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reperfusion injury
Free Radical Biology & Medicine, Vol. 7, pp. 45-52, 1989 Printed in the USA. All rights reserved.
0891-5849/89 $3.00+ .00 © 1989 Pergamon Press plc
Original Contribution I
EARLY TREATMENT WITH DEFEROXAMINE LIMITS MYOCARDIAL ISCHEMIC/REPERFUSION INJURY
B. RAMESH REDDY, ROBERT A . KLONER,* a n d KARIN PRZYKLENK*'t Ischemia Laboratory, Division of Cardiology, Harper Hospital and Wayne State University, Detroit, Michigan, U.S.A. (Received 20 July 1988; Revised 11 October 1988; Accepted 30 January 1989)
Abstract--Oxygen-derived free radicals (the superoxide anion 02- and hydroxyl radical "OH) have been implicated in myocardial injury associated with coronary artery occlusion followed by reperfusion. Transition metals (such as iron or copper) are needed to catalyze the formation of the .OH radical and subsequent .OH-mediated lipid peroxidation, yet the role of these transition metals in the pathogenesis of myocyte necrosis remains undefined. To address this issue, 21 dogs underwent 2 h of coronary artery occlusion and 4 h of reperfusion. Each animal was randomly assigned into 1 of 3 treatment groups: 7 received the iron chelator deferoxamine beginning 30 min preocclusion, 7 received deferoxamine beginning 5 min prior to reperfusion, while 7 dogs served as saline controls. Deferoxamine effectively chelated free iron in both treatment groups (total urine iron content averaged 42 -4- 16, 662 +-- 177' and 803 --- 205* micrograms in control, pretreated, and deferoxamine at reperfusion groups respectively; *p < 0.05), but had no significant effect on in vivo area at risk (AR), hemodynamic parameters, collateral blood flow during occlusion, or myocardial blood flow following reperfusion. Area of necrosis (AN) in dogs pretreated with deferoxamine (34.6 --- 3.7%* of the AR; *p < 0.05) was significantly smaller than that observed in the saline control group (55.4 + 4.7% of the AR). Deferoxamine administered at the time of reperfusion, however, had no significant effect on infarct size (AN/AR = 54.3 +-- 8.7%, p = NS vs. controls). Thus, early treatment with the iron chelator deferoxamine acutely reduced the extent of myocyte necrosis produced by 2 h of transient coronary artery occlusion in the canine model. These data support the hypothesis that iron catalyzed production of oxygen derived free radicals contribute to myocyte necrosis in the setting of ischemia-reperfusion. Keywords--Myocardial infarct size, Coronary occlusion, Reperfusion, Oxygen free radicals, Free radicals
dependent upon the presence of transitional metal ions such as Fe +++ and Cu++. 2,~4 In addition, the transitional metals also catalyze the .OH-mediated lipid peroxidation of cellular membranes, leading to the production of alkoxy- and peroxyradicals and subsequent increase in membrane fluidity and loss of membrane integrity. 2 This would suggest that the formation of free radicals and/or the perpetuation of free radicalmediated tissue injury could be aborted, or at least retarded, by chelating the free metal ions available to catalyze these reactions. The highly specific iron chelating agent deferoxamine has been shown to offer protection from ischemia/hypoxia-reperfusion injury in isolated rat heart preparations~5-w; however, the importance of free iron in an in vivo model of myocardial ischemia-reperfusion injury has not yet been evaluated. We therefore sought to determine whether the iron chelator deferoxamine, administered either prior to occlusion or at the time of reperfusion, could acutely reduce
INTRODUCTION
Oxygen-derived free radicals (the superoxide anion 02- and hydroxyl radical .OH) are cytotoxic and highly reactive molecules that are thought to be involved in the pathogenesis of myocardial ischemic-reperfusion injury. ~-3 Although results to date have been controversial, 4-7 several studies have demonstrated that timely administration of free radical scavenging agents and antioxidants such as superoxide dismutase plus catalase, allopurinol and oxypurinol can limit myocardial injury following prolonged periods of transient coronary occlusion. 8-~3. Production of the highly cytotoxic .OH radical is
*Current affiliation: Heart Institute, Hospital of the Good Samaritan, 616 South Witmer Street, Los Angeles, CA 90017, U.S.A. tCorrespondence should be addressed to: Karin Przyklenk, PhD, Heart Institute/Research, Hospital of the Good Samaritan, 616 South Witmer Street, Los Angeles, CA 90017, U.S.A. 45
46
B.R. REDDY et al.
the extent of myocyte necrosis in an in vivo canine model of prolonged (2 h) coronary artery occlusion followed by 4 h of reperfusion.
ued for the remainder of the study (10 mg/kg for the first hour, followed by a maintenance dose of 1.5 mg/ kg/hr).
