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High- and low-dose superoxide dismutase plus catalase does not reduce myocardial infarct size in a subhuman primate model
High-
and
l;o;w-&se
slu,peroai&
dismiutase
plus ca.ta.1as.e cioes n’ot reduce myocardial infarct size in a subhuman prim,ate model Oxygen free radical scavengers have been found to decrease infarct size in dogs subjected to myotardial ischemia-reperfusion injury. A baboon open-chest model was used to determine if superoxide dismutase (SOD), an ,oxygen free ridical scavenger, together with cataiase would be equally effective in subhuman primates (baboons). The left anterior descending coronary artery (LAD) was ligated for 2 hours. Before reperfusion, the animals received the following: Group 1 (low-dose SODlcatalase; n = 5) received 15,000 W/kg of SOD and SF,000 IWkg of catalase IV over 1 hour, 15 minutes before reperfusion. Group 2 (high-dose human SOD [h-SOD]/catalase; n = 5) received an intraatrial bolus of 400,000 IU of recombinant h-SOD and 27,500 Ill/kg of catalase over 30 seconds, followed by 300,000 IU of h-SOD and 55,000 Ill/kg of catalase over 1 hour, beginning 15 seconds befobe reperfusipn. Group 3 (n = 8) were control animals. Baboons were put to death 22 hours after reperfusion. Their hearts were excised and sectioned after the perfusion bed distal to the site of ligation was delineated with micrdvascular dye. The infarct zone was determined histologically. Areas of the perfusion bed and infarct zone were measured by planimetry. Infarct size did not differ significantly, between, the three groups: control, 88 -t 7%; low-dose SODlcatalase, 88 + 5%; and highhdose h-SODlcatqlase, 74 + 4%. In this model, highand low-dose SOD with catalase did not result in any significant reduction in infarct size. (AM HEART J 1993;126:840-846.)
Brian I. Watanabe, MD, Shyamal Premaratne, MD, PhD, Whitney Limm, MD, Mark M. Mugiishi, MD, and J. Judson McNamara, MD Honolulu, Hawaii
The production of free radicals has been implicated in myocardial ischemia-reperfusion injury.l? 2 Numerous investigators have used oxygen free radical scavengers in their attempts to reduce the possible free radical-mediated myocardial injury in the ischemia-reperfusion setting. 3-5 Superoxide dismutase (SOD) is one of the most widely studied free radical scavengers. It is able to catalyze the dismutation of the superoxide radical, producing hydrogen peroxide and oxygen. SOD alone or in combination with catalase has produced conflicting results with respect to the limitation of infarct size and other evidences of myocardial protection. Some studies4-6 have shown From the Cardiovascular Queens Medical Center, Hawaii.
Research Laboratory, John A. Burns School
This study was supported by Grant stitutes of Health, Bethesda, Md., ican Heart Association. Received Reprint versity
for publication
of Surgery, University of
No. HL-34647-03 from the National Inand by a Grant-in-Aid from the Amer-
Jan. 4, 1993; accepted
March
24, 1993.
requests: J. Judson McNamara, MD, Department of Surgery, of Hawaii, 1356 Lusitana St., Honolulu, HI 96813.
Copyright @ 1993 by Mosby-Year Book, 0002-8703/93/$1.00 + .lO 4/l/48380
840
Department of Medicine,
Inc.
Uni-
beneficial effects with SOD intervention while others7-g have shown no protection with it. These conflicting findings may in part be the result of differences in the species of animals used and in the experimental protocols employed. The purpose of this study was to determine if high-dose human recombinant SOD plus catalase (high-dose SOD/cat) and low-dose SOD plus catalase (low-dose SOD/cat) given just before reperfusion would reduce myocardial infarct size in a subhuman primate (baboon) model of ischemia-reperfusion injury. Catalase was given with SOD to degrade any hydrogen peroxide that might have been produced from the dismutation of the superoxide radical. No studies on the effect of SOD plus catalase on a subhuman primate have been’reported thus far. The baboon (like the human) has little coronary collateral blood supply, making it a good model for the human condition. METHODS Experimental
preparation. All animals were cared for in accordance with the guidelines of the American Physiological Society. Eighteen baboons (Papio anubis) of either sex
Volume 126, Number 4 American Heart Journal
weighing 9.1 to 17.0 kg were fasted for 24 hours before surgery. They were assigned to control and treatment groups using a table of randomized numbers. The animals were initially sedated with intramuscular ketamine hydrochloride (10 mg/kg), Anesthesia was induced and maintained with an intravenous infusion of thiopental sodium (2 mg/ kg). The animals were intubated with a cuffed endotracheal tube and ventilated using a Harvard NSH-34RH volume ventilator (Harvard Apparatus Company, South Natick, Mass.). Supplemental oxygen was administered as needed and the ventilation was maintained to keep the oxygen saturation not less than 95 % and the PCOZat less than 45 mm Hg. The pH was maintained between 7.53 and 7.36. The bladder was catheterized with an 8F feeding tube to monitor the urine output. A nasogastric tube was inserted and placed on low wall suction. Normothermia was maintained with the use of a heating blanket. Electrocardiograms (ECGs) were monitored throughout the procedure using limb leads. A sterile cutdown was performed and SF catheters were inserted into the femoral artery and vein for pressure monitoring and fluid/drug administration, respectively. A 2.5F thermodilution probe (American Edwards Laboratories, Irvine, Calif.) was placed into the opposite femoral artery with its tip positioned just distal to the renal arteries to measure the cardiac output (CO). An injectate catheter was placed in the femoral vein and positioned at the right atrium. Measurements of CO were made using a lung water computer (American Edwards Model 9310, American Edwards Laboratories). The animals were hydrated with intravenous 5 % dextrose in lactated Ringer’s solution (D5LR). A left thoracotomy was performed in the fourth intercostal space and. the heart was suspended in a pericardial cradle. The left anterior descending coronary artery (LAD) was isolated just below the first diagonal branch and a 3-O suture snare was loosely passed around it. A 1.7 mm outer diameter polyetlhelene catheter (Intramedics PE 190, Clay Adams, Parsippany, N.J.) was inserted into the left atrial appendage for the measurement of left atria1 pressure (LAP). A 5F Mil.lar micromanometer tip catheter pressure transducer (model VPC-663A, Millar Instruments Inc., Houston, Texas) was placed through the left ventricular apex for the measurement of left ventricular pressure and left ventricular contractility (dP/dt). Two rows of six 0.11 mm diameter Teflon-coated silver wires spaced 0.5 cm apart were placed on the anterior epicardium for the direct measurement of an epicardial ECG. These epicardial electrodes were placed over the area at risk for ischemia as determined by the darkening of the epicardium during a 30-second trial ligation and release of the LAD. Baseline limb ECG, mean arterial pressure (MAP), LAP, dP/dt, and CO were recorded on a six-channel recorder (Gould Brush Model 260, Gould Incorporated, Oxnard, Calif.). Epicardial ECGs were recorded with an eight-channel recorder (Hewlett-Packard Model 7758B, Hewlett-Packard, Medical Electronics Division, Waltham, Mass.). Experimental protocol. Following baseline hemody-
Watanabe et al.
