated group compared with the saline-treated
controls (DMTU=42+4% versus saline =59+4%, p<0.01). There were no differences between
the groups in risk area as a percent of the left ventricle (DMTU=25+2 % versus saline=21+2%,
p=NS), in LAD endocardial flow during ischemia (DMTU=3.5+0.4 versus saline=4.0+0.5
ml/100 g/min, p=NS), in LAD transmural flow during ischemia (DMTU=13.3+1.3 versus
saline=12.0±1.5 ml/100 g/min,p=NS), or in the heart rate-blood pressure product. Histological and in vitro biochemical analyses confirmed that the tetrazolium method accurately
delineated infarct size in saline- and DMTU-treated dogs. Thus, DMTU given near the onset of
reperfusion dramatically reduced infarct size measured after an interval sufficient for
irreversibly damaged cells to become necrotic. The efficacy of DMTU in reducing infarct size
may reflect its rapid entry into myocardial cells as well as continued protection for a prolonged
period during reperfusion. (Circulation Research 1991;68:1652-1659)
T here has been considerable concern that early
reperfusion of ischemic myocardium, which
terminates damage due to ischemia, may
cause further damage through a different mechanism, resulting in suboptimal myocardial salvage.1'2
This "reperfusion injury" appears to be mediated by
the formation of reactive oxygen metabolites (includSupported by grants from the National Institutes of Health
(HL-41661) and the American Heart Association, Colorado Affiliate.
Address for correspondence: Lawrence D. Horwitz, MD, Cardiology, Box B-130, University of Colorado Health Sciences Center, 4200 E. Ninth Avenue, Denver, CO 80262.
Received October 24, 1990; accepted February 19, 1991.
ing H202, '02-, OH-, and others)3-5 that are toxic to
myocardial cells.6-8 Analysis by spin-trap techniques
has generally concluded that a large burst of reactive
oxygen metabolites is produced immediately after
hypoxic or ischemic myocardium is reoxygenated.59
However, another study'0 has observed production of
reactive oxygen metabolites for several hours after
the onset of reperfusion.
Therapeutic strategies have focused on treatment
with enzymatic scavengers of reactive oxygen metabolites or with iron chelators in animal models of
ischemia and reperfusion.11-1 Most of these agents
have serum half-lives on the order of minutes16 and
do not readily cross cell membranes. Administration
Carrea et al Dimethylthiourea and Myocardial Infarct Size
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
of a superoxide anion scavenger, superoxide dismutase, has generally resulted in short-term myocardial
salvage,11,12 but when reperfusion is carried out for
longer periods (24 hours to 7 days), myocardial
salvage has not been consistently observed.17-19
This discrepancy between short- and long-term
results may be due to at least two factors. Continued
production of reactive oxygen metabolites during the
first 24 hours of reperfusion10 could cause continuing
damage during this period. This ongoing production
probably extends well beyond the time in which many
agents demonstrate active scavenging activity.16
Alternatively, the methodology used to determine
myocardial infarct size (tetrazolium staining) may fail
to identify all irreversibly injured tissue early in
reperfusion,20 leading to an underestimation of the
amount of necrosis that will eventually occur.
Another disappointing aspect of efforts to reduce
reperfusion injury has been that agents that appear to
reduce myocardial injury if given before ischemia have
generally failed to do so if they are given near the
onset of reperfusion.14,15,17-19 This is troubling, since
clinical application of an intervention to prevent
reperfusion injury after acute myocardial infarction
would involve administration after ischemia is well
established. The poor results demonstrated by most
agents with administration after ischemia has begun
may hinge on their ability to enter cells. Antioxidant
scavenger enzymes, such as catalase and superoxide
dismutase, or the iron chelator, deferoxamine, do not
readily enter myocardial cells, particularly when regional myocardial perfusion is diminished.15,16,20
A pharmacological intervention that is useful in
preventing reperfusion injury by reactive oxygen
metabolites should ideally be capable of entering
myocardial cells rapidly to counter the burst of reactive oxygen metabolites generated early in reperfusion. It should also maintain sufficient myocardial
levels to afford protection against low levels of oxidant
production during the subsequent hours.10 Dimethylthiourea (DMTU) is an agent that is highly diffusible,
has a long half-life, and is an effective scavenger of
hydrogen peroxide, hydroxyl radical, and hypochlorous acid.21,22 In this study, we administered DMTU
after regional myocardial ischemia was well established and were able to demonstrate a 30% reduction
in infarct size at 48 hours of reperfusion.
Materials and Methods
Instrumentation
Nineteen male mongrel dogs (25.8+0.9 kg) were
anesthetized with thiamylal sodium (20 mg/kg i.v.)
followed by a-chloralose (100 mg/kg i.v. supplemented as needed). The dogs were intubated and
ventilated with a respirator (Harvard Apparatus,
South Natick, Mass.) using a mixture of 35% oxygen
and 65% nitrogen, which maintains arterial blood
gases in the usual physiological range at Denver's
altitude.
1653
By use of a sterile technique, a left thoracotomy
was performed. The heart was suspended in a pericardial cradle. Catheters were inserted into the proximal descending aorta and left atrium. A snare
occluder was placed loosely around the left anterior
descending coronary artery (LAD) distal to its first
diagonal branch (2-3 cm from the LAD origin).
