Salvage of ischemic myocardium by ibuprofen during infarction in the conscious dog

Salvage of lschemic Myocardium by Ibuprofen During Infarction in the Conscious Dog

BODH I. JUGDUTT, MB, ChB GROVER M. HUTCHINS, MD BERNADINE H. BULKLEY, MD, FACC LEWIS C. BECKER, MD, FACC Baltimore, Maryland

From the Cardiovascular Division of the Department of Medicine and the Department of Pathology of the Johns Hopkins Medical Institutions, Baltimore, Maryland. This study was supported in part by U.S. Public Health Service Grants 1 RO 1 HL 19937-01 and P50-l-IL-17655-05 from the National Heart, Lung, and Blood institute, National Institutes of Health, Bethesda, Maryland. Manuscript received May 1, 1979; revised manuscript received December 18, 1979, accepted December 19, 1979. Address for reprints: Bodh Jugdutt, MD, 6-l 19 Clinical Sciences Building, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2G3, Canada.

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Because nonsteroidal anti-inflammatory drugs dtffer in potency and degree of prostaglandin inhibition, they may have different effects on ischemic myocardium. The effect of ibuprofen, an agent of this type, on myocardial infarct size was measured 2 days after occlusion of the left circumflex coronary artery in conscious dogs. Treatment was randomized in dogs after occlusion: Intravenous infusions of ibuprofen (6.25 mg/kg per hour) were administered to 13 dogs and saline solution (0.9 percent) to 13 control dogs over a period of 6 hours. The boundary of the occluded coronary bed, or anatomic risk region, was defined by postmortem coronary arteriography. Masses of infarct and occluded bed were measured by planimetry of wetghed transverse sections of the left ventricle. Ibuprofen decreased infarct size compared wtth that in control dogs, both as percent of the left ventricle (mean f standard error of the mean 7.5 f 1.4 versus 15.2 f 3.1, p <0.05) and as percent of the occluded bed (16.3 f 2.3 versus 36.6 f 5.7, p
Certain similarities between acute myocardial infarction and acute inflammatory reactions have led to the notion that steroidal and nonsteroidal anti-inflammatory agents might protect ischemic myocardium.iJ Although the nonsteroidal anti-inflammatory drugs appear to act by inhibition of prostaglandin biosynthesis, they differ in anti-inflammatory potency and degree of prostaglandin inhibition.3-5 These drugs might therefore have different effects on ischemic myocardium. Ibuprofen is a nonsteroidal anti-inflammatory drug but a weak prostaglandin inhibitor in the doses used to treat rheumatoid arthritis in human subjects.3-6 Doses in this range (12.5 mg/kg body weight) have been reported to decrease myocardial ischemic injury in the cat2 and to reduce myocardial infarct size in the rat1 and dog.s In contrast, indomethacin, another nonsteroidal anti-inflammatory drug with more potent prostaglandin inhibition2 had no effect on myocardial ischemic injury in the cat2 and increased myocardial ischemic injury in the anesthetized dog.g In our laboratory, indomethacin in doses (10 mg/kg) similar to those used by the previous investigators2yg was found to in-

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MYOCARDIAL SALVAGE BY IBUPF~OFEN-J~_~CDUJ-~ ET AL.

crease infarct size relative to the size of the occluded bed done to in conscious dogs. lo This study was therefore verify whether ibuprofen might reduce infarct size in the same conscious animal model.

Methods Instrumentation: Thirty-three mongrel dogs weighing 18 to 22 kg were placed under general anesthesia and fitted with instruments through a left lateral thoracotomy. An occluder snare was placed around the left circumflex coronary artery, just past the first large marginal branch. The snare consisted of a no. 1 silk thread, of which one end was secured to a plastic tube near the coronary artery and the other end looped around the artery and exited through the plastic tube, which was filled with bacitracin ointment. Plastic catheters were placed in the external jugular vein, common carotid artery and left atrium. The distal ends of the catheters and the snare were exteriorized at the back of the neck through a subcutaneous tunnel. Penicillin (1 million units) and streptomycin (1 g) were given intramuscularly after surgery and the catheters were filled with 1,000 units of heparin in 0.9 percent saline solution. The operative mortality rate was 9 percent (three dogs). Experiments: Ten days later, experiments were done on 30 surviving dogs while they were conscious and standing in a sling for support. Morphine (0.25 mg/kg) was administered intravenously for sedation and analgesia and free flow was established in the catheters. Lead II of the electrocardiogram and left atria1 and aortic pressures (using Statham P23Db transducers) were recorded continuously on a pen recorder. Myocardial blood flow was measured by standard techniques’l using radioactive microspheres, 7 to 9 pm in diameter, with Tween-80@ added and labeled with iodine-125, cerium-141, strontium-85, niobium-95 or scandium-46 (3M Company, Minneapolis, Minnesota). Five minutes before each injection, the microspheres were sonicated for 3 to 5 minutes. About 4 million microspheres were injected into the left atrium and flushed with 5 ml of saline solution over 10 seconds. Reference arterial blood samples were withdrawn at a constant rate of 2.17 ml/min on a calibrated Harvard pump, starting 30 seconds before injection of microspheres and continuing for 2 minutes thereafter. Myocardial blood flow was measured 60 minutes after morphine was given. The dogs were then premeditated with intravenous lidocaine (1 mg/kg). Five minutes later, the snare was pulled to occlude permanently the left circumflex coronary artery, a clamp was tightly applied on the plastic tube and the free end of the silk thread