841
namic and ECG measurements, 25 mg of lidocaine hydrochloride was injected prophylactically by the intravenous route. One minute later the suture snare was tightened and the LAD was occluded for 2 hours. Epicardial ECG recordings were made every 15 minutes following occlusion of the LAD, and this was continued into the reperfusion phase. Heart rate (HR), MAP, LAP, dP/dt, CO, and a limb lead ECG were recorded every 30 minutes after occlusion, and this was continued into the reperfusion phase. Two hours after the occlusion of the LAD, all animals received a bolus of 25 mg of lidocaine hydrochloride and the snare was released and reperfusion was begun. Additional lidocaine was administered if premature ventricular contractions exceeded six per minute. The use of prophylactic lidocaine was necessary to ensure that all animals were relatively free of premature vetricular contractions (PVCs). To our knowledge no interactions between lidocaine and SOD/catalase have been reported in the literature. Fifteen minutes before reperfusion, five animals (lowdose SOD/catalase group) received intravenous SOD, 15,000 IU/kg (Sigma Chemical Co., St. Louis, MO.), and catalase, 55,000 IU/kg (Sigma Chemical Co.), premixed in a standard 50 ml saline solution and sterilized through a 0.22 pm filter. This infusion continued for a full hour, ending 45 minutes into the reperfusion phase. At 15 seconds before the release of the ligature and the onset of reperfusion, a second group of five animals (high-dose SOD/catalase group) received 400,000 IU of human recombinant SOD (Bio-Technology General Corporation, New York, N.Y.) and 27,500 IU/kg of catalase mixed in 10 ml of normal saline via the left atria1 catheter as a bolus over 30 seconds. This bolus was followed with a l-hour intravenous infusion of 300,000 IU of human recombinant SOD and 55,000 IU/kg of catalase mixed in 50 ml of normal saline. The control group of eight animals underwent the same protocol except that they did not receive any therapeutic adjunct. Instead, they received a continous intravenous infusion of D5LR at 50 ml/hr. At 6 hours postligation (4 hours of reperfusion), the left atrial and left ventricular lines as well as the epicardial electrodes were removed and the chest was closed after the restoration of negative pleural pressure. At the conclusion of the 24-hour monitoring period (22 hours of reperfusion), 3060 units of heparin was administered as an intravenous bolus and the animal was put to death using a saturated solution of potassium chloride. Perfusion bed and infarct size evaluation. Once the hearts were excised, a 0.965 mm outer diameter polyethylene catheter (Intramedics PE 50, Clay Adams) was passed into the LAD and, advanced to the level of previous ligation. The catheter was secured and a colored silicon rubber microvascular dye (Microfil, Canton Bio-Medical Products Incorporated, Boulder, Colo.) was injected into the LAD to delineate the perfusion bed at risk for infarction. Welo have previously shown that this dye injection technique allows accurate delineation of the perfusion bed subserved by an occluded artery. The hearts were then submerged in a 10 % formalin fixative for 3 days and were then sliced parallel to the minor
842
Watemabe et al.
Table
I. Hemodynamic
American
values: Controls
Baseline
30 minutes
and
30 minutes
dP/dt Hglsec)
108 f 6 109 f 9 115 f 6
4.6 t 0.5 4.2 k 0.4 4.4 * 0.7
1.1 + 0.2 1.2 f 0.2 1.6 f 0.3
1994 rf 181 2481 I 300 1703 i 315
127 c 12 116 i 13 119 i 4
113 t 7 108 f 6 113 + 7
8.1 t 1.8 9.3 * 1.7 5.0 k 0.5
1.2 + 0.2 1.3 f 0.2 1.4 k 0.2
2045 2490 1999
f 164 f 482 + 392
127 f
116 rl- 11 121 of- 08
109 i- 6 106 k 7 109 c 11
6.6 i 2.4 8.1 t 1.2 6.3 f 0.8
1.1 i- 0.2 1.1 * 0.2 1.1 * 0.2
1799 2538 1674
+- 206 f 468 f 301
126 + 11 108 t 12 131 I 12
111 * 7 106 f 8 103 * 9
7.9 f 1.9 13.4 I! 2.7 6.6 -c 0.7
1.2 * 0.2 1.2 + 0.2 1.0 z!z0.2
1786 2100 1832
i 162 zk 243 t 416
130 i- 12 122 + 17 143 * 11*
109 f 6 101 * 7 100 * 10
7.2 + 2.1 11.3 zk 2.5 6.9 t 0.7*
1.2 * 0.2 1.7 It: 0.3 1.4 _t 0.3
1746 2185 1894
i 180 i 242 + 296
129 + 11 126 f 12 132 rt 11
96 + 4 101 ?I 11 103 + 8
8.1 li7 2.8 7.9 f 1.8 4.5 zk 0.6
1.2 z!z0.2 1.3 f 0.2 1.2 + 0.1
1742 2235 1829
i: 174 t 288 c 339
131 f 12 124 f 14 137 i 11
99 +- 3 101 -t- 9 98 + 11
4.2 f 2.4
1.1 t 0.2 1.3 + 0.2 1.2 f 0.2
1749 2377 1600
+ 189 +- 392 iz 326
13
postocclusion
postocclusion
Control hSOD/cat SOD/cat postocclusion
Control hSOD/cat SOD/cat 6 hours
112 t 11 113 2 4
(mm
postocclusiont
Control hSOD/cat SOD/cat
5 hours
co (Llmin)
postocclusion
Control hSOD/cat SOD/cat
3 hours
LAP (mm Hd
119 t 12
Control hSOD/cat SOD/cat
2 hours
kffw (mm Hd
preocclusion
Control hSOD/cat SOD/cat
2 hours
versus treated
HR (beatslmin)
Time
October 1993 Heart Journal
postocclusion
Control hSOD/cat SOD/cat HR, Heart
9.1 f 2.9 4.6 rf: 0.4
rate; MAP, mean arterial pressure; LAP, left atria1 pressure; CO, cardiac output; dP/dt, cat, high-dose human recombinant superoxide dismutase/catalase; SOD/cat, low-dose superoxide *Significant difference @ < 0.05) compared with corresponding baseline value. TReperfusion begun at 2 hours postocclusion.
axis into 5 mm transverse sections. These cross sections were embedded into paraffin blocks, further sectioned, and stained with hematoxylin-eosin for histologic study. Representative slides from each cross section were then examined microscopically and the infarcted and viable tissue within the perfusion bed were identified. With the use of a microscopic slide projector (M 600, Durst, Tempe, Ariz.), enlargements of the slides were made and tracings of the slides including the infarcted tissue and perfusion bed were done. Planimetry was then performed and the areas of the left ventricle, perfusion bed, and infarcted tissue were determined. By multiplying these areas by the thickness of the corresponding cross section of the tissue, the volumes of the measured areas were established. Summation of the volumes of each tissue section yielded the total volume of the left ventricle (VL,~), the total volume of the perfused bed at risk for infarction (VP,), and the total volume of infarcted tissue (VI). This method of infarct size evaluation and determination has been described in previous stud-
first time derivative dismutase/catalase.
of left ventricular
pressure;
hSODl
ies. %I2 The histologic delineation and planimetry were done by a technician who was blinded to the animal groupings. Statistical analysis. Data are expressed as the mean +: standard error of the mean (SEM). Comparisons of hemodynamic data, volume data, and infarct size were carried out with the use of a one-way analysis of variance. A p value of less than 0.05 was considered significant. RESULTS Hemodynamics.