METHODS
Surgical preparation Fifty-three mongrel dogs of either sex weighing an average of 21 --- 5 kg were anesthetized with sodium pentobarbital (30 mg/kg IV), intubated and ventilated with room air using a Harvard respirator. Cannulae were inserted into the left carotid artery for measurement of systolic and diastolic blood pressure and heart rate, and into the left jugular vein for administration of drugs and fluids. The urinary bladder was then catheterized and urine collected for measurement of urinary iron excretion. A left thoracotomy was performed through the fifth intercostal space, a pericardial cradle was constructed, and the left anterior descending coronary artery (LAD) was isolated for later occlusion, usually distal to its first major diagonal branch. A high fidelity microtipped catheter (Millar Instruments) was advanced into the left ventricular chamber via the left atrial appendage to measure left ventricular pressure and its first derivative, LV dP/dt. The left atrium was cannulated for later injection of radioactive microspheres for measurement of regional myocardial blood flow (RMBF). Protocol After obtaining baseline recordings of hemodynamic parameters, each dog was randomized in a blinded fashion into one of three treatment groups: 1. Deferoxamine pretreatment. Intravenous infusion of deferoxamine (deferoxamine mesylate; Desferal; Ciba-Geigy Ltd.) was initiated 30 min prior to coronary occlusion at an initial dose of 10 mg/kg for the first hour of the protocol, followed by a maintenance dose of 1.5 m g / k g / h r throughout the remainder of the protocol. This dosing regimen of deferoxamine has recently been shown to attenuate postischemic contractile dysfunction in the canine model of the "stunned myocardium ''~8 (a phenomenon thought to be due in part to the action of oxygen-derived free radicals L9-2~) without any apparent effect on hemodynamic parameters or RMBF.18 2. Deferoxamine at reperfusion. Saline was infused for 30 min prior to occlusion and throughout the 2 h period of LAD occlusion. Infusion of deferoxamine was initiated at 5 min prior to reperfusion and contin-
3. Control. Saline was infused for 30 min preocclusion, during LAD occlusion, and throughout reperfusion. All animals were randomized by drawing coded slips of paper from a box. If an experiment was unsuccessful (i.e., due to the death of the animal or exclusion from the protocol), the information was recorded and the coded slip returned to the randomization box. Experiments were continued in this manner until 7 dogs in each group had successfully completed the protocol. The randomization code and information was maintained by one investigator (K.P.) until all analysis had been completed. Thirty minutes after randomization and the onset of treatment, lidocaine (1.5 mg/kg IV) was administered and the LAD was occluded using atraumatic vascular clamps. RMBF was assessed at 30 min postocclusion by injection of microspheres labelled with either 95Nb, 1°3Ru o r 14ICe. Hemodynamic parameters were measured at 30 minutes, 1 h and 2 h postocclusion. After 2 h of occlusion, the LAD was abruptly reperfused by removal of the vascular clamps. Hemodynamic recordings were taken at regular hourly intervals, and a second radiolabelled isotope (i.e., one not chosen for the first injection) was used to measure RMBF at 2 h postreperfusion. At 4 h postreperfusion, the LAD was briefly reoccluded and monastral blue pigment (0.5 ml/kg) was injected into the coronary circulation via the left atrium to delineate the in vivo area at risk (AR). The dogs were then immediately sacrificed by intracardiac injection of KCl (20-40 mEq), and the hearts rapidly excised. Each heart was then cut into 5-7 transverse slices, approximately 5 mm in thickness, parallel to the atrioventricular groove. Epicardial and endocardial contours of the basal surfaces of the heart slices, and the margins of the AR (i.e., the areas unstained by monastral blue dye) were traced onto acetate sheets. The slices were then incubated in a 1% solution of triphenyltetrazolium chloride (TTC) for 10 rain at 37°C to distinguish necrotic from normal myocardium. Validation of the TTC technique has previously been reported. 22"23The heart slices, and boundaries of the area of necrosis (AN) within each slice (i.e., the areas unstained by TTC), were retraced onto the acetate sheets, and the hearts were then fixed by immersion in 10% neutral buffered formalin.
Deferoxamine and infarct size
Analysis After trimming off the right ventricular tissue, each heart slice was weighed. Contours of the LV, AR, and AN were cut from the tracings of the heart slices and weighed. AN and AR were then corrected for the weight of the tissue slice and summed for each heart. AR was then expressed as a percentage of the total LV weight (i.e., AR/LV), while AN was expressed as both a percentage of the AR (AN/AR) and percentage of the total LV (AN/LV). Hemodynamic parameters (heart rate, mean arterial pressure, LV dP/dt) were measured and averaged over 5 continuous cardiac cycles in normal sinus rhythm for each sample period. Approximately 200 ml of urine was collected from each dog during the course of the experiment. Total urinary iron content was measured from these samples by Roche Biomedical Laboratories using the ferrozine binding colorimetric method. 24 For calculation of RMBF, blocks of tissue were cut from both the center of the previously ischemic LAD bed and remote, nonischemic areas. These tissue blocks were then subdivided into endo-, mid-, and epicardial segments and RMBF quantified by the method of Domenech et al.25 All measurements were made by one investigator (B.R.R.) who remained blinded with respect to the treatment groups of each animal until the study had been completed.
Statistics All dogs with high collateral blood flow during coronary occlusion and a small AR (that is, animals that were not sufficiently ischemic to develop myocyte necrosis) were excluded from the final analysis. Specifically, our exclusion criteria, established prior to the onset of the study, were: values of RMBF in the "ischemic" endocardium > 0.15 ml/min/g tissue and/ or AR < 10% of the LV. In addition, dogs which did not appear to reperfuse successfully (i.e. did not exhibit characteristic hyperemic distention of the LAD upon removal of the vascular clamps) were excluded from analysis. No attempt was made to revive dogs that developed ventricular fibrillation during occlusion or during reperfusion. Comparison of hemodynamic parameters, RMBF, infarct size data, and urinary iron concentrations among the three treatment groups were made using analysis of variance (ANOVA). If significant differences among the groups were detected by ANOVA, pairwise comparisons were made using Tukey's test. 26 All values are quoted as mean --- SEM, and values are considered to differ significantly if p < 0.05.
47 RESULTS
Of the 53 dogs entered into the study, 24 died of ventricular fibrillation* during LAD occlusion. Specifically, 11 of 24 dogs (or 46%) initially randomized to receive deferoxamine preocclusion, 6 of 13 animals (46%) assigned to receive deferoxamine at reperfusion, and 6 of 15 randomized to the saline control group (40%) fibrillated during the ischemic period. In addition, 2 dogs (both pretreated with deferoxamine) died during reperfusion. Thus, treatment with deferoxamine did not protect against ventricular fibrillation due to coronary artery occlusion or reperfusion in this canine model. Four dogs were excluded from the study because they were not " i s c h e m i c " as defined by the exclusion criteria: mean endocardial RMBF during coronary occlusion was 0.35 ml/min/g tissue, and AN averaged < 1% of the LV in these animals. In addition, 2 dogs were excluded from analysis because they did not appear to reperfuse successfully upon removal of the vascular clamps, probably due to thrombus formation at the site of the occlusion. No hyperemic distention of the LAD was observed in these 2 animals at the time the clamps were removed, infarcts in both animals were essentially transmural (suggesting a period of occlusion > 2 h), and values of endocardial RMBF measured after 2 h of " r e f l o w " in these 2 dogs remained ischemic by our exclusion criteria, averaging only 0.13 ml/min/g tissue. Thus, a total of 21 dogs (n = 7 in each treatment group) are included in the final analysis.
Hemodynamic parameters (Table 1) Baseline values of heart rate, mean arterial pressure and LV dP/dt were comparable among the three groups prior to randomization and treatment. Infusion of deferoxamine had no apparent effect on any of these hemodynamic parameters, as heart rate, arterial pressure and dP/dt did not differ significantly among the three groups either preocclusion, during LAD occlusion, or following reperfusion.
Regional myocardial blood flow (Table 2) Values of RMBF in the ischemic LAD bed did not differ significantly among the three treatment groups in any myocardial layer. For example, RMBF to the ischemic endocardium at 30 min postocclusion averaged 0.01 +- 0.01, 0.04 +_ 0.02 and 0.03 -+ 0.02 ml/
*The assigned treatment group of one dog that fibrillated during occlusion was not recorded.