was firmly secured around the clamp. A second microsphere flow measurement was made 20 seconds after occlusion. The 30 dogs were then randomly allocated into two treatment groups: Fourteen were treated with ibuprofen and 16 (control group) with saline solution. The total dose of ibuprofen was based on that used by other investigators,7*8 (that is 37.5 mg/kg), and was given in a volume of 150 ml of 0.9 percent saline solution over 6 hours, at a constant rate of 6.25 mg/kg per hour using a Harvard pump. The control group received 150 ml of 0.9 percent saline solution alone over 6 hours. Infusions were begun about 3 minutes after occlusion and continued for 6 hours. Further microsphere flow measurements were made at 15 minutes, 1 hour and 6 hours after occlusion. Phasic and mean arterial and left atria1 pressures, as well as the six limb lead electrocardiogram, were recorded before and after flow measurements. No attempt was made to suppress arrhythmias after occlusion. Two dogs died of ventricular fibrillation within 5 minutes after occlusion (one receiving ibuprofen, one control dog receiving saline solution); two others (both in the control group) died of ventricular fibrillation over the next 5 hours. The 26 surviving dogs (13 receiving ibuprofen, 13 control dogs receiving saline solution) were brought back 2 days later for electrocardiographic and hemodynamic recordings in the fully conscious state. The dogs were then given a lethal dose of anesthetic agent; the hearts were removed, washed free of blood and weighed. Measurement of the occluded bed size and infarct size: Postmortem coronary arteriography10J2J3 was performed to visualize both occluded and unoccluded coronary beds by simultaneous injections of a barium sulfate-gelatin mass, under a controlled pressure of 160 mm Hg, by way of cannulas placed at the origins of the right, left anterior descending and left circumflex coronary arteries. The volume of the injections totaled about 5 ml and resulted in a mean weight gain of 3.5 g (range 2 to 5). The viscosity of the injectate was such that there was no penetration beyond the precapillary level. The hearts were packed with gauze to maintain diastolic relations, fixed in 20 percent formalin solution and radiographed stereoscopically (Fig. 1A). Completeness of the occlusion was confirmed by finding an abrupt interruption in the arteriogram of the left circumflex vessel, with nonfilling of a short segment. However, the bed distal to the occlusion was visualized in the radiographs as a result of filling through collateral channels. The hearts were cut into five transverse sections (1 to 1.5 cm), beginning at the level of the occlusion, from the base to

the apex pig. 2). Paired stereoscopic radiographs

of all the

FIGURE 1. A, representative example of a postmcrtem coronary arteriogram of ths whole heart. The site of the occlusion of the left circumflex coronary artery is indicated by the arrowhead. 6, radiograph of a transverse section of the heart with the outlines of the anatomic risk region or occluded bed (dotted liner). The boundary lines were determined by following the course of each vessel in the whole heart radiograph from ring to ring and identifying the origin of the small intramural arteries. The location of this section on the whole heart is identified by the full lines in panel A. There was no infarction in this heart. Transmural radfopaque markers were removed for clarity.

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SNARE

R,SK REGION

SEPTUM

FIGURE 2. Technique of sectioning the whole heart and sampling for myocardial blood flow from a transverse sectiin. Samples were taken within the risk region or occluded bed around the posterior papillary muscle, from the center (C) and margins (M) of the infarct (hakhad) and its normal looking borders (B) (rtlppled). The nonischemk region was sampled around the anterior papillary muscle. LAD = left anterior descending coronary artery: LC = left circumflex coronary artery.

sections were made and coded. The anatomic boundaries of the occluded coronary arterial bed (anatomic “risk region”) were marked on the coded radiographs of transverse sections (Fig. 1B) by two independent observers using a stereoscopic viewer. The major coronary arterial branches were identified in whole heart radiographs as originating from normal vessels or from the circumflex artery distal to the occlusion. The epicardial and transmural course of each branch was then followed from section to section, and markings were made at the “watershed” between the interdigitating terminal ramifications of the occluded and unoccluded arteries on each transverse section. The process of identifying the origins of terminal branches in transverse sections was aided by reference to the whole heart radiographs (Fig. 1). In all hearts, the occluded bed filled through collateral channels from the nonoccluded vessels. Vessels at the lateral regions of the occluded bed were well opacified, allowing good definition of the occluded bed boundary, although there was a variable zone in the center of the occluded bed around the posterior papillary muscle, which did not opacify fully when infarcts were large. Because of the depth of the sections (average 1 to 2 cm) each marked boundary represented an average through the thickness of the section. The extent of overlap of vessels in the occluded bed and adjacent nonoccluded bed across the marked boundaries was assessed to 1 to 2 mm in the three basal sections and as 2 to 4 mm in the last two apical sections. Good interobserver reproducibility was found in marking the boundaries of the occluded bed on paired coded radiographs; markings by two observers differed by an average of 1.02 f 2.1 mm (standard deviation), which represent 2 percent of the width of the occluded region at the base and 5 percent at the apex (n = 150 observations).