Table I summarizes the hemodynamic data for the control and SQD/catalase-treated groups. Representative time intervals are shown beginning before the onset of ischemia and continuing into the ischemic and reperfusion phases. There were no statistically significant differences (p > 0.05) noted between the groups in HR, LAP, CO, MAP, and dP/dt. Within the high-dose SOD/
Volume 126, Number 4 American Heart Journal
catalase group, measurements of these hemodynamic variables did not show any significant difference at the various time intervals. Within the low-dose SOD/catalase group, the 3-hour LAP and HR were significantly greater than the corresponding baseline values (p < 0.05). Even with the rise in LAP within the low-dose SOD/catalase group, the LAP values still remained within normal limits with no other hemodynamic evidence of cardiovascular compromise. Thus the difference in the LAP does not appear to have significantly affected the outcome. Histologic exalmination. Microscopic examination of the stained tissue showed that the infarcted necrotic tissue produced the characteristic changes of pyknosis, karyolysis, and the loss of cross striations. Areas of infarction were contained within the perfusion bed. Histologic data shown in Table II include VLV, Vpg, and VP Several quotients of these values were calculated, with the most emphasis placed on the ratio v~/Vpn, which represents the volume of the actual infarct relative to the area at risk. When the volume of infarct with respect to the volume of the areas at risk (perfusion bed) was compared in the various groups, no significant differences could be elicited. Control animals produced a 66 k 7 % volume of infarct, high-dose SOD/catalase produced a 74 i- 4 % volume of infarct, and low-dose SOD/catalase produced a 68’ + 5 % volume of infarct. Similarly, no differences could be found when comparing other ratios including the volume of infarct versus the size of the left ventricle and the volume of perfusion bed versus the size of the left ventricle. Epicardial ST segment changes. Fig. 1 summarizes the data from the epicardial ST segment recordings. The baseline (0 hours) values for the control and treated animals’ do not start at the 0 mV level because of minor injuries sustained by the myocardium during the securing of the epicardial electrodes. With the occlusion of the LAD and the onset of ischemia, there was a rapid rise in ST segments in all groups of animals. This ST segment elevation indicated ischemia of the underlying myocardium. With the release of the LAD ligature and successful reperfusion, there was a rapid drop in ST segments, the appearance of arterialized blood in the previously occluded artery, and a resolution of cyanosis. DISCUSSION
Because of the affinity of molecular oxygen for electrons and its ubiquity in aerobic organisms, oxygen free radicals are often mediators of cellular free radical reactionsi Unfortunately, some of these reactions may be deleterious, and oxygen free radicals have been implicated in a variety of disease states.14
Watanabe et al. Table
II. Histologic data: Control versus treated animals Control (n = 8)
VLV
14.3
k 2.7
VI VPB
3.60 5.90 0.25
f 1.0 k 1.6 k 0.04
VINLV VWNLV VINPB
843
. 100
0.39 i 66 f
0.04 I
hSOD/cat (n = 5) 12.2 ir 1.9 3.80 + 0.42 5.10 0.35
f 0.38 + 0.07
0.46 i 0.07 74 f. 4
SOD/cat
(n = 5)
t test
13.4 _t 2.9 3.0 k 0.87 4.3 k 1.0
NS
0.22 f 0.03 0.32 + 0.03 68 f 5
NS NS
NS NS
NS
VLV, Volume of left ventricle (I&); VI, volume of infarcted tissue (I&); VPB, volume of perfusion bed (cm3); VriVp~ 100, percentage of perfusion bed with infarction; NS, not significant; other abbreviations as in Table I.
Of particular interest is the role of the superoxide and hydroxyl radicals in ischemia-induced pathologic states following reperfusion. The present study found no difference in the size of infarct between the control and treated groups. The use of superoxide and peroxide catabolizing enzymes to reduce infarct size was first reported by Jolly et a1.4 Using a canine open-heart model, the administration of SOD and catalase begining 15 minutes before reperfusion resulted in a significant decrease in infarct size. Several subsequent studies718! I5 with both these agents have failed to reproduce these results. Ambrosio et a1.5 were able to reduce infarct size in dogs subjected to 90 minutes of ischemia and 48 hours of reperfusion by the administration of highdose recombinant SOD. The present experiment was unable to show a reduction of infarct size in animals given the same-dose of human recombinant SOD as in their study. Since the animal species and the period of ischemia were different in these two experiments, it is not possible to come to any definitive conclusions. It is known that a 24-hour period of ischemia in the baboon will produce a 94 -t 3.5% infarct of the perfusion bed, and that 2 hours of isclremia followed by 22 hours of reperfusion’can significantly reduce infarct size.l” Therefore the 2-hour ischemic interval as used in our study is not the most extreme model. Experiments carried out with SOD in attempts to decrease the size of infarct in the ischemia-reperfusion setting have produced conflicting results. There are some possible reasons for the lack of benefit with SOD/catalase intervention in the present study. Many of the studies showing a reduction in infarct size have used triphenyltetrazolium chloride (TTC) staining in the evaluation of infarct size.6 17-B Q ues t’ ions have been raised regarding the accuracy of using TTC staining in SOD experiments.7, 20,21 TTC may underestimate the actual in-
844
Watanabe
et al.
0 - Control e - hSOD/cat 77 - SOD/cat
0
1
2 HOURS
3
4
5
6
POST-OCCLUSION
1. Mean of the sum of epicardial ST segment changes after occlusion in control (open circles), highdose SOD/catalase (filled circles), and low-dose SOD/catalase-treated animals (open triangles).
Fig.
farct size when used with SOD, especially at reperfusion periods of24 hours or less.22 However, there have been studies that produced no reduction of infarct size when TTC and short perfusion periods have been used.23-25 Thus the use of TTC alone cannot explain all of the conflicting results. Another explanation related to the length of reperfusion is that because of the short half-life of SOD, it is unable to protect the myocardium against continued free radical production during prolonged periods of reperfusion. This was the rationale for giving high doses of SOD intraatrially 15 seconds before reperfusion. This method would also ensure rapid delivery of the drug to the infarct zone and minimal dilution. With low doses of SOD, we gave the enzyme 15 minutes before reperfusion to ensure adequate perfusion of the infarct zone and to enable the drug to have adequate blood/tissue levels. Studies conducted with SOD conjugated to polyethylene glycol (PEG) to extend the half-life of the former have shown a reduction in the size of infarct with reperfusion for up to 4 days.26s27 Further, Lehman et a1.28 have recently shown that high-but not low-dose PEG-SOD, significantly reduces reperfusion injury when administered 24 hours before the initiation of global ischemia. However, these findings do not explain the observations by Ambrosio et a1.5 of a reduction in infarct size with SOD and 48 hours of reperfusion. Further, recent experiments conducted separately by Ooiwa et a1.2g and by Tanaka et a1.20 have not produced a limitation of infa+ct size with PEG-SOD. There were some methodologic and species differ-
ences between these “positive” and “negative” study result with PEG-SOD that may account for the contrasting findings (that is, species used, dosage, rate of reperfusion, method of evaluation of infarct size). Thus the question of whether the lack of benefit from SOD is’ a result of its short half-life remains unanswered. Collateral blood supply may also have a role in affecting the outcome. Collaterals may help keep some tissue viable during periods of ischemia and may aid in the delivery of cardioprotective drugs to the ischemic myocardium. Most experiments with SOD have involved a dog model of myocardial ischemiareperfusion. Dogs have an extensive collateral blood suppl~,~~ which should be taken into account when evaluating interventions in myocardial ischemiareperfusion experiments. Studies with the administration of SOD using a dog model have yielded conflicting results, ranging from dogs with the lowest collateral supply showing the most benefit5 to the possibility that only dogs with the highest collateral flow may benefit.24 While our study showed no benefit with SODlcatalase in an animal with minimal collateral flo~,~’ other experiments have produced a reduction in infarct size in animals with minimal collaterals?? l8 However, these studies used shorter periods of reperfusion and TTC staining. Even with the differences in#‘the duration of reperfusion and the method of evaluatibn of infarct size, a collateral blood supply mdy not, be the whole answer to the ability of SOD to reduce infarct size.