48
B. R. REDDY et al. Table 1. H e m o d y n a m i c P a ra me t e rs Occlusion
I. H e a r t Rate (beats/min): Control (n = 7) DF-Pre (n = 7) DF-at Re p (n = 7) II. Mean Arterial Pressure (ram Hg): Control (n = 7) DF-Pre (n = 7) DF-at R e p (n = 7) III. + LV d P / d t (mm Hg/sec): Control (n = 7) DF-Pre (n = 7) D F - a t R e p ( n = 7)
Reperfusion
Baseline
Pre-Occlusion
30'
120'
60"
120'
141 -+ 5 150 -+ 11 150 -+ 8
149 _+ 6 146 --- 11 150 -+ 8
142 -+ 5 147 _+ 4 151 _+ 7
136 _+ 4 143 -+- 10 155 -+ 6
141 -+ 5 150 -+ 10 152 + 7
144 + 5 147 -+ 7 157 +- 7
147_+ 5 150 + 9 157 +- 9
115 -+ 4 121 -+ 9 120 --- 8
120 -+ 5 123 -+ 10 119 -+ 7
117 -+ 6 121 -+ 10 120 -+ 5
111 _+ 9 117 _+ 9 121 _+ 5
112-8 112- 7 115 + 6
118-+6 112-+ 8 117 -+ 6
119-+6 108-+ 5 114 -+ 6
1443 -+ 142 1364 -+ 133 1467-+ 130
1607 +-- 151 1479 -+ 150 1492-+ 173
1400 _+ 144 1436 -+ 160 1483 _+ 123
1386 + 142 1307-+ 122 1325 - 89
1471 -+ 154 1300-+ 107 1450 + 135
1464 -+ 126 1279-+ 136 1442 + 111
1408 _+ 181 1421 _+ 158 1475-+ 79
240'
DF-Pre = Deferoxamine pretreatment. DF-at Rep = Deferoxamine given at reperfusion.
min/g tissue in the control, deferoxamine pretreatment and deferoxamine at reperfusion groups respectively (p = NS). Values of RMBF in the LAD bed during reperfusion were also comparable among the three treatment groups: in the previously ischemic endocardium, mean RMBF was 0.54 - 0.10, 0.64 + 0.11 and 0.58 -+ 0.09 m l / m i n / g tissue in the 3 groups respectively (p = NS). RMBF was also comparable among all groups in the remote, nonischemic tissue perfused by the circumflex bed, both during LAD occlusion and following reperfusion. Thus, deferoxamine had no significant effect on myocardial blood flow in this canine model. Urinary iron content (Fig. 1)
As anticipated 24, only small amounts of iron (42 --16 micrograms) were detected in the urine of control animals. In contrast, total urine iron content averaged 662 --- 177" micrograms in dogs pretreated with de-
feroxamine and 803 --- 205* micrograms in animals that received deferoxamine at the time of reperfusion (*p < 0.05 vs. controls). Thus, deferoxamine was equally effective in chelating free iron in both drugtreated groups. Infarct size and area at risk (Fig. 2)
Treatment with deferoxamine did not have a significant effect on area at risk in this canine model: AR/ LV was 17.2 +-_ 1.4%, 19.2 + 2.1% and 19.6 -+ 2.6% in the control, deferoxamine pretreatment and deferoxamine at reperfusion groups respectively (p = NS; Fig. 2). Area of necrosis averaged 55.4 + 4.7% of the AR in the saline control group. In contrast, A N / A R was reduced significantly in animals that had been pretreated with deferoxamine, averaging 34.6 + 3.7%* (*p < 0.05 vs. controls by ANOVA; Fig. 2). Deferoxamine given at the time of reperfusion, however,
Table 2. Regional Myocardial Blood Flow ( m l / m i n / g tissue) 30" Postocclusion
I. Ischemic L A D Bed: Control (n = 6) DF-Pre (n = 7) DF-at R e p (n = 7) II. R e m o t e Nonischemic Circumflex Bed: Control (n = 6) DF-Pre (n = 7) DF-at Rep (n = 7)
2 H ours Postreperfusion
ENDO
MID
EP I
ENDO
MID
EP I
0.01 +_ .01 0.04 --+ .02 0.03 --_ .02
0.04 4- .02 0.07 -+ .02 0.04 _+ .02
0.12 +- .05 0.24 _+ .06 0.15 -+ .07
0.54 --- .10 0.64 _+ .11 0.58 + .09
0.44 +- .02 0.64 - .06 0.49 -+ .09
0.59 - .09 0.81 -+ .12 0.89 -+ .12
1.34 _+ .21 1.38 _+ .22 1.62 _+ .23
1.22 _+ .28 1.34 _+ .24 1.68 -+ .24
1.04 -+ .15 1.18 +- .24 1.44 --- .23
1.15 _+ .13 1.32 _+ .18 1.60 -+- .33
1.09 -+ .13 1.26 --- .18 1.69 -+ .32
1.00 _+ .12 1.17 _+ .21 1.70 -+ .34
DF-Pre = Deferoxamine pretreatment. DF-at Rep = Deferoxamine given at reperfusion. ENDO = endocardial; MID = midmyocardial: EPI = epicardial.
Deferoxamine and infarct size URINE IRON LEVELS
E
1200
£
1ooo
,o
E
"-" ~E
£
400
",U-.
200
"6
dence suggests that the formation of the .OH radical and subsequent production of lipid peroxides, rather than O2- per se, may be the primary mediators of tissue injury in the myocardium. 29'3° One major pathway of •OH production is the two-step metal catalyzed reaction: 2
800 02
600 c-
49
0
+ Fe+++/Cu ÷÷
Fe++/Cu+ + HzO2
T
p.-
r---I CONTROLS
DF: PREOCCLUSION
1771 DF: AT REPERFUSION
Fig. 1. Totalurine ironcontent in controlanimals, dogsthat received deferoxamine preocclusion, and those treated with deferoxamine at the time of reperfusion. DF = deferoxamine;*p < 0.05 vs. controls.
had no significant effect on infarct size: mean A N / A R was 54.3 +-- 8.7% (p = NS vs. controls; Fig. 2). In summary, the iron chelator deferoxamine administered prior to two hours of coronary artery occlusion acutely reduced infarct size, assessed at 4 h postreperfusion, in the absence of any apparent effect on hemodynamic parameters, the degree of collateral blood flow during occlusion, or RMBF following reperfusion. Although deferoxamine given at the time of reperfusion also chelated free iron, this " d e l a y e d " treatment had no significant beneficial effect on myocyte necrosis in this model.