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The left ventricular sections were freed of the right heart chambers, atria, connective and fatty tissue and large epicardial vessels and were weighed. Outlines of the top and bottom surfaces of each left ventricular ring and their infarcts, identified by gross inspection, were traced on a transparent plastic overlay. The tracings of these rings were then superimposed on the marked radiographs and aligned using pairs of radiopaque metallic markers inserted through the walls of the rings as well as natural markers (papillary muscles, cavity and wall contours). The markings of the boundaries of the occluded bed were copied and transferred to the left ventricular rings to facilitate sampling for blood flow. Areas of the left ventricular rings, risk regions and infarcts were measured with electronically performed planimetry. Masses of the infarcts and occluded beds were then computed by relating the average areas (top and bottom surfaces of each left ventricular section) to the weights of the rings. In 10 dogs, areas of infarct outlines made by two independent observers differed by an average of 2 percent (n = 45 observations), indicating good interobserver reproducibility. Measurement of regional myocardial blood flow: The arteriographically defined occluded bed, or anatomic risk region, contained clearly visible infarcts centered around the posterior papillary muscle with visibly normal tissue toward the periphery.‘c*ls Sampling for regional myocardial blood flow was made in relation to the boundaries of the infarct and the occluded bed (Fig. 2). From each left ventricular ring, transmural samples (0.5 to 3 g) were taken serially throughout the occluded bed, around the posterior papillary muscle, so as to include: (1) the center of the infarct and the margins of the infarct, at least 2 mm within the endocardial edge of the infarct; and (2) the adjoining normal-looking border tissue, located 6 to 24 millimeters within the lateral boundary of the occluded bed and lateral to the infarct edge. Transmural samples (2 to 3 g) were also taken from the center of the nonoccluded left anterior descending bed, around the anterior papillary muscle. All samples were divided into epicardial and endocardial halves. The upper surfaces of samples within the occluded bed from the mid left ventricular ring of each heart were marked with bromochrome for histologic examination. All samples were placed in plastic vials containing 5 ml of 10 percent formalin solution and counted for radioactivity together with the reference blood samples in a well-type gamma scintillation counter (Packard model 5986) at five energy windows adjusted to the peak emission of the five nuclides. Regional myocardial blood flow was calculated from the formula: F = R X (Cm/Q) (ml/min per g), where F = flow in myocardial tissue sample (ml/min per g), R = withdrawal rate of reference arterial blood (ml/min), Cm = corrected counts per gram in myocardial tissue sample, and Cr = total counts in reference blood samples. Samples from corresponding regions in the left ventricular rings were pooled for each heart to calculate weighted flows in the infarct center (c), infarct margin (m), visually normal border (b) and nonischemic regions. Coronary vascular resistance in each region was calculated by dividing mean flow by mean arterial pressure. Histology: Tissue samples from the infarcted regions of the middle left ventricular rings were then embedded in paraffin and histologic sections were taken in the plane of the planimetered surfaces and stained with hematoxylin-eosin. Microscopic assessment of percent total necrosis per sample was made. Statistical analysis: Paired and unpaired Students’ t tests were used to calculate the significance of differences within and between groups. Linear regression analysis was done by the least square fit method, and the significance of correlation coefficient (r) values, slopes and intercepts calculated. The

MYOCARDIAL SALVAGE BY IBUPROFEN-JUGDUTT ET AL.

TABLE I Hemodynamics in Dogs Given Saline Solution or Ibuprofen

MeanArterial Heart Rate (beats/min)

Timing

Pressure

Mean Left Atrial Pressure

(mm Hg)

(mm Hg)

Thirteen Dogs Given Saline Solution Before occlusion After occlusion 20 s 15 min :::

104f4

126f3

7fl

129 f 4’ 122 f 3 116f2 117f3

122f5 124f3 119f4 116f3

11 f 11 f 10f 10f

A::

Infarct Mass Risk Region Mass (g) (9)

LV Mass

Infarct/LV

(9)

(%)

Infarct/Risk Region (%)

Thirteen Dogs Given Saline Solution 1’ 1 1 1

Thirteen Dogs Treated With Ibuprofen Before occlusion After occlusion 20 s 15 min

TABLE II Effect of Ibuprofen and Saline Solution on Infarct Size in Conscious Dogs