Volume 126, Number 4 American
Watanabe et al.
Heart Journal
It has been suggested that high doses of SOD may be ineffective or even detrimental to the ischemicreperfused heart.31 While high doses of SOD were of no benefit in the present study, lower doses that were similar to dosages used in other studies that reported a reduction in infarct size*? 6 also showed no benefit. Hence the dose of SOD/catalase used in the present study does not appear to have played a telling role in the results obtained. Questions have also been raised about the ability of SOD and catalase to gain access to some sites of free radical production because of their large molecular weights.8 A lack of access could theoretically decrease the effectiveness of SOD and catalase in the ischemia-reperfusion setting. In this context, one may also speculate that injecting 400,000 IU of SOD into the left atrium over 30 seconds starting 15 seconds before reperfusion may not have the same effect as giving the adequate doses of SOD directly (intracoronarily) distal to the occlusion at the time of reperfusion. This would allow maximum availability of SOD when the concentration of oxygen free radicals is maximal. Questions have been raised regarding the generation of oxygen free radicals with ischemia and reperfusion in the human heart. There is indirect evidence for the generation of oxygen free radicals in humans placed on cardiopulmonary bypass machines.32> 33 The role of xanthinoxidase in the generation of free radicals in the human heart is controversial. Of course there are several other methods for the formation of these, such as through activated neutrophils, the electron transport chain, and the arachidonic acid cascade. Many questions remain unanswered, one of which is whether the size of the infarct should be determined much later than at 24 hours, perhaps as late as 1 week later. The number of animals used in this experiment, though small, was chosen for the various groups using randomized tables. Since no reduction in the infarct size was seen to any statistical significance, it was not necessary to sacrifice more animals at great expens’e. One of the most important issues that continues to be assessed is whether sustained treatment with oxygen free radical scavengers such as SOD can be effective in reducing infarct size. Numerous studies in which oxygen free radical scavengers were given as a brief treatment have produced negative results. Perhaps prolonged treatment with free radical scavengers (such as a continous 24-hour infusion or an increase in half-life) that are able to reach both intraand extracellular sites will be more effective in consistently reducing ischemia-reperfusion injury. Through the years, studies on SOD with and without catalase have given conflicting and at times confus-
845
ing results. To date, no one explanation seems to adequately answer all of the questions of the ability (or inability) of SOD to decrease infarct size. It can be concluded that high- and low-dose SOD with catalase was of no benefit in reducing the infarct size in this subhuman primate model. REFERENCES
1. McCord JM. Ox,ygen derived free radicals in post-ischemic tissue iniurv. N Ens1 J Med 1985:312:159-63. their involvement in disease processes. 2. Lunec J.“Free radic&: Ann Clin Biochem 1990;27:173-82. 3. Magovern GJ, Bolling SF, Casale AS, Bulkley BH, Gardner TJ. The mechanism of mannitol in reducing ischemic injury: hyperosmolerity or hydroxyl scavenger? Circulation. 1984; 7O(suppl 1):191-5. 4. Jolly SR, Kane ‘WJ, Bailie MB, Abrams GD, Lucchesi BR. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984;54:277-85. 5. Ambrosio G, Becker LC, Hutchins GM, Weisman HF, Weisfeldt ML. Reduction in experimental infarct size by recombinant human superoxide dismutase: insights into the pathophysioplogy of reperfusion injury. Circulation 1986;74:142433. 6. Downey JM, Miura CE, Eddy LJ, Chambers DE, Mellert T, Hearse DJ, Yellon DM. Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart. J Mol Cell Cardiol 1987;19:1053-60. 7. Richard VJ, Murry CE, Jennings RB, Reimer RA. 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;78:473-80. 8. Gallagher KP, Buda AJ, Pace D, Gerren RA, Shlafer M. Failure of superoxide dismutase and catalase to alter the size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation 1986;73:1065-76. 9. Uraizee A, Reimer KA, Murry CE, Jennings RB. Failure of superoxide dismutase to limit size of myocardial infarction after 40 minutes of ischemia and 4 days of reperfusion in dogs. Circulation 1987;75:1237-48. 10. Geary GG, Smith GT, McNamara JJ. Defining the anatomic bed of an occluded coronary artery and the region at risk to infarction. Am J Cardiol 1981;47:1240-47. 11. Geary GG, Smith GT, Suehiro GT, Zeman C, Siu B, McNamara JJ. Quantitative assessment of infarct size reduction by coronary venous retroperfusion in baboons. Am J Cardiol 1982;50:1424-30. 12. Geary GG, Smith GT, Suehiro GT, McNamara JJ. Failure of mfedipine therapy to reduce myocardial infarct size in the baboon. Am J Cardiol 1982:49:331-S. BA, Crapo JD. Free radicals and tissue injury. Lab 13. Freeman Invest 1982;47:412-26. 14. Southorn PA, Powis G. Free radicals in medicine. II. Involvement in human disease. Mayo Clin Proc 1988;63:390-408. 15. Nejima J, Knight DR, Fallon JT, Uemura N, Manders WT, Canfield DR, Cohen MV, Vatner SF. Superoxide dismutase reduces reperfusion arrhythmias but fails to salvage regional function of myocardium at risk in conscious dogs. Circulation 1989;79:143-53. 16. Geary GG, Smith GT, McNamara JJ. Quantitative effect of early coronary artery reperfusion in baboons. Extent of salvage of perfusion bed of an occluded artery. Circulation 1982;66:391-6. 17. Werns SW, Shea MJ, Driscoll EM. The independent effects of oxygen radical scavengers on canine infarct size. Circ Res 1985;56:895-8. 18. Naslund U, Haggmark S, Johansson G, Marklund S, Reiz S, Oberg A. Superoxide dismutase and catalase reduces infarct size m a porcine myocardial occlusion-reperfusion model. J Mol Cell Cardiol 1986;18:1077-84.
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Chqnbers DE, Parks DA, Patterson G, Roy R, I&Cord JM, Yoshida S, Parmley LF, Downey JM. Xanthine oxidase as a source of free radical damage in myocardial ischemia. J Mol Cell Cardiol 1985;17:145-52. 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;67:636-44. Yellon DM. Myocardial reperfusion and reperfusion injury. Bratisl Lek Listy 1991;92:66-76. 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;21:1187-93. Vanhaecke J, Van de Werf F, Ronaszeki A, Flameng W, Lesaffre E, De Geest H. Effect of superoxide dismutase on infarct size and post ischemic recovery of myocardial contractility and metabolism in dogs. J Am Co11Cardiol1991;18:224-30. Przyklenk K, Kloner RA. “Reperfusion injury” by oxygen derived free radicals? Effect of superoxide dismutase plus catalase given at the time of reperfusion on myocardial infarct size, contractile function, coronary microvasculatrure and regional myocardial flow. Circ Res 1989;64:86-96. Pate1 BS, Jeroudi MO, O’Neill PG, Roberts R, Bolli R. Effect of human recombinant superoxide dismutase on canine myocardial infarction. Am J Physiol 1990;258:H369-80. Tamura Y, Chi L, Driscoll EM, 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;63:944-59.