) Fe++/Cu ÷ + 02 >
Fe+++/Cu ++ + OH- + .OH
There are no specific physiologic or enzymatic defenses that act to scavenge the excess quantities of .OH thought to be produced in pathologic conditions such as ischemia-reperfusion. 2 The highly reactive .OH radicals may then attack unsaturated fatty acid side chains of cellular membranes and undergo numerous chain reactions, resulting in the formation of lipid radicals, organic oxygen radicals, and lipid peroxides. Lipid peroxides can, in the presence of transition metals such as iron, further decompose to form alkoxy- and peroxyradicals. 2 These free radical mediated alterations in the polyunsaturated side chains of the lipid components of cell membranes result in an increase in membrane fluidity and permeability and decrease in membrane integrity. 2 Iron and/or copper clearly play an important theoretical role in the mechanisms of tissue injury by free radicals: in the absence of transition metals, the rate constant for the metal catalyzed formation of .OH is essentially zero. 14 Deferoxamine, a trihydroxamic INFARCT SIZE AND AREA AT RISK
DISCUSSION
70
Considerable recent attention has been focused on the possible role of O2- (the superoxide anion), H202 (hydrogen peroxide) and .OH (the hydroxyl radical) in the pathogenesis of myocardial ischemia-reperfusion injury. To address this issue, several investigators have attempted to reduce the extent of myocyte necrosis by administration of free radical scavenging agents and antioxidants. 4-~3 While results have been controversial, 4-v early administration of superoxide dismutase (SOD) + catalase, 8 SOD alone, 9-~ allopurinol 1°42 and oxypurinop3--either prior to or during the period of ischemia--have been shown to acutely reduce infarct size produced by 6 0 - 9 0 min of ischemia and 4 - 2 4 h of reflow. Although treatment with the specific O2- scavenger SOD has been shown to have beneficial effects in both in v i v o 9-11 and in vitro models 27,28of ischemia/hypoxia followed by reperfusion/reoxygenation, in vitro evi-
60
T
50 40
2O 10 0
AN/AR (g)
F"-I CONTROLS
AN/LV (~)
AR/LV (~)
DF: PREOCCLUSION
17"-21 DF: AT REPERFUSION
Fig. 2. Area of necrosis (AN) expressed as a percentage of the area at risk (AR), AN expressed as a percentage of the total LV weight, and AR expressed as a percentage of the LV for control animals, dogs that receiveddeferoxaminepreocclusion, and those treated with deferoxamine at the time of reperfusion. DF = deferoxamine; *p < 0.05 vs. controls.
50
B . R . REDDY et al.
acid, is a potent chelator of free iron. 31 It is commonly used in the clinical treatment of acute iron intoxication and chronic iron overload and, in spite of some instances of deferoxamine toxicity, 32 is generally well tolerated. 33,34In addition to its capacity to chelate free iron, deferoxamine also inhibits lipid peroxidation 32'35 and acts as a scavenger of free radicals? 2'36 Thus, timely administration of this potent chelating and free radical scavenging agent may attenuate tissue injury in pathologic conditions associated with increased free radical production such as ischemia followed by reperfusion. Deferoxamine given at the time of cardiac resuscitation has previously been shown to reduce ischemiareperfusion injury to the brain and improve survival in an anesthetized rat model of cardiac arrest. 37 Similar beneficial results (i.e., reduced levels of conjugated dienes and malondialdehyde, a degradation product of lipid peroxidation) were noted in brain tissue of dogs resuscitated from cardiac arrest? 8 More recently, pretreatment with deferoxamine has been shown to enhance recovery of regional contractile function following a brief (15 min) period of transient coronary artery occlusion in the canine model of the stunned myocardium, ~8"39 a phenomenon thought to be due at least in part to the action of free radicals. ~9--'~However, iron chelation and its possible beneficial effects in the setting of prolonged periods of myocardial ischemia/ hypoxia followed by reflow have, to date, only been studied using isolated perfused heart models. For example, Myers et al. J5 found that deferoxamine, given during a 60 min period of global normothermic hypoxia, significantly reduced the release of creatine kinase following reoxygenation. Similar in vitro studies have demonstrated that " d e l a y e d " treatment with deferoxamine (i.e., added to the perfusate at the time of reoxygenation) lowered vascular resistance,~6 blunted release of creatine kinase, ~6 increased myocardial phosphocreatine content j7 and enhanced recovery of left ventricular developed pressure 17 when compared with controls. In addition, Badylak et a1.16 noted that the ultrastructure of the mitochondria, endoplasmic reticulum, and cell membranes were in part preserved by " d e l a y e d " administration of deferoxamine. Our observation that pretreatment with deferoxamine acutely limited the extent of myocyte necrosis produced by 2 h of regional ischemia and 4 h of reflow is in good agreement with these previous in vitro studies, L5-17and is similar to the beneficial results obtained using early treatment with free radical scavengers and antioxidants. 8-~3 Furthermore, our data suggest that iron may play an important role in the pathogenesis of myocyte necrosis in this canine model. What is the source of free iron in the setting of
myocardial ischemia/reperfusion? Iron is released from ferritin under anaerobic conditions; 4° thus, free iron may also accumulate during prolonged periods of coronary artery occlusion. In fact, an increase in nonprotein bound low molecular weight iron has been documented in the myocardium of dogs subjected to 2 h of regional ischemia. 4~ In addition, superoxide anions release iron from ferritin. 42 As both direct 43'44 and indirect evidence 45~46suggests that oxygen free radicals are generated during coronary artery occlusion, this superoxide-mediated iron release may be an important factor in our model. The precise source of free iron in myocardia ischemia and reperfusion is, however, speculative. While pretreatment with deferoxamine reduced infarct size produced by 2 h of coronary occlusion and 4 h of reflow, deferoxamine given at the time of reperfusion had no significant effect on the extent of myocyte necrosis when compared with saline controls. These data differ from previous in vitro results, in which " d e l a y e d " administration of deferoxamine preserved myocardial function, high energy phosphate stores and morphology in isolated perfused hearts. 16,17 The relative efficacy of " e a r l y " treatment (prior to or during occlusion) versus " d e l a y e d " treatment (at the time of reflow) on myocardial ischemia-reperfusion injury is an issue of both experimental interest and potential clinical importance. While treatment with scavenging agents or antioxidants prior to or during 60-90 min of coronary artery occlusion has, in most studies, resulted in an acute reduction in infarct size measured 4 - 2 4 hours after reperfusion, 8-1°12'~3 treatment at the time of reflow has produced conflicting results, j~.47.48 How can the difference in efficacy of deferoxamine pretreatment vs. deferoxamine given at reperfusion be explained? Our data may suggest that: 1) oxygen free radicals formed during the ischemic episode contribute to myocyte necrosis, whereas iron-catalyzed production of .OH and lipid peroxides at the time of reperfusion are not a significant cause of myocyte death in this model. Deferoxamine pretreatment may have reduced infarct size by chelating free iron released during ischemia, 3~ scavenging free radicals produced during coronary occlusion, 3z-36 and blunting the superoxidemediated release of iron from ferritin. 42 " D e l a y e d " treatment with deferoxamine at the time of reflow clearly would not have affected free radical production during the period of coronary artery occlusion. Alternatively, 2) the burst of free radicals thought to occur within seconds of reperfusion 49'5° may cause lethal myocyte injury. Treatment prior to occlusion would ensure that deferoxamine was " o n board" at the time of reflow. However, deferoxamine given only 5 min prior