Experimental Protocol
During a 15-minute postinstrumentation equilibration period, an injection of 0.2 ml Tween 80 in 2 ml of
0.9% saline was performed to test for adverse hemodynamic effects. Before LAD occlusion, 1-2 million
15-,.tm-diameter radiolabeled microspheres were injected into the left atrium for a baseline regional
myocardial blood flow measurement. Subsequently, a
30-second test occlusion was performed. Epicardial
cyanosis was apparent in all dogs during the test
occlusion.
Aortic and left atrial pressures were recorded
using Statham P23Db transducers (Gould, Cleveland, Ohio). The lead II electrocardiogram was recorded with subcutaneous needle electrodes. Blood
pressure, left atrial pressure, and a lead II electrocardiogram were continuously monitored on an oscillograph (model R 612, Beckman Instruments, Inc.,
Fullerton, Calif.). The heart rate-blood pressure
product was calculated by multiplying heart rate
times mean aortic pressure.
Complete blood counts were obtained from arterial samples before occlusion, at the end of ischemia,
at 2 hours of reperfusion, and at 48 hours of reperfusion. Tidal volume, respiratory rate, and end-expiratory pressure were adjusted to maintain a physiological pH and Po2.
LAD ligation was performed by tightening the snare
for 90 minutes. Epicardial cyanosis was apparent in all
dogs. At 60 minutes of occlusion, a second set of
microspheres was injected into the left atrium. The
treatment phase of the protocol was begun at 75
minutes of occlusion. Dogs were randomized to receive either DMTU (500 mg/kg i.v. in 100 ml of 0.9%
NaCl) or saline (100 ml of 0.9% NaCl) during the last
15 minutes of ischemia and the first 15 minutes of
reperfusion. After 90 minutes of ischemia, the LAD
occluder was released. A third set of microspheres was
injected into the left atrium at 2 hours of reperfusion.
The left atrial and aortic lines were externalized using
subcutaneous tunnels, and the chest was closed using
sterile technique. Ampicillin (500 mg i.v.) was administered as a prophylactic antibiotic to all dogs. Assisted
ventilation was continued until adequate spontaneous
respirations returned.
The dogs were returned to the kennel, where they
were fed standard chow for 48 hours. Morphine sulfate
was administered intramuscularly as deemed appropriate for pain control. On day 2, the dogs were again
anesthetized with thiamylal sodium (20 mg/kg i.v.)
followed by a-chloralose (100 mg/kg i.v.). Hemodynamics were recorded, and blood samples were obtained for complete blood counts and arterial blood
1654
Circulation Research Vol 68, No 6 June 1991
gases. A fourth injection of microspheres was then
performed. Subsequently, the thoracotomy incision was
reopened. Heparin (5,000 IU i.v.) was then administered., and the heart and proximal aorta were removed.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Measurement of Infarct Size
The heart and aorta were connected to a dualperfusion apparatus. The aortic root and LAD immediately distal to the site of prior occlusion were
cannulated. The proximal LAD was ligated at the site
of prior occlusion. This allowed perfusion of the
circumflex territory with Evan's blue dye through the
aortic root while the LAD territory was perfused
through the LAD catheter with a 2% solution of
2,3,5 -triphenyltetrazolium chloride (TTC) at 37°C.
Perfusion pressure was equal in both cannulas and
was maintained at 100 mm Hg.23-25
After 5 minutes, the heart was removed from the
perfusion apparatus. The left ventricle was isolated
and weighed. Five to seven transverse sections of
-1-cm thickness were then obtained by sectioning
parallel to the atrioventricular groove. The slices
were weighed and traced onto clear acetate sheets to
demarcate 1) remote normal (blue stain), 2) ischemic, noninfarct (TTC-positive, red stain), or 3)
ischemic, infarct (TTC-negative, unstained).
Regional Myocardial Blood Flow
Regional myocardial blood flow was assessed before occlusion, at 75 minutes of occlusion, at 60
minutes of reperfusion, and at 48 hours of reperfusion using 15-,um-diameter microspheres (Dupont,
Wilmington, Del.) containing either 57Co, 46Sc, 85Sr,
or ll'3Sn.1s One to 2 million microspheres in a 0.01%
Tween 80 suspension (in 10% dextran) were diluted
to a final volume of 2 ml with 0.9% NaCl. This
suspension was agitated vigorously, injected through
the left atrial catheter, and flushed in with 6 ml of
0.9% NaCl. Starting at 10 seconds before injection of
the microspheres and continuing until 3 minutes
after the injection, a reference sample was withdrawn
from the aortic catheter at a rate of 2 ml/min.15 This
procedure was repeated at each time point using a
different isotope.