American
actober 1993 Heart Journal
27. Chi L, Tamura Y, Hoff PT, Macha M, Gallagher KP, Schork MA, Luchessi 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;64:665-75. 28. Lehman JD, Dyke C, Abd-Elfattah A, Yeh T, Ding M, Ezrin A, Wechsler AS. Polyethylene glycol-conjugated superoxide dismutase attenuates reperfusion injury when administered twenty four hours before ischemia. 3 Thorac Cardiovasc Surg 1992;104:1597-601. 29. Ooiwa H, Stanley A, Felaneous-Byland A, Wilborn W, Downey JM. Superoxide dismutase conjugated to polyethylene glycol fails to limit myocardial infarct size after 30 minutes ischemia followed by 72 hours of reperfusion in the rabbit. J Mol Cell Cardiol 1991;23:119-25. 30. Maxwell MP, Hearse DJ, Yellon DM. Species variation in the coronary collateral circulation during regional myocardial ischemia: a critical determinant of the rate of evolution and extent of myocardial infarction. Cardiovasc Res 1987;21:737-46. 31. Omar BA, Gad NM, Jordan MC, Striplin SP, Russell WJ, Downey JM, McCord JM. Cardioprotection by Cu, Zn-superoxide dismutase is lost at high doses in the reoxygenated heart. Free Radic Bioi Med 1990;9:465-71. 32. Menasche P, Piwnica A. Free radicals and myocardial protection: a surgical viewpoint. Ann Thorac Surg 1989;47:939-45. 33. Zweir JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Nat1 Acad Sci USA 1987:84:1404-8.
and
l;o;w-&se
slu,peroai&
dismiutase
plus ca.ta.1as.e cioes n’ot reduce myocardial infarct size in a subhuman prim,ate model Oxygen free radical scavengers have been found to decrease infarct size in dogs subjected to myotardial ischemia-reperfusion injury. A baboon open-chest model was used to determine if superoxide dismutase (SOD), an ,oxygen free ridical scavenger, together with cataiase would be equally effective in subhuman primates (baboons). The left anterior descending coronary artery (LAD) was ligated for 2 hours. Before reperfusion, the animals received the following: Group 1 (low-dose SODlcatalase; n = 5) received 15,000 W/kg of SOD and SF,000 IWkg of catalase IV over 1 hour, 15 minutes before reperfusion. Group 2 (high-dose human SOD [h-SOD]/catalase; n = 5) received an intraatrial bolus of 400,000 IU of recombinant h-SOD and 27,500 Ill/kg of catalase over 30 seconds, followed by 300,000 IU of h-SOD and 55,000 Ill/kg of catalase over 1 hour, beginning 15 seconds befobe reperfusipn. Group 3 (n = 8) were control animals. Baboons were put to death 22 hours after reperfusion. Their hearts were excised and sectioned after the perfusion bed distal to the site of ligation was delineated with micrdvascular dye. The infarct zone was determined histologically. Areas of the perfusion bed and infarct zone were measured by planimetry. Infarct size did not differ significantly, between, the three groups: control, 88 -t 7%; low-dose SODlcatalase, 88 + 5%; and highhdose h-SODlcatqlase, 74 + 4%. In this model, highand low-dose SOD with catalase did not result in any significant reduction in infarct size. (AM HEART J 1993;126:840-846.)
Brian I. Watanabe, MD, Shyamal Premaratne, MD, PhD, Whitney Limm, MD, Mark M. Mugiishi, MD, and J. Judson McNamara, MD Honolulu, Hawaii
The production of free radicals has been implicated in myocardial ischemia-reperfusion injury.l? 2 Numerous investigators have used oxygen free radical scavengers in their attempts to reduce the possible free radical-mediated myocardial injury in the ischemia-reperfusion setting. 3-5 Superoxide dismutase (SOD) is one of the most widely studied free radical scavengers. It is able to catalyze the dismutation of the superoxide radical, producing hydrogen peroxide and oxygen. SOD alone or in combination with catalase has produced conflicting results with respect to the limitation of infarct size and other evidences of myocardial protection. Some studies4-6 have shown From the Cardiovascular Queens Medical Center, Hawaii.
Research Laboratory, John A. Burns School
This study was supported by Grant stitutes of Health, Bethesda, Md., ican Heart Association. Received Reprint versity
for publication
of Surgery, University of
No. HL-34647-03 from the National Inand by a Grant-in-Aid from the Amer-
Jan. 4, 1993; accepted
March
24, 1993.
requests: J. Judson McNamara, MD, Department of Surgery, of Hawaii, 1356 Lusitana St., Honolulu, HI 96813.
Copyright @ 1993 by Mosby-Year Book, 0002-8703/93/$1.00 + .lO 4/l/48380
840
Department of Medicine,
Inc.
Uni-
beneficial effects with SOD intervention while others7-g have shown no protection with it. These conflicting findings may in part be the result of differences in the species of animals used and in the experimental protocols employed. The purpose of this study was to determine if high-dose human recombinant SOD plus catalase (high-dose SOD/cat) and low-dose SOD plus catalase (low-dose SOD/cat) given just before reperfusion would reduce myocardial infarct size in a subhuman primate (baboon) model of ischemia-reperfusion injury. Catalase was given with SOD to degrade any hydrogen peroxide that might have been produced from the dismutation of the superoxide radical. No studies on the effect of SOD plus catalase on a subhuman primate have been’reported thus far. The baboon (like the human) has little coronary collateral blood supply, making it a good model for the human condition. METHODS Experimental
preparation. All animals were cared for in accordance with the guidelines of the American Physiological Society. Eighteen baboons (Papio anubis) of either sex
Volume 126, Number 4 American Heart Journal
weighing 9.1 to 17.0 kg were fasted for 24 hours before surgery. They were assigned to control and treatment groups using a table of randomized numbers. The animals were initially sedated with intramuscular ketamine hydrochloride (10 mg/kg), Anesthesia was induced and maintained with an intravenous infusion of thiopental sodium (2 mg/ kg). The animals were intubated with a cuffed endotracheal tube and ventilated using a Harvard NSH-34RH volume ventilator (Harvard Apparatus Company, South Natick, Mass.). Supplemental oxygen was administered as needed and the ventilation was maintained to keep the oxygen saturation not less than 95 % and the PCOZat less than 45 mm Hg. The pH was maintained between 7.53 and 7.36. The bladder was catheterized with an 8F feeding tube to monitor the urine output. A nasogastric tube was inserted and placed on low wall suction. Normothermia was maintained with the use of a heating blanket. Electrocardiograms (ECGs) were monitored throughout the procedure using limb leads. A sterile cutdown was performed and SF catheters were inserted into the femoral artery and vein for pressure monitoring and fluid/drug administration, respectively. A 2.5F thermodilution probe (American Edwards Laboratories, Irvine, Calif.) was placed into the opposite femoral artery with its tip positioned just distal to the renal arteries to measure the cardiac output (CO). An injectate catheter was placed in the femoral vein and positioned at the right atrium. Measurements of CO were made using a lung water computer (American Edwards Model 9310, American Edwards Laboratories). The animals were hydrated with intravenous 5 % dextrose in lactated Ringer’s solution (D5LR). A left thoracotomy was performed in the fourth intercostal space and. the heart was suspended in a pericardial cradle. The left anterior descending coronary artery (LAD) was isolated just below the first diagonal branch and a 3-O suture snare was loosely passed around it. A 1.7 mm outer diameter polyetlhelene catheter (Intramedics PE 190, Clay Adams, Parsippany, N.J.) was inserted into the left atrial appendage for the measurement of left atria1 pressure (LAP). A 5F Mil.lar micromanometer tip catheter pressure transducer (model VPC-663A, Millar Instruments Inc., Houston, Texas) was placed through the left ventricular apex for the measurement of left ventricular pressure and left ventricular contractility (dP/dt). Two rows of six 0.11 mm diameter Teflon-coated silver wires spaced 0.5 cm apart were placed on the anterior epicardium for the direct measurement of an epicardial ECG. These epicardial electrodes were placed over the area at risk for ischemia as determined by the darkening of the epicardium during a 30-second trial ligation and release of the LAD. Baseline limb ECG, mean arterial pressure (MAP), LAP, dP/dt, and CO were recorded on a six-channel recorder (Gould Brush Model 260, Gould Incorporated, Oxnard, Calif.). Epicardial ECGs were recorded with an eight-channel recorder (Hewlett-Packard Model 7758B, Hewlett-Packard, Medical Electronics Division, Waltham, Mass.). Experimental protocol. Following baseline hemody-
Watanabe et al.