Deferoxamine and infarct size to reperfusion m a y not have entered the ischemic area in sufficient concentrations to e f f e c t i v e l y chelate iron and limit the burst o f iron c a t a l y z e d .OH production in the initial m o m e n t s f o l l o w i n g r e o x y g e n a t i o n . Finally, 3) recent e v i d e n c e indicates that d e f e r o x a m i n e m a y have a b i p h a s i c , a n t i o x i d a n t / p r o o x i d a n t effect. 5~ That is, d e f e r o x a m i n e chelates free iron and reduces free r a d i c a l - m e d i a t e d injury when given in low doses. H o w e v e r , d e f e r o x a m i n e appears to p a r a d o x i c a l l y amplify or i n c r e a s e o x i d a t i v e d a m a g e in the presence o f reducing agents 5~ ( i . e . , such as 0 2 - ) in a d o s e - d e p e n dent manner. 51 In the present study, the same dose o f d e f e r o x a m i n e was used in both treatment groups; in fact, the total c u m u l a t i v e dose given during the entire protocol was l o w e r in animals treated at the time o f reflow than in animals that r e c e i v e d d e f e r o x a m i n e prior to c o r o n a r y occlusion. H o w e v e r , Borg et al. 5~ have postulated that d e f e r o x a m i n e m a y exhibit a p r o o x i d a tive effect in instances o f p r o l o n g e d and intense ischemia. That is, the o b s e r v e d reduction in infarct size in animals pretreated with d e f e r o x a m i n e vs. the absence of a beneficial effect in dogs treated at the time o f reperfusion m a y reflect a biphasic antioxidative effect if given p r e o c c l u s i o n versus p r o o x i d a t i v e effect if given f o l l o w i n g 2 h of c o r o n a r y artery occlusion. This study has focused on the potential role of iron in the p a t h o g e n e s i s o f i s c h e m i c / r e p e r f u s i o n injury. It is important to note, h o w e v e r , that free c o p p e r can also act as the transition metal catalyst for the production o f .OH. A l t h o u g h the total p h y s i o l o g i c concentration o f iron in the human (and perhaps canine) b o d y is c o n s i d e r a b l y greater than that o f c o p p e r , 52 c o p p e r is both more labile and more soluble than iron. 52 Thus, higher levels o f c o p p e r m a y be available to participate in metal c a t a l y z e d production o f "OH. 52 In addition, the rate constant for the reaction between Fe ÷ ÷ and U 2 0 2 is higher than the c o r r e s p o n d i n g rate constant for Cu ÷.52 C o p p e r clearly m a y also be an important mediator o f free r a d i c a l - i n d u c e d m y o c y t e injury, yet the effect o f c o p p e r chelating agents in the setting of isc h e m i a and reperfusion remains to be evaluated. In c o n c l u s i o n , results o f our r a n d o m i z e d and blinded study indicate that the potent iron chelator deferoxamine, a d m i n i s t e r e d prior to c o r o n a r y artery o c c l u s i o n , acutely r e d u c e d the extent o f m y o c y t e necrosis produced by two hours o f transient coronary artery occlusion and 4 h o f reperfusion in the anesthetized openchest dog, p r e s u m a b l y by limiting the i r o n - c a t a l y z e d f o r m a t i o n o f c y t o t o x i c h y d r o x y l radicals and lipid peroxides during occlusion a n d / o r during reperfusion. W h e t h e r this acute salutary effect o f d e f e r o x a m i n e pretreatment represents an absolute reduction in infarct s i z e - - o r m e r e l y d e l a y s the d e v e l o p m e n t of tissue nec r o s i s - r e m a i n s to be investigated. In contrast, defer-
51
o x a m i n e initiated at the time o f reperfusion had no significant beneficial effect on infarct size. A l t h o u g h d e f e r o x a m i n e given at the time o f reflow chelated free iron (as indicated by the total urine iron content in these animals), this " d e l a y e d " treatment may have been ineffective because 1) the i r o n - c a t a l y z e d production of .OH at the time of reperfusion m a y not be an important cause o f m y o c y t e necrosis in this canine model; 2) d e f e r o x a m i n e m a y not have entered the ischemic area in sufficient quantities to chelate the free iron prior to reflow; or 3) d e f e r o x a m i n e given at the time of reflow m a y have exerted a p a r a d o x i c a l prooxidant (rather than antioxidant) effect. The precise m e c h a n i s m o f action o f d e f e r o x a m i n e given preocclusion versus at the time o f reperfusion remains to be resolved. REFERENCES
I. McCord, J. M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159-163; 1985. 2. Thompson, J. A,; Hess, M. L. The oxygen free radical system: a fundamental mechanism in the production of myocardial necrosis. Prog. Cardiovasc. Diseases 28:449-462; 1986. 3. Werns, S. W.; Shea, M. J.; Lucchesi, B. R. Free radicals and myocardial injury: pharmacologic implications. Circulation 74:1-5; 1986. 4. Reimer, K. A.; Jennings, R. B. Failure of the xanthine oxidase inhibitor allopurinol to limit infarct size after ischemia and reperfusion in dogs. Circulation 71:1069-1075; 1985. 5. Puett, D. W.; Forman, M. B.; Cares, C. U.; Wilson, B. H.; Hande, K. R.; Friesinger. G. C.; Virmain, R. Oxypurinol limits myocardial stunning but does not reduce infarct size after reperfusion. Circulation 76:678-686; 1987. 6. Gallagher, K. P.; Buda. A. J.; Pace, D.; Gerren R. A.; Schlafer, M. Failure of superoxide dismutase and catalase to alter infarct size in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation 73:1065-1076; 1986. 7. Uraizee, A.; Reimer, K. A.; Murry, C. E.; Jennings, R. B. Failure of superoxide dismutase to limit size of myocardial infarction after 40 minutes of ischemia and 4 days of reperfusion in dogs. Circulation 75:1237-1248; 1987. 8. Jolly, S. R.; Kane, W. J.; Bailie, M. B.; Abrams, G. D.; Lucchesi, B. R. Canine myocardial reperfusion injury: its reduction by the combined administration of superoxide dismutase and catalase. Circ. Res. 54:277-285; 1984. 9. Werns, S. W.; Shea, M. J.; Driscoll, E. M.; Cohen, C.; Abrams, G. D.; Pitt, B.; Lucchesi, B. R. The independent effects of oxygen radical scavengers on canine infarct size: reduction by superoxide dismutase but not by catalase. Circ. Res. 56:895898; 1986. 10. Chambers, D. E.; Parks, D. A.; Patterson, G.; Roy, R.; McCord, J. M.; Yoshida, S.; Parmley, L. F.; Downey, J. M. Xanthine oxidase as a source of free radical damage in myocardial ischemia. J. Mol. Cell. Cardiol. 17:145-152; 1985. 11. Ambrosio, G.; Becker, L. C.; Hutchins, G. M.; Weisman, H. F.; Weisfeldt, M. L. Reduction in experimental infarct size by recombinant human superoxide dismutase: insights into the pathophysiology of reperfusion injury. Circulation 74:14241433; 1986. 12. Werns, S. W.; Shea, M. J.; Mitsos, S. E.; Dysko, R. C.; Fatone, J. C.; Shork, M. A. ; Abrams, G. D. ; Pitt, B.; Lucchesi, B. R. Reduction of the size of infarction by allopurinol in the ischemic-reperfused canine heart. Circulation 73:518-524; 1986. 13. Werns, S. W.; Grum, C. M.; Ventura, A.; Lucchesi, B. R. Effects of allopurinol or oxypurinol on myocardial reperfusion injury. Circulation 76(suppl IV):IV-197 abstr. (1987).