107 f 4

131 f 4

126 f 4’ 118f5

125 f 5 124f4

10f llfl

1’

117f5 116f5

122 121 f 3 5

10f

1

7fl

14.4 f 3.0 (O-39.2)

33.3 f 3.0 (20.4-60.0)

93.5 f 3.1 (76.0-118.7)

15.2 f 3.1 (O-41.4)

38.6 f 5.7 (O-67.5)

Thirteen Dogs Treated With Ibuprofen 7.2 f 1.4’ (O-15.1)

35.8 f 3.1 (18.3-52.8)

90.1 f 4.9 (70.2-118.5)

7.5 f 1.4’ (O-15.2)

16.3 f 2.57 (O-28.8)

p < 0.05; + p < 0.005, respectively, comparing dogs given ibuprofen or saline solution. Values are expressed as mean f standard error of the mean; values in parentheses represent range. LV = left ventricle. l

p I 0.05 versus value before occlusion (paired t test). Values are expressed as mean f standard error of the mean. l

2 by 2 chi square test was used to assess the significance of differences in event frequency between groups. The significance of sequential flows and hemodynamic measurements after occlusion was assessed by an analysis of variance with orthogonal contrast within groups, whereas groups were compared by trend analysis. Values are given as mean f standard error of the mean.

Results Mortality: Excluding the two dogs (one each from the control and treatment groups) that died from ventricular fibrillation within 5 minute of occlusion, none of the 13 remaining dogs receiving ibuprofen died before 2 days whereas 2 of the 15 dogs receiving saline solution died from ventricular fibrillation within the first 5 hours. The difference in mortality between the two groups was not statistically significant (chi square = 1.87, probability [p] <0.25). Postmortem coronary arteriography was performed in all 30 hearts and the arteriograms showed satisfactory visualization of both the unoccluded and occluded bed, through collateral channels (Fig. 1). The size of the risk regions among the early deaths were within the range of values for the two groups. Data in the 26 dogs (13 control dogs given saline solution and 13 treated with ibuprofen) who survived 2 days form the basis of this report. Hemodynamic changes: These changes were similar in the two groups (Table I). Heart rate, mean arterial pressure and mean left atria1 pressure were similar before occlusion. After coronary occlusion, heart rate and left atria1 pressure increased significantly (p
aVf were seen in seven saline treated versus three ibuprofen-treated dogs (chi square = 3.23, p
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1

50

loo-

2 t 40

80 -

;

30

60-

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Ibuprofen altered the relation of the infarct to the respective occluded beds or risk regions in each left

ventricular ring from the base to the apex of the heart (Fig. 5). In constructing these maps showing the spatial relation of infarcts to occluded beds for the two groups (Fig. 5), the two hearts of dogs with no infarction (one receiving saline solution, one receiving ibuprofen) were excluded, leaving 12 hearts with infarction in each group (Fig. 4). In both groups, the infarct and occluded region were larger in the basal and middle portions of the heart and tapered toward the apex. Also the infarct/occluded bed ratio was greater in the basal than in the apical rings in both the ibuprofen-treated dogs (19.3 f 3.4 versus 5.6 f 3.0 percent, p <0.05) and the control group (46.0 f 5.2

slope 0.99

. SALINE o Ii3UPROfEN

FIGURE 3. Ibuprofen (IBU, hatched bars) reduced infarct size compared with that in control dogs (C, white bars) in grams (probability [p] cO.05) (left) and as percent of the left ventricle (p <0.05) or percent of the risk region or occluded bed (p <0.005) (right). The masses of the risk region and left ventricle did not differ significantly in the two groups. Normalization of infarct size relative to the risk region (rlgM panel) affords more complete separation of the two groups than absolute masses (left panel). Mean values f standard error of the mean are shown.

the notion that after administration of ibuprofen larger occluded regions are required before infarction is seen.

zontal axis intercepts, we reanalyzed the data after excluding such hearts. Because the largest occluded bed size with no infarction was 22 g or 28 percent of the left ventricle (Fig. 4), we replotted linear regression after arbitrarily excluding hearts with occluded beds less than 28 percent of the left ventricle (three hearts treated with ibuprofen, one treated with saline solution); we found that with ibuprofen the new extrapolated intercept was significantly (p <0.05) displaced to the right compared with saline solution for plots of both absolute masses (21.6 g versus 13.9 g) and masses normalized to the left ventricle (25.3 versus 13.9 percent). The new slopes and r values of the linear regressions did not change significantly from previous values when small occluded regions were excluded. Although extrapolated data must be interpreted cautiously, the shift in intercept (of ibuprofen versus saline solution) to the right supports

50

p z P
LEFT VENTRICLE

1

or93

0.56

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096

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40 z E e 2 I- 30 is e z

.

p<

0.001

/

I

B 20 IBUPROFEN

% % IO

0

0

IO

20

30

40

50

60

MASS OF RISK REGION (grams)