841
namic and ECG measurements, 25 mg of lidocaine hydrochloride was injected prophylactically by the intravenous route. One minute later the suture snare was tightened and the LAD was occluded for 2 hours. Epicardial ECG recordings were made every 15 minutes following occlusion of the LAD, and this was continued into the reperfusion phase. Heart rate (HR), MAP, LAP, dP/dt, CO, and a limb lead ECG were recorded every 30 minutes after occlusion, and this was continued into the reperfusion phase. Two hours after the occlusion of the LAD, all animals received a bolus of 25 mg of lidocaine hydrochloride and the snare was released and reperfusion was begun. Additional lidocaine was administered if premature ventricular contractions exceeded six per minute. The use of prophylactic lidocaine was necessary to ensure that all animals were relatively free of premature vetricular contractions (PVCs). To our knowledge no interactions between lidocaine and SOD/catalase have been reported in the literature. Fifteen minutes before reperfusion, five animals (lowdose SOD/catalase group) received intravenous SOD, 15,000 IU/kg (Sigma Chemical Co., St. Louis, MO.), and catalase, 55,000 IU/kg (Sigma Chemical Co.), premixed in a standard 50 ml saline solution and sterilized through a 0.22 pm filter. This infusion continued for a full hour, ending 45 minutes into the reperfusion phase. At 15 seconds before the release of the ligature and the onset of reperfusion, a second group of five animals (high-dose SOD/catalase group) received 400,000 IU of human recombinant SOD (Bio-Technology General Corporation, New York, N.Y.) and 27,500 IU/kg of catalase mixed in 10 ml of normal saline via the left atria1 catheter as a bolus over 30 seconds. This bolus was followed with a l-hour intravenous infusion of 300,000 IU of human recombinant SOD and 55,000 IU/kg of catalase mixed in 50 ml of normal saline. The control group of eight animals underwent the same protocol except that they did not receive any therapeutic adjunct. Instead, they received a continous intravenous infusion of D5LR at 50 ml/hr. At 6 hours postligation (4 hours of reperfusion), the left atrial and left ventricular lines as well as the epicardial electrodes were removed and the chest was closed after the restoration of negative pleural pressure. At the conclusion of the 24-hour monitoring period (22 hours of reperfusion), 3060 units of heparin was administered as an intravenous bolus and the animal was put to death using a saturated solution of potassium chloride. Perfusion bed and infarct size evaluation. Once the hearts were excised, a 0.965 mm outer diameter polyethylene catheter (Intramedics PE 50, Clay Adams) was passed into the LAD and, advanced to the level of previous ligation. The catheter was secured and a colored silicon rubber microvascular dye (Microfil, Canton Bio-Medical Products Incorporated, Boulder, Colo.) was injected into the LAD to delineate the perfusion bed at risk for infarction. Welo have previously shown that this dye injection technique allows accurate delineation of the perfusion bed subserved by an occluded artery. The hearts were then submerged in a 10 % formalin fixative for 3 days and were then sliced parallel to the minor
842
Watemabe et al.
Table
I. Hemodynamic
American
values: Controls
Baseline
30 minutes
and
30 minutes
dP/dt Hglsec)
108 f 6 109 f 9 115 f 6
4.6 t 0.5 4.2 k 0.4 4.4 * 0.7
1.1 + 0.2 1.2 f 0.2 1.6 f 0.3
1994 rf 181 2481 I 300 1703 i 315
127 c 12 116 i 13 119 i 4
113 t 7 108 f 6 113 + 7
8.1 t 1.8 9.3 * 1.7 5.0 k 0.5
1.2 + 0.2 1.3 f 0.2 1.4 k 0.2
2045 2490 1999
f 164 f 482 + 392
127 f
116 rl- 11 121 of- 08
109 i- 6 106 k 7 109 c 11
6.6 i 2.4 8.1 t 1.2 6.3 f 0.8
1.1 i- 0.2 1.1 * 0.2 1.1 * 0.2
1799 2538 1674
+- 206 f 468 f 301
126 + 11 108 t 12 131 I 12
111 * 7 106 f 8 103 * 9
7.9 f 1.9 13.4 I! 2.7 6.6 -c 0.7
1.2 * 0.2 1.2 + 0.2 1.0 z!z0.2
1786 2100 1832
i 162 zk 243 t 416
130 i- 12 122 + 17 143 * 11*
109 f 6 101 * 7 100 * 10
7.2 + 2.1 11.3 zk 2.5 6.9 t 0.7*
1.2 * 0.2 1.7 It: 0.3 1.4 _t 0.3
1746 2185 1894
i 180 i 242 + 296
129 + 11 126 f 12 132 rt 11
96 + 4 101 ?I 11 103 + 8
8.1 li7 2.8 7.9 f 1.8 4.5 zk 0.6
1.2 z!z0.2 1.3 f 0.2 1.2 + 0.1
1742 2235 1829
i: 174 t 288 c 339
131 f 12 124 f 14 137 i 11
99 +- 3 101 -t- 9 98 + 11
4.2 f 2.4
1.1 t 0.2 1.3 + 0.2 1.2 f 0.2
1749 2377 1600
+ 189 +- 392 iz 326
13
postocclusion
postocclusion
Control hSOD/cat SOD/cat postocclusion
Control hSOD/cat SOD/cat 6 hours
112 t 11 113 2 4
(mm
postocclusiont
Control hSOD/cat SOD/cat
5 hours
co (Llmin)
postocclusion
Control hSOD/cat SOD/cat
3 hours
LAP (mm Hd
119 t 12
Control hSOD/cat SOD/cat
2 hours
kffw (mm Hd
preocclusion
Control hSOD/cat SOD/cat
2 hours
versus treated
HR (beatslmin)
Time
October 1993 Heart Journal
postocclusion
Control hSOD/cat SOD/cat HR, Heart
9.1 f 2.9 4.6 rf: 0.4
rate; MAP, mean arterial pressure; LAP, left atria1 pressure; CO, cardiac output; dP/dt, cat, high-dose human recombinant superoxide dismutase/catalase; SOD/cat, low-dose superoxide *Significant difference @ < 0.05) compared with corresponding baseline value. TReperfusion begun at 2 hours postocclusion.
axis into 5 mm transverse sections. These cross sections were embedded into paraffin blocks, further sectioned, and stained with hematoxylin-eosin for histologic study. Representative slides from each cross section were then examined microscopically and the infarcted and viable tissue within the perfusion bed were identified. With the use of a microscopic slide projector (M 600, Durst, Tempe, Ariz.), enlargements of the slides were made and tracings of the slides including the infarcted tissue and perfusion bed were done. Planimetry was then performed and the areas of the left ventricle, perfusion bed, and infarcted tissue were determined. By multiplying these areas by the thickness of the corresponding cross section of the tissue, the volumes of the measured areas were established. Summation of the volumes of each tissue section yielded the total volume of the left ventricle (VL,~), the total volume of the perfused bed at risk for infarction (VP,), and the total volume of infarcted tissue (VI). This method of infarct size evaluation and determination has been described in previous stud-
first time derivative dismutase/catalase.
of left ventricular
pressure;
hSODl
ies. %I2 The histologic delineation and planimetry were done by a technician who was blinded to the animal groupings. Statistical analysis. Data are expressed as the mean +: standard error of the mean (SEM). Comparisons of hemodynamic data, volume data, and infarct size were carried out with the use of a one-way analysis of variance. A p value of less than 0.05 was considered significant. RESULTS Hemodynamics.