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14. Halliwell, B.; Gutteridge, J. M. C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219:1-14; 1984. 15. Myers, C. L.; Weiss, S. J.; Kirsh, M. M.; Schlafer, M. Involvement of hydrogen peroxide and hydroxyl radical in the 'oxygen paradox': reduction of creatine kinase release by catalase, allopurinol or deferoxamine, but not by superoxide dismutase. J. Mol. Cell. Cardiol. 17:675-684; 1985. 16. Badylak, S. F.; Simmons, A.; Turek, J.; Babbs, C. F. Protection from reperfusion injury in the isolated rat heart by postischemic deferoxamine and allopurinol administration. Cardiovasc. Res. 21:500-506; 1987. 17. Ambrosio, G.; Zweier, J. L.; Jacobus, W. E.; Weisfeldt, M. L.; Flaherty, J. T. Improvement of postischemic myocardial function and metabolism induced by administration of desferoxamine at the time of reflow: the role of iron in the pathogenesis of reperfusion injury. Circulation 76:906--915; 1987. 18. Bolli, R.; Patel, B. S.; Zhu, W. X.; O'Niell, P. G.; Charlat, M. L.; Roberts, R. The iron chelator desferrioxamine attenuates postischemic ventricular dysfunction. Am. J. Physiol. 253:HI372-H 1380; 1987. 19. Przyklenk, K.; Kloner, R. A. Superoxide dismutase plus catalase improve contractile function in the canine model of the "stunned myocardium." Circ. Res. 58:148-156; 1986. 20. Myers, M. L.; Bolli, R.; Lekich, R. F.; Hartley, C. J.; Roberts, R. Enhancement of recovery of myocardial function by oxygen free radical scavengers after reversible regional ischemia. Circulation 72:915-921; 1985. 21. Gross, G. J.; Farber, N. E.; Hardman, H. F.; Warltier, D. C. Beneficial actions of superoxide dismutase and catalase in stunned myocardium of dogs. Am. J. Physiol. 250:H372-H377; 1986. 22. Fishbein, M. C.; Meerbaum, S.; Rit, J.; Lando, U.; Kanmatsuse, K.; Mercier, J. C.; Corday, E.; Ganz, W. Early phase acute myocardial infarct size quantification: validation of the triphenyltetrazolium chloride tissue enzyme staining technique. Am. Heart. J. 101:593-600; 1981. 23. Vivaldi, M. T.; Kloner, R. A.; Schoen, F. J. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am. J. Pathol. 121:522-530; 1985. 24. van Stekelenburg, G. J.; Valk, C.; de Boer, G. J. Determination of deferoxamine chelated iron. Clin. Chem. 28:2328-2329: 1982. 25. Domenech, R. J.; Hoffman, J. E.; Noble, M. M.; Saunders, K. B.; Henson, R. J.; Subyanto, S. Total and regional coronary blood flow measured by radioactive microspheres in conscious and anesthetized dogs. Circ. Res. 25:581-596; 1969. 26. Wallenstein, S.; Zucker, C. L.; Fleiss, J. L. Some statistical methods useful in circulation research. Circ. Res. 47:1-9; 1980. 27. Burton, K. P. Superoxide dismutase enhances recovery following myocardial ischemia. Am. J. Physiol. 248:H637-H643; 1985. 28. Stewart, J. R.; Blackwell, W. H.; Crute, S. L.; Loughlin, V.; Hess, M. L.; Greenfield, L. J. Prevention of myocardial ischemic reperfusion injury with oxygen free radical scavengers. Surg. Forum. 33:317-320; 1982. 29. Burton, K. P.; McCord, J. M.: Ghai, G. Myocardial alterations due to free radical generation. Am. J. Physiol. 246:H776-H783; 1984. 30. Blaustein, A. S.; Schine, L.; Brooks, W. W.; Feinburg, B. L.; Bing, O. H, L. Influence of exogenously generated oxidant species on myocardial function. Am. J. Physiol. 250:H595H599; 1986. 31. Keberle, H. The biochemistry of desferrioxamine and its relation to iron metabolism. Ann. N.Y. Acad. Sci. 119:758-768; 1964. 32. Editorial. Metal chelation therapy, oxygen radicals, and human disease. Lancet 1:143-145; 1985. 33. McEnry, J. T. Hospital management of acute iron ingestion. Clin. Toxicol. 4:603-613; 1971.