After the hearts were perfused with dye and sliced,
each slice was divided into regions of nonischemic
(blue) and ischemic (red and white) flow, based on
staining characteristics. These were then sectioned
into endocardial, midmyocardial, and epicardial
zones. At least two samples (0.5-1.5 g) from each zone
were then separated and weighed for radionuclide
counting in a three-channel gamma spectrophotometer (model 5000, Packard Instrument Co., Inc., Meriden, Conn.) using appropriate energy windows.15
Background and crossover counts from other isotopes
were accounted for, and blood flow measurements
(ml/100 g/min) were obtained. To avoid any error
from apparent microsphere leakage or tissue edema,
flows to the ischemic zone segments during ischemia
and day 1 reperfusion were multiplied by a correction
factor. This factor was calculated as the ratio of
preocclusion blood flow in the nonischemic myocardial segment to the preocclusion blood flow in a
segment subsequently made ischemic.26
Serum DMTU Analysis
Plasma samples were obtained before and at the
end of the 30-minute DMTU infusion and at 2 hours
of reperfusion, 24 hours of reperfusion, and 48 hours
of reperfusion for subsequent analysis of DMTU
content by high-performance liquid chromatography.
The plasma samples were centrifuged in a micropartition system (Amicon, Beverly, Mass.) with 500molecular weight filters at 8,500 rpm (6,600g) for 90
minutes. This filtrate contains most of the plasma
liquid plus all free components with <500 molecular
weight. Aliquots of an internal standard, 0.1% dimethyl sulfoxide, were added to the plasma samples.
Ten-microliter samples were then injected in a mobile phase of deionized water by an autosampler
(model 9095, Varian Associates, Inc., Palo Alto,
Calif.) using a pump (model 510, Waters Instruments, Inc., Rochester, Minn.). Each sample was
passed through a Hypersil ODS, 5-gm, 15-cm column (Jones Chromatography USA, Inc., Littleton,
Col.) to a variable wavelength spectrophotometer
(model 481, Waters Instruments) set at 230 nm. The
data were transferred to a data module (model 740,
Waters Instruments). DMTU concentrations were
determined using the DMTU/dimethyl sulfoxide ratio from a standard curve.2728
Histopathology
The TTC stain is an established method of assessing
viable versus infarcted tissue at times greater than 3-6
hours, although some antioxidant strategies are reported to alter the tissue-staining characteristics of
ITC.20 Therefore, we tested the ability of TTC staining to distinguish viable, previously ischemic tissue
from infarcted tissue in our preparation. Myocardium
that remained after sections were removed for determination of myocardial blood flow was stored in 10%
buffered formalin. Sections of myocardium from the
center of TTC-positive (red), TTC-negative (unstained), and Evan's blue-stained segments were randomly selected from three DMTU-treated dogs and
three saline-treated dogs. These segments were labeled and reviewed in a blinded manner by a cardiac
pathologist (R.H.S.). The segments were embedded in
paraffin, and three serial sections were stained with
hematoxylin and eosin, Masson's trichrome, or phosphotungstic acid-hematoxylin. The sections were
semiquantitatively assessed for coagulative necrosis,
contraction-band necrosis, interstitial edema, hemorrhage, and interstitial neutrophil infiltration.
Presence of coagulative necrosis characterized by
changes in staining qualities and loss of myocyte
subcellular structural detail was scored as 0=none
(normal), 1=mild, 2=moderate, and 3=severe.
Presence of contraction band necrosis characterized by myocyte contraction bands was scored as
0= none (normal), 1 = occasional, 2 = moderate,
Carrea et al Dimethylthiourea and Myocardial Infarct Size
1655
M DMTU
SALINE
150
RMBF
(mIl100gm/min)
1
1x
±SE
NS vs SALINE
1
N0 1 *
1 2 3 4
BASELINE
2-MID-MYOCARDIAL|
1 2 3 4
75 MIN
ISCHEMIA
1
01 2 3 4
120 MIN REP
1.III~ .PC4-TRANSMURAL
1 2 3 4
48 HOURS
REPERFUSION
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
3=frequent. Interstitial edema was scored as
0=normal, 1=focal and mild loosening of interstitial connective tissue stroma, 2=diffuse and moderate loosening of interstitial connective tissue
stroma, 3=diffuse and severe loosening of interstitial connective tissue stroma. Hemorrhage was
scored as 0=absent, 1 =focal and mild extravasation
of red blood cells, 2=focal or diffuse but moderate
extravasation of red blood cells, and 3=diffuse and
severe extravasation of red blood cells. Neutrophil
infiltration was scored as 0=absent, 1 =present in a
few high-power fields, 2=present in approximately
half of high-power fields, and 3=present in most
high-power fields.
In Vitro Assessment on the Lack of Effect of DMTU
on TTC Staining
The TTC stain is a histochemical stain that requires dehydrogenase activity to reduce TTC to
formazan. In addition to histological examination, we
performed studies in vitro to exclude an effect of
experimentally relevant concentrations of DMTU on
TTC staining. The staining characteristics of ITC
with and without addition of DMTU were assessed
by modification of techniques reported by Singh et
a129 and by Green and Narahara.30 Approximately
500 mg of normal canine myocardium was homogenized (Polytron, Brinkmann Instruments, Inc., Westbury, N.Y.) in 5 vol of 10 mM potassium phosphate
buffer (pH 7.8) containing 0.1 mM EDTA. After
centrifugation at 800g for 10 minutes at 40C, the
supernatant was used for enzymatic analysis. The
basic assay materials were 0.05 ml of 20 mM TTC,
0.05 ml of 10 mM NaN3, and 0.2 ml tissue extract in
5-ml test tubes. Each assay condition required one of
the following additions: phosphate buffer alone, 1
mM DMTU, 10 mM DMTU, 100 mM DMTU, 1
mg/ml NAD plus 100 mM DMTU, 0.1 ml sodium
succinate, or 0.1 ml sodium succinate plus 100 mM
DMTU. All test tubes were then brought to a final
volume of 0.5 ml with 20 mM phosphate buffer (pH
7.5) and incubated at 37°C for 60 minutes. After
incubation, 1.5 ml of 95% ethanol was added to each
tube. After mixing thoroughly, they were centrifuged
at 2,500g for 10 minutes at 4°C. The supernatants
were then passed through a 0.22-,m filter (Millipore
Corp., Bedford, Mass.). The absorbance of the fil-
FIGURE 1. Bar plot demonstrating
similar regional myocardial blood
flow (RMBF) in dimethylthiourea
(DMTU)-treated and saline-treated
(control) groups at baseline, 75
minutes of left anterior descending
coronary artery ischemia, 120 minutes of reperfusion (REP), and 48
hours of reperfusion.