Table I summarizes the hemodynamic data for the control and SQD/catalase-treated groups. Representative time intervals are shown beginning before the onset of ischemia and continuing into the ischemic and reperfusion phases. There were no statistically significant differences (p > 0.05) noted between the groups in HR, LAP, CO, MAP, and dP/dt. Within the high-dose SOD/
Volume 126, Number 4 American Heart Journal
catalase group, measurements of these hemodynamic variables did not show any significant difference at the various time intervals. Within the low-dose SOD/catalase group, the 3-hour LAP and HR were significantly greater than the corresponding baseline values (p < 0.05). Even with the rise in LAP within the low-dose SOD/catalase group, the LAP values still remained within normal limits with no other hemodynamic evidence of cardiovascular compromise. Thus the difference in the LAP does not appear to have significantly affected the outcome. Histologic exalmination. Microscopic examination of the stained tissue showed that the infarcted necrotic tissue produced the characteristic changes of pyknosis, karyolysis, and the loss of cross striations. Areas of infarction were contained within the perfusion bed. Histologic data shown in Table II include VLV, Vpg, and VP Several quotients of these values were calculated, with the most emphasis placed on the ratio v~/Vpn, which represents the volume of the actual infarct relative to the area at risk. When the volume of infarct with respect to the volume of the areas at risk (perfusion bed) was compared in the various groups, no significant differences could be elicited. Control animals produced a 66 k 7 % volume of infarct, high-dose SOD/catalase produced a 74 i- 4 % volume of infarct, and low-dose SOD/catalase produced a 68’ + 5 % volume of infarct. Similarly, no differences could be found when comparing other ratios including the volume of infarct versus the size of the left ventricle and the volume of perfusion bed versus the size of the left ventricle. Epicardial ST segment changes. Fig. 1 summarizes the data from the epicardial ST segment recordings. The baseline (0 hours) values for the control and treated animals’ do not start at the 0 mV level because of minor injuries sustained by the myocardium during the securing of the epicardial electrodes. With the occlusion of the LAD and the onset of ischemia, there was a rapid rise in ST segments in all groups of animals. This ST segment elevation indicated ischemia of the underlying myocardium. With the release of the LAD ligature and successful reperfusion, there was a rapid drop in ST segments, the appearance of arterialized blood in the previously occluded artery, and a resolution of cyanosis. DISCUSSION
Because of the affinity of molecular oxygen for electrons and its ubiquity in aerobic organisms, oxygen free radicals are often mediators of cellular free radical reactionsi Unfortunately, some of these reactions may be deleterious, and oxygen free radicals have been implicated in a variety of disease states.14
Watanabe et al. Table
II. Histologic data: Control versus treated animals Control (n = 8)
VLV
14.3
k 2.7
VI VPB
3.60 5.90 0.25
f 1.0 k 1.6 k 0.04
VINLV VWNLV VINPB
843
. 100
0.39 i 66 f
0.04 I
hSOD/cat (n = 5) 12.2 ir 1.9 3.80 + 0.42 5.10 0.35
f 0.38 + 0.07
0.46 i 0.07 74 f. 4
SOD/cat
(n = 5)
t test
13.4 _t 2.9 3.0 k 0.87 4.3 k 1.0
NS
0.22 f 0.03 0.32 + 0.03 68 f 5
NS NS
NS NS
NS
VLV, Volume of left ventricle (I&); VI, volume of infarcted tissue (I&); VPB, volume of perfusion bed (cm3); VriVp~ 100, percentage of perfusion bed with infarction; NS, not significant; other abbreviations as in Table I.
Of particular interest is the role of the superoxide and hydroxyl radicals in ischemia-induced pathologic states following reperfusion. The present study found no difference in the size of infarct between the control and treated groups. The use of superoxide and peroxide catabolizing enzymes to reduce infarct size was first reported by Jolly et a1.4 Using a canine open-heart model, the administration of SOD and catalase begining 15 minutes before reperfusion resulted in a significant decrease in infarct size. Several subsequent studies718! I5 with both these agents have failed to reproduce these results. Ambrosio et a1.5 were able to reduce infarct size in dogs subjected to 90 minutes of ischemia and 48 hours of reperfusion by the administration of highdose recombinant SOD. The present experiment was unable to show a reduction of infarct size in animals given the same-dose of human recombinant SOD as in their study. Since the animal species and the period of ischemia were different in these two experiments, it is not possible to come to any definitive conclusions. It is known that a 24-hour period of ischemia in the baboon will produce a 94 -t 3.5% infarct of the perfusion bed, and that 2 hours of isclremia followed by 22 hours of reperfusion’can significantly reduce infarct size.l” Therefore the 2-hour ischemic interval as used in our study is not the most extreme model. Experiments carried out with SOD in attempts to decrease the size of infarct in the ischemia-reperfusion setting have produced conflicting results. There are some possible reasons for the lack of benefit with SOD/catalase intervention in the present study. Many of the studies showing a reduction in infarct size have used triphenyltetrazolium chloride (TTC) staining in the evaluation of infarct size.6 17-B Q ues t’ ions have been raised regarding the accuracy of using TTC staining in SOD experiments.7, 20,21 TTC may underestimate the actual in-
844
Watanabe
et al.
0 - Control e - hSOD/cat 77 - SOD/cat
0
1
2 HOURS
3
4
5
6
POST-OCCLUSION
1. Mean of the sum of epicardial ST segment changes after occlusion in control (open circles), highdose SOD/catalase (filled circles), and low-dose SOD/catalase-treated animals (open triangles).