34. Propper, R.; Nathan, D. Clinical removal of iron. Ann. Rev. Med. 33:509-519; 1982. 35. Willis, E. D. Lipid peroxide formation of microsomes: the role of non-haem iron. Biochem. J. 113:325-332; 1969. 36. Halliwell, B. Use of desferrioxamine as a probe for iron-dependent formation of .OH. Biochem. Pharmacol. 34:229-233; 1985. 37. Babbs, C. E Role of iron ions in the genesis of reperfusion injury following successful cardiopulmonary resuscitation: preliminary data and a biochemical hypothesis. Ann. Emerg. Med. 14:777-783: 1985. 38. Komara, J. S.; Nayini, N. R.; Bialick, H. A.; Indrieri, R. J.; Evans, A. T.; Garritano, A. M.; Hoehner, T. J.: Jacobs, W. A.; Huang, R. R.; Krause, G. S.: White, B. C.; Aust, S. D. Brain iron delocalization and lipid peroxidation following cardiac arrest. Ann. Emerg. Med. 15:384-389; 1986. 39. Farber, N. E.: Vercelloti, G. M.; Pieper, G. M.: Gross, G. J. Enhancement of postischemic metabolic and functional recovery of the stunned myocardium by desferoxamine. Circulation 76(suppl IV):IV-230 abstr. (1987). 40. Sirivech, S.; Frieden, E.; Osaki, S. The release of iron from horse spleen by reduced flavins. Biochem. J. 143:311-315; 1974. 41. Holt, S.; Gunderson, M.; Joyce, K. M.; Nayini, N. R.; Eyster, G. F.; Garritano, A. M.; Zonia, C. L.; Krause, G. S.: Aust, S. D.; White, B. C. Myocardial tissue iron delocalization and evidence for lipid peroxidation after two hours of ischemia. Ann. Emerg. Med. 14:499 abstr. (1985). 42. Biemond, P.; Swaak, A. J. G.; van Eijk, H. G.; Koster, J. F. Superoxide dependent iron release from ferritin in inflammatory diseases. Free Radical Biol. Med. 4:185-198; 1988. 43. Arroyo, C. M.; Kramer, J.; Lieboff, R.; Mergner, G.; Dickens, B.; Weglicki, W. Detection by electron spin resonance (ESR) spectroscopy with spin trapping of oxygen- and carbon-centered free radicals in ischemic canine myocardium. Fed. Proc. 46:410 abstr. (1987). 44. Kramer, J. A.; Arroyo, C. M.; Weglicki, W. B. Direct observation of free radical generation in ischemic hearts. Circulation 76(suppl IV):IV-199 abstr. (1987). 45. Akizuki, S.; Yoshida, S.; Chambers, C. E.; Eddy, L. J.; ParmIcy, L. F.; Yellon, D. L.; Downey, J. M. Infarct size limitation by the xanthine oxidase inhibitor allopurinol in closed-chest dogs with small infarcts. Cardiovasc. Res. 19:686-692: 1985. 46. Przyklenk, K.; Kloner, R. A. Effect of oxygen-derived free radical scavengers on infarct size following six hours of permanent coronary artery occlusion: salvage or delay of myocyte necrosis? Basic. Res. Cardiol. 82:146-158; 1987. 47. Nejima, J.; Canfield, D. R.; Manders, W. T.; Knight, D. R.; Cohen, M. V. ; Fallon, J. T.; Vatner, S. F. Failure of superoxide dismutase and catalase to alter size of infarct and functional recovery in conscious dogs with reperfusion. Circulation 76(suppl IV):IV-198 abstr. (1987). 48. Przyklenk, K.; Kloner, R. A. "Reperfusion injury" by oxygenderived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature, and regional myocardial blood flow. Circ. Res. 64:86-96; 1989. 49. Kramer, J. A.; Arroyo, C. M.; Dickens, B. F.; Weglicki, W. B. Spin-trapping evidence that graded myocardial ischemia alters postischemic superoxide production. Free Radical Biol. Med. 3:153-159; 1987. 50. Garlick, P. B.; Davies, M. J.; Hearse, D. J.; Slater, T. F. Direct detection of free radicals in reperfused rat heart using electron spin resonance spectroscopy. Circ. Res. 61:757-760; 1987. 51. Borg, D. C.; Schaich, K. M. Prooxidant action of desferrioxamine: fenton-like production of hydroxyl radicals by reduced ferriosamine. J. Free Radical Biol. Med. 2:237-243; 1986. 52. Chevion, M. A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals. Free Radical Biol. Med. 5:27-37; 1988.
0891-5849/89 $3.00+ .00 © 1989 Pergamon Press plc
Original Contribution I
EARLY TREATMENT WITH DEFEROXAMINE LIMITS MYOCARDIAL ISCHEMIC/REPERFUSION INJURY
B. RAMESH REDDY, ROBERT A . KLONER,* a n d KARIN PRZYKLENK*'t Ischemia Laboratory, Division of Cardiology, Harper Hospital and Wayne State University, Detroit, Michigan, U.S.A. (Received 20 July 1988; Revised 11 October 1988; Accepted 30 January 1989)
Abstract--Oxygen-derived free radicals (the superoxide anion 02- and hydroxyl radical "OH) have been implicated in myocardial injury associated with coronary artery occlusion followed by reperfusion. Transition metals (such as iron or copper) are needed to catalyze the formation of the .OH radical and subsequent .OH-mediated lipid peroxidation, yet the role of these transition metals in the pathogenesis of myocyte necrosis remains undefined. To address this issue, 21 dogs underwent 2 h of coronary artery occlusion and 4 h of reperfusion. Each animal was randomly assigned into 1 of 3 treatment groups: 7 received the iron chelator deferoxamine beginning 30 min preocclusion, 7 received deferoxamine beginning 5 min prior to reperfusion, while 7 dogs served as saline controls. Deferoxamine effectively chelated free iron in both treatment groups (total urine iron content averaged 42 -4- 16, 662 +-- 177' and 803 --- 205* micrograms in control, pretreated, and deferoxamine at reperfusion groups respectively; *p < 0.05), but had no significant effect on in vivo area at risk (AR), hemodynamic parameters, collateral blood flow during occlusion, or myocardial blood flow following reperfusion. Area of necrosis (AN) in dogs pretreated with deferoxamine (34.6 --- 3.7%* of the AR; *p < 0.05) was significantly smaller than that observed in the saline control group (55.4 + 4.7% of the AR). Deferoxamine administered at the time of reperfusion, however, had no significant effect on infarct size (AN/AR = 54.3 +-- 8.7%, p = NS vs. controls). Thus, early treatment with the iron chelator deferoxamine acutely reduced the extent of myocyte necrosis produced by 2 h of transient coronary artery occlusion in the canine model. These data support the hypothesis that iron catalyzed production of oxygen derived free radicals contribute to myocyte necrosis in the setting of ischemia-reperfusion. Keywords--Myocardial infarct size, Coronary occlusion, Reperfusion, Oxygen free radicals, Free radicals
dependent upon the presence of transitional metal ions such as Fe +++ and Cu++. 2,~4 In addition, the transitional metals also catalyze the .OH-mediated lipid peroxidation of cellular membranes, leading to the production of alkoxy- and peroxyradicals and subsequent increase in membrane fluidity and loss of membrane integrity. 2 This would suggest that the formation of free radicals and/or the perpetuation of free radicalmediated tissue injury could be aborted, or at least retarded, by chelating the free metal ions available to catalyze these reactions. The highly specific iron chelating agent deferoxamine has been shown to offer protection from ischemia/hypoxia-reperfusion injury in isolated rat heart preparations~5-w; however, the importance of free iron in an in vivo model of myocardial ischemia-reperfusion injury has not yet been evaluated. We therefore sought to determine whether the iron chelator deferoxamine, administered either prior to occlusion or at the time of reperfusion, could acutely reduce
INTRODUCTION
Oxygen-derived free radicals (the superoxide anion 02- and hydroxyl radical .OH) are cytotoxic and highly reactive molecules that are thought to be involved in the pathogenesis of myocardial ischemic-reperfusion injury. ~-3 Although results to date have been controversial, 4-7 several studies have demonstrated that timely administration of free radical scavenging agents and antioxidants such as superoxide dismutase plus catalase, allopurinol and oxypurinol can limit myocardial injury following prolonged periods of transient coronary occlusion. 8-~3. Production of the highly cytotoxic .OH radical is
*Current affiliation: Heart Institute, Hospital of the Good Samaritan, 616 South Witmer Street, Los Angeles, CA 90017, U.S.A. tCorrespondence should be addressed to: Karin Przyklenk, PhD, Heart Institute/Research, Hospital of the Good Samaritan, 616 South Witmer Street, Los Angeles, CA 90017, U.S.A. 45
46
B.R. REDDY et al.