trate at 458 nm, which represents formazan production, was recorded.
Statistical Analysis
Data reported are mean+SEM. All DMTU versus
saline statistical comparisons at particular time
points were performed using an unpaired t test. All
within-group analyses over time were performed
using a two-way analysis of variance. The StudentNewman-Keuls test was used for multiple comparisons.31 Collateral ischemic blood flow versus infarct
size as a percent of risk area (MI/RISK) was assessed
using an analysis of covariance (ANCOVA), with
LAD endocardial (or transmural) blood flow during
ischemia assigned as the independent variable and
MI/RISK assigned as the dependent variable.25,32 A
value of p<0.05 was considered significant.
Results
Study Group
Sixteen of the 19 dogs were included in the final
analysis. Two dogs were excluded due to ischemic
zone collateral flow >30 ml/100 g/min and failure to
demonstrate an infarct by T1C (one DMTU- and
one saline-treated dog). One dog (saline-treated)
died between 8 and 16 hours after reperfusion and
was excluded. Ischemic ST segment changes and
reperfusion ventricular tachyarrhythmias occurred in
all dogs included in the final analysis. During the
early reperfusion period, ventricular fibrillation requiring defibrillation occurred in four DMTUtreated dogs and three saline-treated dogs.
Regional Myocardial Blood Flow
Ischemic zone endocardial blood flow during occlusion (DMTU=3.5+0.4 versus saline=4.0+0.5 ml/100
g/min,p=NS) and ischemic zone epicardial blood flow
(DMTU=26.2+3.1 versus saline=23.7±2.2 ml/100
g/min, p=NS) were similar in both groups (Figure 1).
Ischemic zone transmural blood flow during ischemia
was also similar (DMTU=13.3±1.3 versus saline=
12.0±1.5 ml/100 g/min, p=NS). Blood flows were not
significantly different between the DMTU and saline
groups at any time. Substantial and similar ischemia
was produced by LAD ligation in both groups.
1656
Circulation Research Vol 68, No 6 June 1991
135.7+6.7, p =NS). Thus, there was a significant reduction in infarct size after DMTU treatment that did
not appear to be influenced by differences in left
ventricular mass, risk area, or ischemic coronary collateral blood flow.
0.77
MI/RISK
0.6
0.5
0.4
0.3
0.2
0.1
-
-
Hemodynamics
There were no significant differences between the
DMTU and saline groups at any time during the study
in rate-pressure product, heart rate, mean arterial
pressure, or left atrial pressure. The rate-pressure
product at 48 hours was lower in each group than
during the measurements obtained on the initial day of
the study. The mean values for the rate-pressure
product are shown in Figure 4. We conclude that
DMTU caused no apparent hemodynamic effect that
could explain its effect on myocardial infarct size.
±SE
p<.01
0.0
DMTU
SALINE
(n=8)
(n=8)
0.5
RISK/LV
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Hematologic Parameters
The hematologic parameters are summarized in
Table 1. Hemoglobin levels did not differ between
the two groups and did not change during the study.
White blood cell counts were similar in both groups
at all times but did show a trend toward increased
values after 48 hours of reperfusion.
±SE
p=NS
SALINE
DMTU
(n=8)
(n=8)
FIGURE 2. Top panel: Bar plot relating weight of infarcted
tissue to weight of tissue in region at risk (MI/RISK) in the
saline-treated (control) group versus the dimethylthiourea
(DMTU)-treated group. Bottom panel: Bar plot relating
weight of tissue in the risk region to the total left ventricular
weight (RISK/LV) in the saline-treated (control) group versus
the dimethylthiourea (DMTU)-treated group.
DMTU Levels
DMTU levels were 6.2±0.1 mM at the end of the
infusion, 4.8+0.2 mM at 2 hours of reperfusion,
3.0±0.6 mM at 24 hours of reperfusion, and 2.2±0.7
mM at 48 hours of reperfusion (Figure 5). Assuming
first-order kinetics, the serum half-life is 43.7 hours.