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O

IO

20

30

40

50

RISK REGION AS PERCENT OF LEFT

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FIGURE 4. Infarct size directly related to the size of the risk region (or occluded bed). Masses are plotted in grams (left) and as percent of left ventricle (right). Ibuprofen alters the slope of the relation seen in control dogs, so that there is less infarct for similar risk regions with ibuprofen. There is no infarct for risk regions less than about 20 g or 20 percent of the left ventricle in control dogs. With ibuprofen, the risk regions are all greater than 20 percent of the left ventricle (rlght panel). Although inspection of the plot on the right suggests that the extrapolated intercept might be displaced to the right for ibuprofen compared with the intercept in control dogs, no significant statistical difference was found between these intercepts (25.8 versus 20.3, p
MYOCARDIAL SALVAGE BY IBUPROFEN-JUGDUTT ET AL.

FIGURE5. The spatial geometry of the infarct within the risk region (or occluded bed) from base (1) to apex (5) of the left ventricle is similar for the group receiving saline solution (top)andthe group receiving ibuprofen (bottom). Infarct mass as percent of risk region is indicated for each left ventricular ring by numbers within the infarcts (atippled area). In both groups, risk regions taper to the apex and there is more infarct at the base than at the apex. Ibuprofen increased the area of rnyocardlal salvage within the occluderj bed from base to apex of the left ventricle. These maps of infarcts within the risk regions were accurately reconstructed for the two groups from measurements made between the points, shown here, on each corresponding left ventricular ring from every heart. Thus, each point represents average data from all hearts with infarcts within each group.

SALINE I (Basei

i Cm‘cmI

CONTROLS

(N=12) 4

3

2

5 (Apex)

A*-

.-w*

i_

IM

IBUPROFEN

versus 23.0 f 8.2 percent, p CO.05). Ibuprofen decreased (p <0.05) the absolute mass of the infarct in all rings of the left ventricle versus those of control dogs (that is base, 2.5 f 0.5 versus 5.0 f 0.5 g; apex, 0.1 f 0.1 versus 0.9 f 0.4 g). Ibuprofen also decreased (p <0.05) the infarct/occluded bed ratio in all left ventricular rings compared with that of control dogs (Fig. 5). When the reconstructed spatial maps of the infarcts within the occluded beds for the two groups were compared, it was apparent that ibuprofen salvaged myocardium in both lateral and subepicardial directions and did so uniformly from basal to apical regions of the occluded bed. Regional myocardial blood flow (Table III): The changes in regional myocardial blood flow were similar in the 10 control and 9 ibuprofen-treated dogs. Blood flow measurements from 2 of the original 13 dogs given saline solution and 1 of the original 13 dogs treated with ibuprofen were excluded because of arrhythmias during flow measurements; also, blood flows from another four dogs (one receiving saline solution, three receiving

(N= 12)

ibuprofen) were analyzed separately because these dogs had very small or no infarcts. In all dogs with infarcts, myocardial blood flow decreased significantly (p
TABLE Ill Regional Myocardial Blood Flows in Dogs Glven Saline Solutlon or Ibuprofen Regional Myocardial Blood Flow (ml/min per g) Infarct Margin