Fig.
farct size when used with SOD, especially at reperfusion periods of24 hours or less.22 However, there have been studies that produced no reduction of infarct size when TTC and short perfusion periods have been used.23-25 Thus the use of TTC alone cannot explain all of the conflicting results. Another explanation related to the length of reperfusion is that because of the short half-life of SOD, it is unable to protect the myocardium against continued free radical production during prolonged periods of reperfusion. This was the rationale for giving high doses of SOD intraatrially 15 seconds before reperfusion. This method would also ensure rapid delivery of the drug to the infarct zone and minimal dilution. With low doses of SOD, we gave the enzyme 15 minutes before reperfusion to ensure adequate perfusion of the infarct zone and to enable the drug to have adequate blood/tissue levels. Studies conducted with SOD conjugated to polyethylene glycol (PEG) to extend the half-life of the former have shown a reduction in the size of infarct with reperfusion for up to 4 days.26s27 Further, Lehman et a1.28 have recently shown that high-but not low-dose PEG-SOD, significantly reduces reperfusion injury when administered 24 hours before the initiation of global ischemia. However, these findings do not explain the observations by Ambrosio et a1.5 of a reduction in infarct size with SOD and 48 hours of reperfusion. Further, recent experiments conducted separately by Ooiwa et a1.2g and by Tanaka et a1.20 have not produced a limitation of infa+ct size with PEG-SOD. There were some methodologic and species differ-
ences between these “positive” and “negative” study result with PEG-SOD that may account for the contrasting findings (that is, species used, dosage, rate of reperfusion, method of evaluation of infarct size). Thus the question of whether the lack of benefit from SOD is’ a result of its short half-life remains unanswered. Collateral blood supply may also have a role in affecting the outcome. Collaterals may help keep some tissue viable during periods of ischemia and may aid in the delivery of cardioprotective drugs to the ischemic myocardium. Most experiments with SOD have involved a dog model of myocardial ischemiareperfusion. Dogs have an extensive collateral blood suppl~,~~ which should be taken into account when evaluating interventions in myocardial ischemiareperfusion experiments. Studies with the administration of SOD using a dog model have yielded conflicting results, ranging from dogs with the lowest collateral supply showing the most benefit5 to the possibility that only dogs with the highest collateral flow may benefit.24 While our study showed no benefit with SODlcatalase in an animal with minimal collateral flo~,~’ other experiments have produced a reduction in infarct size in animals with minimal collaterals?? l8 However, these studies used shorter periods of reperfusion and TTC staining. Even with the differences in#‘the duration of reperfusion and the method of evaluatibn of infarct size, a collateral blood supply mdy not, be the whole answer to the ability of SOD to reduce infarct size.
Volume 126, Number 4 American
Watanabe et al.
Heart Journal
It has been suggested that high doses of SOD may be ineffective or even detrimental to the ischemicreperfused heart.31 While high doses of SOD were of no benefit in the present study, lower doses that were similar to dosages used in other studies that reported a reduction in infarct size*? 6 also showed no benefit. Hence the dose of SOD/catalase used in the present study does not appear to have played a telling role in the results obtained. Questions have also been raised about the ability of SOD and catalase to gain access to some sites of free radical production because of their large molecular weights.8 A lack of access could theoretically decrease the effectiveness of SOD and catalase in the ischemia-reperfusion setting. In this context, one may also speculate that injecting 400,000 IU of SOD into the left atrium over 30 seconds starting 15 seconds before reperfusion may not have the same effect as giving the adequate doses of SOD directly (intracoronarily) distal to the occlusion at the time of reperfusion. This would allow maximum availability of SOD when the concentration of oxygen free radicals is maximal. Questions have been raised regarding the generation of oxygen free radicals with ischemia and reperfusion in the human heart. There is indirect evidence for the generation of oxygen free radicals in humans placed on cardiopulmonary bypass machines.32> 33 The role of xanthinoxidase in the generation of free radicals in the human heart is controversial. Of course there are several other methods for the formation of these, such as through activated neutrophils, the electron transport chain, and the arachidonic acid cascade. Many questions remain unanswered, one of which is whether the size of the infarct should be determined much later than at 24 hours, perhaps as late as 1 week later. The number of animals used in this experiment, though small, was chosen for the various groups using randomized tables. Since no reduction in the infarct size was seen to any statistical significance, it was not necessary to sacrifice more animals at great expens’e. One of the most important issues that continues to be assessed is whether sustained treatment with oxygen free radical scavengers such as SOD can be effective in reducing infarct size. Numerous studies in which oxygen free radical scavengers were given as a brief treatment have produced negative results. Perhaps prolonged treatment with free radical scavengers (such as a continous 24-hour infusion or an increase in half-life) that are able to reach both intraand extracellular sites will be more effective in consistently reducing ischemia-reperfusion injury. Through the years, studies on SOD with and without catalase have given conflicting and at times confus-
845
ing results. To date, no one explanation seems to adequately answer all of the questions of the ability (or inability) of SOD to decrease infarct size. It can be concluded that high- and low-dose SOD with catalase was of no benefit in reducing the infarct size in this subhuman primate model. REFERENCES
1. McCord JM. Ox,ygen derived free radicals in post-ischemic tissue iniurv. N Ens1 J Med 1985:312:159-63. their involvement in disease processes. 2. Lunec J.“Free radic&: Ann Clin Biochem 1990;27:173-82. 3. Magovern GJ, Bolling SF, Casale AS, Bulkley BH, Gardner TJ. The mechanism of mannitol in reducing ischemic injury: hyperosmolerity or hydroxyl scavenger? Circulation. 1984; 7O(suppl 1):191-5. 4. Jolly SR, Kane ‘WJ, Bailie MB, Abrams GD, Lucchesi BR. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984;54:277-85. 5. Ambrosio G, Becker LC, Hutchins GM, Weisman HF, Weisfeldt ML. Reduction in experimental infarct size by recombinant human superoxide dismutase: insights into the pathophysioplogy of reperfusion injury. Circulation 1986;74:142433. 6. Downey JM, Miura CE, Eddy LJ, Chambers DE, Mellert T, Hearse DJ, Yellon DM. Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart. J Mol Cell Cardiol 1987;19:1053-60. 7. Richard VJ, Murry CE, Jennings RB, Reimer RA. 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;78:473-80. 8. Gallagher KP, Buda AJ, Pace D, Gerren RA, Shlafer M. Failure of superoxide dismutase and catalase to alter the size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation 1986;73:1065-76. 9. Uraizee A, Reimer KA, Murry CE, Jennings RB. Failure of superoxide dismutase to limit size of myocardial infarction after 40 minutes of ischemia and 4 days of reperfusion in dogs. Circulation 1987;75:1237-48. 10. Geary GG, Smith GT, McNamara JJ. Defining the anatomic bed of an occluded coronary artery and the region at risk to infarction. Am J Cardiol 1981;47:1240-47. 11. Geary GG, Smith GT, Suehiro GT, Zeman C, Siu B, McNamara JJ. Quantitative assessment of infarct size reduction by coronary venous retroperfusion in baboons. Am J Cardiol 1982;50:1424-30. 12. Geary GG, Smith GT, Suehiro GT, McNamara JJ. Failure of mfedipine therapy to reduce myocardial infarct size in the baboon. Am J Cardiol 1982:49:331-S. BA, Crapo JD. Free radicals and tissue injury. Lab 13. Freeman Invest 1982;47:412-26. 14. Southorn PA, Powis G. Free radicals in medicine. II. Involvement in human disease. Mayo Clin Proc 1988;63:390-408. 15. Nejima J, Knight DR, Fallon JT, Uemura N, Manders WT, Canfield DR, Cohen MV, Vatner SF. Superoxide dismutase reduces reperfusion arrhythmias but fails to salvage regional function of myocardium at risk in conscious dogs. Circulation 1989;79:143-53. 16. Geary GG, Smith GT, McNamara JJ. Quantitative effect of early coronary artery reperfusion in baboons. Extent of salvage of perfusion bed of an occluded artery. Circulation 1982;66:391-6. 17. Werns SW, Shea MJ, Driscoll EM. The independent effects of oxygen radical scavengers on canine infarct size. Circ Res 1985;56:895-8. 18. Naslund U, Haggmark S, Johansson G, Marklund S, Reiz S, Oberg A. Superoxide dismutase and catalase reduces infarct size m a porcine myocardial occlusion-reperfusion model. J Mol Cell Cardiol 1986;18:1077-84.
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19.
20.
21. 22.
23.
24.
25. 26.
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