Myocardial Infarct Size
Infarct size (MI/RISK) and risk area (RISK/LV)
are shown in Figure 2. MI/RISK was reduced by
DMTU therapy compared with saline (DMTU=
42+4% versus saline=59+4%, p<0.01). MI/RISK
versus transmural ischemic zone flow is shown in
Figure 3. DMTU-treated dogs had decreased MI/
RISK as a function of LAD endocardial or transmural
ischemic flow by ANCOVA (both p<0.05). Risk area
as a percentage of the total left ventricle was similar in
both groups (DMTU= 25 + 2% versus saline = 21 + 2%,
p=NS). Total left ventricular mass was similar in
both groups (DMTU=136.1+7.7 g versus saline=
0.8
0.7 0.6-
0
0
Lack of Effect of DMTU on TTC Staining
The reduction of TTC to formazan as quantified by
absorbance at 458 nM is shown in Table 2. There was
neither inhibition nor accentuation of absorbance at
458 nM by experimentally relevant levels of DMTU.
This suggests that DMTU does not artifactually
influence the conversion of ITC to formazan in vitro.
Thus, the observed differences in infarct size are
unlikely to be due to an effect of DMTU on the TTC
staining characteristics of canine myocardium.
0
00
0.5'
0
0l.
MI/RISK 0.4
0.3 0.20.1
0.0
p<.05 (BY ANALYSIS OF
COVARIANCE)
0
10
20
TRANSMURAL
ISCHEMIC ZONE FLOW
(MLf100GIMIN)
30
EJC SALINE|
| DMTU
l
FIGURE 3. Scattergram of infarct size as a percent
of risk area (MI/RISK) plotted against transmural
blood flow during occlusion in the ischemic zone.
SALINE, saline-treated (control) group; DMTU,
dimethylthiourea-treated group.
Carrea et al Dimethylthiourea and Myocardial Infarct Size
RATE
PRESSURE
PRODUCT
(x 1O4)
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1657
-
FIGURE 4. Line plot of rate-pressure product
(calculated as mean arterial pressure times heart
rate) versus time. DMTU, dimethylthiourea-treated
group; SALINE, saline-treated (control) group;
isch, ischemia.
*
ISCHEMIA
=p<.05
vs
isch
REPERFUSION
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Histopathology
The results of the histopathologic scoring are shown
in Table 3. In both groups, there were no abnormal
histological findings in the ischemic-viable (TTC-positive [red]) or normal remote zones (Evan's blue).
Tissue that was identified as infarcted by lack of ITC
staining had coagulation necrosis, contraction-band necrosis, interstitial edema, hemorrhage, and neutrophil
infiltration. There were no significant differences within
infarcted tissue when the DMTU- and saline-treated
groups were compared. Thus, ITC staining accurately
delineated infarcted from viable tissue at 48 hours
according to our histological analysis.
Discussion
DMTU is a derivative of thiourea but does not
precipitate the pulmonary edema associated with thiourea and has no cardiac toxicity in intact animals.22 It is
a highly diffusible, low molecular weight compound
that readily enters myocardium and other tissues in
vivo.22,33 We found that DMTU has a serum half-life of
43 hours in our canine model. In previous reports,
DMTU reduced reactive oxygen metabolite-mediated
injury in lung,22'34 kidney,35 brain,36 and skeletal muscle.37 Other investigators have observed diminished
canine myocardial stunning with DMTU.38-40
We previously reported that DMTU administration
at the end of a 90-minute ischemic period reduced
infarct size measured at 3 hours of reperfusion by
40%.34 However, recent work has cast doubt on the
accuracy and meaning of 'ITC delineation of infarct
TABLE 1. Hematologic Parameters in Ischemic/Reperfused Dogs
Baseline
Occlusion
(90 min)
WBC (x 103/mm3)
DMTU
10.1+1.1
Saline
9.9+2.3
15.6±3.2
13.9±+2.4
DMTU
O SALINE
Reperfusion
120 min
48 hr
14.6 ± 1.8
17.6±4.1
17.1±+ 1.6
18.1±2.6
Hgb (g/dl)
13.7±0.8 14.1±0.4 12.8±1.1
DMTU
13.0±1.8
Saline
13.0±0.6 12.6±0.6 12.6±0.5
12.4+1.0
Values are mean±SEM. WBC, white blood cell count; DMTU,
dimethylthiourea-treated dogs; Saline, saline-treated (control)
dogs; Hgb, hemoglobin. Allp=NS DMTU vs. saline.
size with such a short reperfusion period. There is
reason to believe that some tissue that appears to be
viable by TTC staining very early in reperfusion may be
irreversibly injured and will undergo necrosis by 24
hours of reperfusion.15,17-19 It is also possible that some
tissue that is viable at 3-6 hours of reperfusion may be
subsequently injured over 24-48 hours of reperfusion
by further generation of reactive oxygen metabolites.10
Therefore, we decided to reevaluate the efficacy of
DMTU in preventing injury during ischemia and reperfusion by studying myocardial infarction size after 48
hours of reperfusion. We chose this time point to avoid
doubts regarding the reliability of T1C staining and
because it permitted detection of late injury due to
ongoing generation of reactive oxygen metabolites.
We found that peripheral intravenous infusion of
DMTU near the onset of reperfusion produced substantial improvement in myocardial infarct size measured at 48 hours. This protection was apparent at all
levels of collateral blood flow and was not due to
changes in hemodynamic parameters during ischemia, white blood cell counts, or tissue neutrophil
infiltration. There were no alterations in regional
myocardial blood flow. DMTU did not cause artifactual changes in TTC staining characteristics. The
histopathologic examination confirmed that TTC
staining accurately distinguished necrotic from viable
tissue at 48 hours.