Infarct Center Timing

inner

Outer

-

Inner

Border Region

Outer

Inner

Outer

Nonischemic Region inner

Outer

Ten Dogs Given Saline Solutior Before occlusion After occlusion 20 s 15 min t%

0.95 f 0.05

Before occlusion After occlusion 20 ?I 15 min lh 6h

0.96 f 0.08

0.91 f 0.08

0.94 f 0.10

0.05 f 0.01*+ 0.09 f 0.02

0.10 0.18 0.14 0.19

0.14 0.23 0.21 0.19

0.83 f 0.09

0.03 f O.Ol*+ 0.10 f 0.02*+ 0.06 f 0.01 0.15 f 0.02 0.06 f 0.01 0.16 0.15 f 0.02

0.90 f 0.05

0.86 f 0.07

0.99 f 0.11

0.91 f 0.10

1.03 f 0.09

1.02 f 0.11

0.12 f 0.03’+ 0.16 f 0.03 0.18 0.16 f 0.04 0.03

0.20 f 0.04*+ 0.24 f 0.03 0.26 0.24 f 0.05 0.04

0.47 f 0.05’+ 0.56 f 0.06 0.61 0.56 f 0.07 0.06

0.73 f 0.12’+ 0.82 f 0.11 0.79 0.78 f 0.08 0.10

0.98 f 0.07 1.16 f 0.13 0.93 0.98 f 0.10 0.07

1.04 f 0.09 1.10 f 0.11 0.88 0.97 f 0.08 0.06

0.89 f 0.08

1.07 f 0.05

0.93 f 0.05

0.59 f 0.06

1.11 1.27 1.07 0.84

1.00 1.14 1.02 0.83

Nine Dogs Treated With Ibuprofen

f f f f

0.02*+ 0.05 0.04 0.06

f f f f

0.93 f 0.10

0.96 f 0.07

0.04++ 002; 2 o”.o”fI= + 0.05 0.06 0137 f 0107 0.03 0.31 f 0.06 0.41 f 0.05

f f f f

0.11 0.16 0.13 0.11

f f f f

0.09 0.13 0.13 0.12

p
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assessed by an analysis of variance with orthogonal breakdown. A trend analysis did not reveal a significant difference in changes in sequential blood flows after occlusion in the two groups. Flows in the border region in the group receiving ibuprofen were less (p <0.05) than those in the group receiving saline solution; this difference may be a result of myocardial salvage in the group receiving ibuprofen, causing normal border tissue in this group to be sampled closer to the center of the occluded bed and further away from its lateral boundaries than border tissue samples in the control group. Blood flow in the nonoccluded bed did not change significantly over the 6 hours after occlusion in either group. In the four dogs (one receiving saline solution, three receiving ibuprofen) with very small (less than 2 g or 2 percent of the left ventricle) or no infarcts (Fig. 41, the size of the occluded bed ranged from 26 to 28 percent of left ventricular mass. In the one dog treated with saline solution with no infarct, blood flows in the center of the occluded bed before occlusion and at 20 seconds, 15 minutes, 1 hour and 6 hours after occlusion were 1.67, 0.97,0.92, 1.37 and 0.74 ml/min per g, respectively. In the three dogs treated with ibuprofen (one with no infarct, two with infarcts of 1 and 1.5 percent of the left ventricle, respectively), the flows in the center of the occluded bed at the corresponding times were 1.09 f 0.08,0.54 f 0.06,0.76 f 0.02,0.86 f 0.08 and 0.64 f 0.07 ml/min per g, respectively. Histology: The border samples in both groups were visually normal and no necrosis was found in any of the 40 hearts examined histologically. The inner halves of both the infarct center and the infarct margin regions had 100 percent histologic necrosis. The outer halves of these two regions had 55 and 20 percent histologic necrosis, respectively.

Discussion Infarct size after ibuprofen: The major finding in our study was that ibuprofen reduced infarct size after permanent coronary occlusion in the conscious dog. The volume of noninfarcted myocardium within the OCeluded bed was increased, and salvage was found in both epicardial and lateral regions of the occluded bed from the base to the apex of the left ventricle. The demonstration in the same model that prostaglandin inhibition with large doses of indomethacin (10 mg/kg) caused infarct extension in lateral and subepicardial regions of the occluded bed,10 provides evidence in favor of a functional border zone. In our dog model, we used postmortem coronary arteriography to define the size of the occluded bed or the anatomic risk region. lo,13 The analysis of infarct size as a function of the region at risk has been used by other investigators.l* The distribution of necrosis within the risk region corresponded to a gradient of collateral flow from peripheral to more central regions. Although the spared lateral borders were well within the boundaries of the occluded bed defined arteriographically, the possibility that inclusion of some normal tissue from the

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unoccluded beds might explain in part the larger blood flows in the border samples cannot be excluded.15 Validity of microsphere flow measurements: Recently, the validity of microsphere flow measurements made in regions of infarcted myocardium has been questioned.16-18 Edema in the infarct might produce an apparent loss of microspheres, which would be expected to reduce artificially absolute values of flow. In addition, infarcted myocardium might lead to true physical loss of microspheres, resulting in further errors. However, we managed the treatment and control groups similarly and measured flows over a limited 6 hour period after occlusion, 42 to 48 hours before death. It is likely that microspheres injected over this short a period of time would be lost to a similar extent, making comparison of relative differences in flow over that interval between groups still valid. Furthermore,the changes in flow were directionally similar in necrotic and nonnecrotic regions of the occluded bed, where no sphere loss is expected, for both the dogs treated with saline solution and those treated with ibuprofen. Mechanism of ibuprofen’s myocardial protective effect: Our finding of myocardial salvage with ibuprofen supports observations made by other investigators using different animal models,2g7*8 although the mechanism responsible for the protective effect of ibuprofen is unknown. The decrease in infarct size is probably not explained by effects on myocardial oxygen demands because changes in heart rate, arterial pressure and left atria1 pressure were similar in the treated and control groups. Although one might have expected a decrease in collateral blood flow from prostaglandin inhibition,g the changes in collateral flow were similar in both groups. Thus, protection by ibuprofen may have been due to cellular effects. The notion that nonsteroidal anti-inflammatory drugs might be beneficial in myocardial infarction is based on their ability to inhibit certain similar reactions that occur in acute infarction and inflammatory reactions. First, leukocytes and other inflammatory cells are known to become trapped in ischemic regions.lg Normally, the release from these cells of lysosomal enzymes such as hydrolases 2~~3 and proteases2* assists in their However, these phagocytic and “scavenger ” functions. enzymes might also be harmful to nonnecrotic cells in the occluded coronary bed. Thus, nonsteroidal antiinflammatory drugs, including ibuprofen, by inhibiting the migration of inflammatory cells25 and the release of harmful lysosomal enzymes,20-23 might protect myocardial tissue in the occluded bed. Second, platelets also become trapped in ischemic regions.lg Such platelet trapping, especially at the margins of infarcts, has been suggested as a reason for evolving necrosis in the marginal zone and has been shown to be reduced by administration of aspirin.26 Ibuprofen may act like aspirin and reduce platelet trapping in marginal zones. This effect may be due to the inhibition of platelet aggregation,6T27,28 presumably through inhibition of thromboxane formation.2g~Z10 Thromboxane AZ is a potent platelet aggregator and vasoconstrictor.2g When platelets aggregate, they se-