TABLE 2. Reduction of 2,3,5-Triphenyltetrazolium Chloride to
Formazan Quantified by Absorbance at 458 nM
Absorbance
Contents of assay
at 458 nM
Tissue extract alone
62.5+1
Tissue extract
Plus 1 mM DMTU
62.0±0.8*
Plus 10 mM DMTU
65.0±3.0*
Plus 100 mM DMTU
61.0±0.8*
Plus 1 mg/ml NAD and 100 mM DMTU
64.5±1.0*
Plus succinate
150.0+1.5t
Plus 50 mM succinate and 100 mM DMTU
152-2.7t
Values are mean+SEM; n=4. DMTU, dimethylthiourea.
*p=NS and tp<0.05 vs. tissue extract alone; 4p=NS vs. tissue
extract plus succinate.
1658
Circulation Research Vol 68, No 6 June 1991
TABLE 3. Histopathologic Scoring in Tissue From Ischemic/Reperfused Dogs
Contraction
Interstitial
Coagulative
necrosis
band necrosis
edema
Zone
DMTU-treated dogs (n=3)
1.7+0.3
2.3+0.3
Infarct
2.7+0.3
0+0
0+0
0+0
Ischemic-viable
0±0
0+0
0+0
Remote
Saline-treated dogs (n=3)
Infarct
Ischemic-viable
2.0+0.3
0+0
0+0
Neutrophil
Hemorrhage
2.3+0.3
0+0
0)0
infiltration
2±0.6
0+0
0±0
3.0±0
2.3±0.3
1.3+0.3
0+0
0+0
0+0
0+0
0+0
0+0
Remote
Values are mean ± SEM. DMTU, dimethylthiourea. All p = NS saline value vs. corresponding DMTU value. (Refer
to "Materials and Methods" for explanation of scoring.)
1.9+0.2
0+0
0+0
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Results with other antioxidant measures in preventing myocardial damage in similar models of ischemia
and reperfusion have been controversial. Some recent
studies17-19 with superoxide dismutase, a scavenger of
superoxide anion, have failed to confirm the benefits
reported in earlier work. Superoxide dismutase is
poorly diffusible and may not enter cells readily.16'39 In
addition, it has a very short half-life, limiting the
duration of protection it affords. Bovine superoxide
dismutase has not generally been efficacious in studies
performed after 48 hours or more of reperfusion.18
Although human recombinant superoxide dismutase
reduced TTC-measured infarct size in one study done
at 48 hours of reperfusion,12 it has recently been
reported that this preparation of the enzyme alters
TTC staining characteristics, which could lead to
inaccuracy in assessing damage with the dye.20 Superoxide dismutase conjugated with polyethylene glycol
has a long half-life.1625 Chi et a125 reported that
polyethylene glycol-superoxide dismutase (1,000
units/kg) reduced myocardial infarct size in a canine
model of 6 hours of ischemia followed by 24 hours of
reperfusion but that regular superoxide dismutase was
ineffective. Tamura et al,41 using the same dose of
polyethylene glycol-superoxide dismutase in a canine
model of 90 minutes of ischemia, demonstrated protection at 6 hours and 96 hours of reperfusion. However, Tanaka et a142 reported that an unusually high
dose of polyethylene glycol-superoxide dismutase
(10,000 units/kg) given together with catalase had no
protective benefit in a canine model of 90 minutes of
ischemia and 96 hours of reperfusion. It is possible
that the higher dose may have been detrimental, since
evidence has been published that manganese-superoxide dismutase may reduce damage in ischemic/
reperfused rabbit hearts when given at low doses but
exacerbate damage at higher doses.43
DMTU scavenges H202, OH, and HOCI, but not
02-. It is possible that there is greater benefit from
neutralizing certain reactive oxygen metabolites than
others. However, in contrast to many other potential
therapeutic agents, DMTU enters tissues readily and
has a long half-life in vivo. These properties (rapid
delivery to the site under attack and prolonged effectiveness for at least 24 hours) may be necessary to
accomplish meaningful reduction in myocardial necrosis with antioxidants. Agents with these properties,
including DMTU, warrant further evaluation for potential clinical application. In general, clinical usefulness probably requires that an agent be capable of
reducing injury with administration after ischemia is
well established.
Acknowledgments
The authors wish to acknowledge the superb technical assistance of Julie L. Peach, Terri S. Hogue,
and Holly S. Coller.
7
End
6
5
References
Infusion
ours
4
DMTU CONC 3
(mM)
24 Hours
48 Hours
2
ft
1
TIME POST INFUSION
INFUSION
FIGURE 5. Line plot showing serum dimethylthiourea levels
(DMTU CONC) at end of infusion, 2 hours after infusion, 24
hours after infusion, and 48 hours after infusion. The calculated serum half-life is 43.7 hours.