MYOCARDIAL

Crete certain granular constituents including coagulation factors, vasoconstrictors and other substances that promote further aggregation and thrombus formation.27,28 Platelet clumps may thus interfere with collateral development and function within the occluded bed, and initiate a vicious cycle of plug formation, vasoconstriction, thrombosis and more necrosis, especially in marginal regions. Third, prostaglandins are known to be released during myocardial ischemias1*32 and nonsteroidal antiinflammatory drugs inhibit their biosynthesis.3-6 However, these drugs differ in their degrees of inhibition of the various prostaglandins.4s5 Although some vasoactive prostaglandins would be expected to be beneficial in the acute ischemic setting, others appear to be detrimental.3” Although ibuprofen may have inhibited the synthesis of vasodilator and vasoconstrictor prostaglandins by different degrees, we detected neither a favorable nor a harmful effect on collateral flow. Ibuprofen may have affected the synthesis of other vasoactive substances, such as prostacyclin,34*35 kinins, angiotensin and thromboxanes2g*30 so that, on balance, flow was maintained. To complicate matters, there are problems related to dose and effect. Thus, ibuprofen in small doses, within the range used in the therapy of arthritis in human subjects, is a weak inhibitor of prostaglandin synthesis34 and has been reported to suppress lysozyme

SALVAGE

BY IBUPROFEN-JUGOUTT

ET AL.

release and stabilize lysosomal membraneG and (information from Upjohn Co.) to prevent platelet aggregation by inhibiting thromboxane synthesis27*28 and to inhibit leukocyte migration.25 However, larger doses of ibuprofen have been reported to produce lysis of lysosomes in vitro.20 The wide variety of actions of nonsteroidal anti-inflammatory drugs may explain the differences in their ability to modify the cellular events of acute myocardial infarction. Further studies are therefore needed before the animal data can be extrapolated to man. Implications: We have shown that ibuprofen given after coronary occlusion in the conscious dog decreases infarct size. This effect is seen in both lateral and subepicardial regions of the occluded bed, and is uniform from the base to the apex of the left ventricle. Neither changes in myocardial oxygen demands nor changes in collateral flow appear to explain this effect. These results suggest that protection of ischemic myocardium by ibuprofen is related to cellular effects associated with its anti-inflammatory and prostaglandin-inhibiting properties. Acknowledgment We are indebted to Patricia Shaw, Anthony DiPaula and Alexander Wright for technical assistance, to Joanna Walton for typing the manuscript and to Robert Rurow for assistance with biostatistics.

References 1. Ubby P, Maroko PR, Bfoor CM, Sobel BE, Braunwakf E. Reduction of experimental myocardial infarct size by corticosteroid administration. J Clin Invest 1973;52:599-607. 2. Gglelree ML, Lefer AM. influence of non-steroidal anti-inflammatory agents on myocardial ischemia in the cat. J Pharmacol Exp Ther 1976;197:562-93. 3. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature [New Biol] 1971;231:2325. 4. Flower R, Gryglewski R, Herbaczynska-Cedro K. Vane JR. Effects of anti-inflammatory drugs on prostaglandin biosynthesis. Nature [New Biol] 1972;238: 104-6. 5. Ferrelra SH, Vane JR. New aspects of the mode of action of nonsteroid anti-inflammatory drugs. Annu Rev Pharmacol 1974; l4:57-73. 6. Davies EF. Avery GS. Ibuprofen: a review of its pharmacological properties and therapeutic efficacy in rheumatic disorders. Drugs 1971;2:416-46. 7. Maclean D, Flshbein MC, Blum RI. Braunwald E, Maroko PR. Long-term preservation of ischemic myocardium by ibuprofen after experimental coronary artery occlusion (abstr). Am J Cardiol t978;41:394. 6. Rlbelro LGT, Yasuda 1, Lowensteln E, Braunwald E, Yaroko PR. Comparative effects on anatomic infarct size qf verapamil, ibuorofen. and morphine-promethazine-chlorpromazine combination [abstr): Am J Cardiol i979;43:396. 9 Kirmser R. Beraer JH. Cohen LS. Wolfson S. Effect of indomethacin, a ;rosGglandin inhibitor, on epicardial ST elevation and myocardial blood flow after coronary occlusion (abstr). Circulation 1976;54:Suppl ll:ll-194. 10. Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC. Effect of indomethacin on collateral blood flow and infarct size in the conscious dog. Circulation 1979;59:734-43. 11. Rudolph AM, Heymann MA. The circulation of the fetus in utero. Methods for studying distribution of blood flow, cardiac output and