1. Kloner RA, Ellis SG, Lange R, Braunwald E: Studies of
experimental coronary artery reperfusion: Effects on infarct
size, myocardial function, biochemistry, ultrastructure and
microvascular damage. Circulation 1983;68(suppl I):I-8-I-15
2. Braunwald E, Kloner RA: Myocardial reperfusion: A double
edged sword. J Clin Invest 1985;76:1713-1719
3. McCord JM: Oxygen-derived free radicals in post-ischemic
tissue injury. N Engl i Med 1985;312:159-163
4. Wems SW, Shea MJ, Lucchesi BR: Free radicals and myocardial
injury: Pharmacologic implications. Circulation 1986;74:1-5
5. Zweier JL, Flaherty JT, Weisfeldt ML: Direct measurement of
free radical generation following reperfusion of ischemic
myocardium. Proc Natl Acad Sci U S A 1984;184:1404-1407
6. Starke PE, Farber JL: Endogenous defenses against the
cytotoxicity of hydrogen peroxide in cultured rat hepatocytes.
J Biol Chem 1985;260:86-92
Carrea et al Dimethylthiourea and Myocardial Infarct Size
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
7. Fridovich I: Superoxide radical: An endogenous toxicant.
Annu Rev Pharmacol Toxicol 1983;23:239-257
8. Thompson JA, Hess ML: The oxygen free radical system: A
fundamental mechanism in the production of myocardial
necrosis. Prog Cardiovasc Dis 1986;28:449-462
9. Lesnefsky EJ, Fennessey PM, VanBenthuysen KM, McMurtry
IF, Travis VL, Horwitz LD: Superoxide dismutase decreases
early reperfusion release of conjugated dienes following
regional canine ischemia. Basic Res Cardiol 1989;84:191-196
10. Kuzuya T, Hoshida S, Kim Y, Nishida M, Fuji H, Kitabatake
A, Tada M, Kamada T: Detection of oxygen-derived free
radical generation in the canine postischemic heart during late
phase of reperfusion. Circ Res 1990;66:1160-1165
11. Jolly SR, Kane WJ, Baillie MB, Abrams GD: Canine myocardial
reperfusion injury: Its reduction by the combined administration
of superoxide dismutase and catalase. Circ Res 1984;54:277-285
12. Ambrosio G, Weisfeldt ML, Jacobus WE, Flaherty JT: Evidence
for a reversible oxygen radical-mediated component of reperfusion injury: Reduction by a recombinant human superoxide
dismutase administered at the time of reflow. Circulation 1987;
75:282-291
13. Ambrosio G, Zweier JL, Jacobus WE, Weisfeldt ML, Flaherty
JT: Improvement of postischemic myocardial function and
metabolism induced by administration of deferoxamine at the
time of reflow: The role of iron in the pathogenesis of
reperfusion injury. Circulation 1987;76:906-915
14. Reddy BR, Kloner RA, Przyklenk K: Early treatment with
deferoxamine limits myocardial ischemic/reperfusion injury.
Free Radic Biol Med 1989;7:45-52
15. Lesnefsky EJ, Repine JE, Horwitz LD: Deferoxamine pretreatment reduces canine infarct size and oxidative injury. J
Pharnacol Exp Ther 1990;253:1103-1109
16. Pyatak PS, Abuchowski A, Davis FF: Preparation of a polyethylene glycol: superoxide dismutase adduct, and an examination of
its blood circulating life and anti-inflammatory activity. Res
Commun Chem Pathol Pharmacol 1980;29:113-127
17. Gallagher KP, Buda AJ, Pace D, Gerren RA, Shlafer M:
Failure of superoxide dismutase and catalase to alter size of
infarction in conscious dogs after 3 hours of occlusion followed
by reperfusion. Circulation 1986;73:1065-1076
18. Nejima J, Knight DR, Falon JT, Uemura N, Manders T, Canfield
DR, Cohen MV, Vatner SF: Superoxide dismutase reduces
reperfusion arrhythmias but fails to salvage regional function or
myocardium at risk in conscious dogs. Circulation 1989;79:
143-153
19. Richard VJ, Murry CE, Jennings RB, Reimer KA: Therapy to
reduce free radicals during early reperfusion does not limit the
size of myocardial infarcts caused by 90 minutes of ischemia in
dogs. Circulation 1988;78:473-480
20. Shirato C, Miura T, Ooiwa H, Toyofuku T, Wilborn WH,
Downey JM: Tetrazolium artifactually indicates superoxide
dismutase-induced salvage in reperfused rabbit heart. J Mol
Cell Cardiol 1989;21:1187-1193
21. Fox RB: Prevention of granulocyte-mediated oxidant lung
injury in rats by a hydroxyl radical scavenger, dimethylthiourea. J Clin Invest 1984;74:1456-1464
22. Fox RB, Harada RN, Tate RM, Repine JE: Prevention of
thiourea-induced pulmonary edema by hydroxyl-radical scavengers. JAppl Physiol 1983;55:1456-1459
23. Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K,
Mercier JC, Corday E, Ganz W: Early phase acute myocardial
infarct size quantification: Validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J
1981;101:593-600
24. Werns SW, Shea MJ, Mitsos SE, Dysko RC, Fantone JC,
Schork MA, Abrams GD, Pitt BP, Lucchesi BR: Reduction of
the size of infarction by allopurinol in the ischemic-reperfused
canine heart. Circulation 1986;73:518-524
25. Chi L, Tamura Y, Hoff PT, Macha M, Gallagher KP, Schork
MA, Lucchesi BR: Effect of superoxide dismutase on myocardial infarct size in the canine heart after 6 hours of regional
ischemia and reperfusion: A demonstration of myocardial
salvage. Circ Res 1989;64:665-675
1659
26. Consigny PM, Verrier ED, Payne BD, Edelist G, Jester J,
Baer RW, Vlahakes GJ, Hoffman JI: Acute and chronic
microsphere loss from canine left ventricular myocardium. Am
J Physiol 1982;242(Heart Circ Physiol 3):H392-H404
27. Kobayashi H, Matano 0, Goto S: Simultaneous quantification
of thioureas in rat plasma by high-performance liquid chromatography. J Chromatogr 1981;207:281-285
28. Curtis WE, Muldrow ME, Parker NB, Barkley R, Linas SL,
Repine JE: N,N'-Dimethylthiourea dioxide formation from
N,N'-dimethylthiourea reflects hydrogen peroxide concentrations in simple biological systems. Proc Natl Acad Sci U S A
1988;85:3422-3425
29. Singh A, Lee KJ, Goldfarb RD, Tsan MF: Relation between
myocardial glutathione content and extent of ischemiareperfusion injury. Circulation 1989;80:1795-1804
30. Green JD, Narahara HT: Assay of succinate dehydrogenase
activity by the tetrazolium method: Evaluation of an improved
technique in skeletal muscle fractions. J Histochem Cytochem
1980;28:408-412
31. Steel RGD, Torrie JH: Principles and Procedures of Statistics.
New York, McGraw-Hill Book Co, 1960, pp 110, 111, 114
32. Wilkinson L: SYSTAT: The System for Statistics. Evanston, Ill,
Systat, Inc, 1988
33. Portz SJ, Lesnefsky EJ, VanBenthuysen KM, Repine JE,
Parker NB, McMurtry IF, Horwitz LD: Dimethylthiourea, but
not dimethylsulfoxide, reduces canine myocardial infarct size.
Free Radic Biol Med 1989;7:53-58
34. Paull DE, Keagy BA, Kron EJ, Wilcox BR: Improved lung
preservation using a dimethylthiourea flush. J Surg Res 1989;46:
333-338
35. Linas SL, Shanley PF, White CW, Parker NP, Repine JE:
Oxygen metabolite mediated injury in perfused kidneys is
reflected by consumption of DMTU and glutathione. Am J
Physiol 1987;253(Renal Fluid Electrolyte Physiol 22):F692-F701
36. Patt A, Harken AH, Burton LK, Rodell TC, Peiermattei D,
Schorr WJ, Parker NB, Berger EM, Horesh IR, Linas SL,
Cheronis JC, Repine JE: Xanthine oxidase derived hydrogen
peroxide contributes to ischemia-reperfusion induced edema
in gerbil brains. J Clin Invest 1988;81:1556-1562
37. McCutchan HJ, Schwappach JR, Enquist EG, Walden DL,
Terada LD, Reiss OK, Leff JA, Repine JE: Xanthine oxidase
derived hydrogen peroxide contributes to reperfusion injury of
ischemic skeletal muscle. Am J Physiol 1990;258(Heart Circ
Physiol 5):H1415-H1419
38. Bolli R, Zhu WX, Hartley CJ, Michael LH, Repine JE, Hess
ML, Kukreja RC, Roberts R: Attenuation of dysfunction in
the postischemic 'stunned' myocardium by dimethylthiourea.
Circulation 1987;76:458-468
39. Beckman JS, Minor RL, White CW, Repine JE, Rosen GM,
Freeman BA: Superoxide dismutase and catalase conjugated
to polyethylene glycol increases endothelial enzyme activity
and oxidant resistance. J Biol Chem 1987;263:6884-6892
40. Brown JM, Terada LS, Grosso MA, Whitmann GJ, Velasco
SE, Patt A, Harken AH, Repine JE: Xanthine oxidase produces hydrogen peroxide which contributes to reperfusion
injury of ischemic isolated perfused rat hearts. J Clin Invest
1988;81:1297-1301
41. Tamura Y, Chi L, Driscoll EM Jr, Hoff PT, Freeman BA,
Gallagher KP, Lucchesi BR: Superoxide dismutase conjugated
to polyethylene glycol provides sustained protection against
myocardial ischemia/reperfusion injury in canine heart. Circ
Res 1988;63:944-959
42. Tanaka M, Stoler RC, FitzHarris GP, Jennings RB, Reimer
KA: Evidence against the "early protection-delayed death"
hypothesis of superoxide dismutase therapy in experimental
myocardial infarction. Circ Res 1990;67:636-644
43. Omar BA, McCord JM: The cardioprotective effect of Mnsuperoxide dismutase is lost at higher doses in the postischemic
isolated rabbit heart. Free Radic Biol Med 1990;9:473-478
KEY WORDS * ischemia/reperfusion * oxygen radicals
hydroxyl radical * reactive oxygen metabolites * hydrogen
peroxide
Reduction of canine myocardial infarct size by a diffusible reactive oxygen metabolite
scavenger. Efficacy of dimethylthiourea given at the onset of reperfusion.
F P Carrea, E J Lesnefsky, J E Repine, R H Shikes and L D Horwitz
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Circ Res. 1991;68:1652-1659
doi: 10.1161/01.RES.68.6.1652
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1991 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/68/6/1652
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/