organ blood flow. Circ Res 1967;21:163-64. 12. Schaper W. The Collateral Circulation of the Heart. Amsterdam: North Holland, 1971: 5-16, 164-77. 13. Jugdult 81, Hutchins GM, Bulkley BH, Becker LC. Relation of collateral blood flow and regional risk to infarct size in the conscious dog. Two dimensional maps of infarcts, regions at risk, and collateral blood flow. Circulation 1979;60:1141-50. 14. Lowe JE, Relmer KA, Jennings RB. Experimental infarct size as a function of the amount of myocardium at risk. Am J Pathol 1976;90:363-76. 15. Hlrzel HO, Sonnenbllck EH. Klrk ES. Absence of a lateral border zone of intermediate creatine phosphokinase depletion surrounding a central infarct 24 hours after acute coronary occlusion in the dog. Circ Res 1977;41:673-63. 16. Whlte FC, Sanders M, Bloor CM. Regional redistribution of myocardial blood flow after coronary occlusion and reperfusion in the conscious dog. Am J Cardiol 1976;42:234-43. 17. Jugdutt BI, Hutchins GM, Bulkley BH, Becker LC. The loss of radioactive microspheres from necrotic myocardium. Circ Res 1979;45:746-56. 16. Capuno NL, Gokfsteln RE, Aamodt R. Smith HJ, Epstein SE. Loss of microspheres from ischemic canine cardiac tissue. An important technical limitation. Circ Res 1979;44:223-7. 19. Sommers HM, Jennings RB. Experimental acute myocardial infarction: histologic and histochemical studies of early myocardial infarcts induced by temporary or permanent occlusion of a coronary artery. Lab Invest 1964;13:1491-503. 20. Lewis DA. The actions of some non-steroidal drugs on lysosomes. J Pharm Pharmacol 1970;22:909-12. 21. Nakanlshl M, Goto K. Inhibitory effects of anti-inflammatory drugs on enzyme release from rabbit polymorphonuclear leukocyte lysosoties. Biochem Pharmacol 1975;24:421-4. 22. Smlth RJ, Sabln C, Gllchrest H, Wllllams S. Effect of anti-inflammatory drugs on lysosomes and lysosomal enzymes from rat liver. Biochem Pharmacol 1976;25:2171-7.

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23. Weiaamann G, Hoffstein S, Kaplan H, Gennaro D, Hirsch J, Fox AC. Early lysosomal disruption in myocardial infarction and protection by methylprednisolone (abstr). Clin Res 1975;23:383A. 24. Lefer AM, Spath JA Jr. Preservation of myocardial integrity by a protease inhibitor during acute myocardial ischemia. Arch Int Pharmacodyn Ther 1974;211:225-6. 25. Brown KA, Collins AJ. Action of non-steroidal, anti-inflammatory drugs on human and rat peripheral leukocyte migration in vitro. Ann Rheum Dis 1977;36:239-43. 26. Ruf W, Lelnberger H, McNamara JJ. Influence of aspirin on platelet trapping in myocardial infarct in baboons (abstr). Am J Cardiol 1978;41:407. 27. Packham MA, Mustard JF. Clinical pharmacology of platelets. Blood 1977;50:555-73. 28. Majerus PW. “Why aspirin?” Circulation 197654:357-g. 29. ElIIs EF, Oswald 0, Roberts LJ II, et al. Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane As. Science 1976;193:1135-7. 30. Needleman P, Kulkarnl PS, Ras A. Coronary tone modulation:

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31.

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formation and actions of prostaglandins, endoperoxides, and thromboxanes. Science 1977:195:409-12. Block AJ, Felnberg H, Herbaczynaka-Cedro K, Vane JR. Anoxia-induced release of prostaglandins in rabbit isolated heart. Circ Res 1975;36:34-42. Ogletree ML, Flynn JT, Feola M, Lefer AM. Early prostaglandin release from the ischemic myocardium in the dog. Surg Gynecol Obstet 1977;144:734-40. Ogletree ML, Lefer AM. Prostaglandin-induced preservation of the ischemic myocardium. Circ Res 1978;42:218-24. Fltzpafrkk TM, Alter A, Corey EJ, Ramwell PW, Rose JC, Kot PA. Cardiovascular responses to PGls (prostacyclin) in the dog. Circ Res 1978;42:192-4. Needleman P, Kaley G. Cardiac and coronary prostaglandin synthesis and function. N Engl J Med 1978;298:1122-8. Needleman P, Key SL, Denny SE, lsakson PC, Marshall GR. Mechanism and modification of bradykinin-induced coronary vasodilation. Proc Nat Acad Sci USA 1975;72:2060-3.