the extent of myocyte necrosis in an in vivo canine model of prolonged (2 h) coronary artery occlusion followed by 4 h of reperfusion.
ued for the remainder of the study (10 mg/kg for the first hour, followed by a maintenance dose of 1.5 mg/ kg/hr).
METHODS
Surgical preparation Fifty-three mongrel dogs of either sex weighing an average of 21 --- 5 kg were anesthetized with sodium pentobarbital (30 mg/kg IV), intubated and ventilated with room air using a Harvard respirator. Cannulae were inserted into the left carotid artery for measurement of systolic and diastolic blood pressure and heart rate, and into the left jugular vein for administration of drugs and fluids. The urinary bladder was then catheterized and urine collected for measurement of urinary iron excretion. A left thoracotomy was performed through the fifth intercostal space, a pericardial cradle was constructed, and the left anterior descending coronary artery (LAD) was isolated for later occlusion, usually distal to its first major diagonal branch. A high fidelity microtipped catheter (Millar Instruments) was advanced into the left ventricular chamber via the left atrial appendage to measure left ventricular pressure and its first derivative, LV dP/dt. The left atrium was cannulated for later injection of radioactive microspheres for measurement of regional myocardial blood flow (RMBF). Protocol After obtaining baseline recordings of hemodynamic parameters, each dog was randomized in a blinded fashion into one of three treatment groups: 1. Deferoxamine pretreatment. Intravenous infusion of deferoxamine (deferoxamine mesylate; Desferal; Ciba-Geigy Ltd.) was initiated 30 min prior to coronary occlusion at an initial dose of 10 mg/kg for the first hour of the protocol, followed by a maintenance dose of 1.5 m g / k g / h r throughout the remainder of the protocol. This dosing regimen of deferoxamine has recently been shown to attenuate postischemic contractile dysfunction in the canine model of the "stunned myocardium ''~8 (a phenomenon thought to be due in part to the action of oxygen-derived free radicals L9-2~) without any apparent effect on hemodynamic parameters or RMBF.18 2. Deferoxamine at reperfusion. Saline was infused for 30 min prior to occlusion and throughout the 2 h period of LAD occlusion. Infusion of deferoxamine was initiated at 5 min prior to reperfusion and contin-
3. Control. Saline was infused for 30 min preocclusion, during LAD occlusion, and throughout reperfusion. All animals were randomized by drawing coded slips of paper from a box. If an experiment was unsuccessful (i.e., due to the death of the animal or exclusion from the protocol), the information was recorded and the coded slip returned to the randomization box. Experiments were continued in this manner until 7 dogs in each group had successfully completed the protocol. The randomization code and information was maintained by one investigator (K.P.) until all analysis had been completed. Thirty minutes after randomization and the onset of treatment, lidocaine (1.5 mg/kg IV) was administered and the LAD was occluded using atraumatic vascular clamps. RMBF was assessed at 30 min postocclusion by injection of microspheres labelled with either 95Nb, 1°3Ru o r 14ICe. Hemodynamic parameters were measured at 30 minutes, 1 h and 2 h postocclusion. After 2 h of occlusion, the LAD was abruptly reperfused by removal of the vascular clamps. Hemodynamic recordings were taken at regular hourly intervals, and a second radiolabelled isotope (i.e., one not chosen for the first injection) was used to measure RMBF at 2 h postreperfusion. At 4 h postreperfusion, the LAD was briefly reoccluded and monastral blue pigment (0.5 ml/kg) was injected into the coronary circulation via the left atrium to delineate the in vivo area at risk (AR). The dogs were then immediately sacrificed by intracardiac injection of KCl (20-40 mEq), and the hearts rapidly excised. Each heart was then cut into 5-7 transverse slices, approximately 5 mm in thickness, parallel to the atrioventricular groove. Epicardial and endocardial contours of the basal surfaces of the heart slices, and the margins of the AR (i.e., the areas unstained by monastral blue dye) were traced onto acetate sheets. The slices were then incubated in a 1% solution of triphenyltetrazolium chloride (TTC) for 10 rain at 37°C to distinguish necrotic from normal myocardium. Validation of the TTC technique has previously been reported. 22"23The heart slices, and boundaries of the area of necrosis (AN) within each slice (i.e., the areas unstained by TTC), were retraced onto the acetate sheets, and the hearts were then fixed by immersion in 10% neutral buffered formalin.
Deferoxamine and infarct size
Analysis After trimming off the right ventricular tissue, each heart slice was weighed. Contours of the LV, AR, and AN were cut from the tracings of the heart slices and weighed. AN and AR were then corrected for the weight of the tissue slice and summed for each heart. AR was then expressed as a percentage of the total LV weight (i.e., AR/LV), while AN was expressed as both a percentage of the AR (AN/AR) and percentage of the total LV (AN/LV). Hemodynamic parameters (heart rate, mean arterial pressure, LV dP/dt) were measured and averaged over 5 continuous cardiac cycles in normal sinus rhythm for each sample period. Approximately 200 ml of urine was collected from each dog during the course of the experiment. Total urinary iron content was measured from these samples by Roche Biomedical Laboratories using the ferrozine binding colorimetric method. 24 For calculation of RMBF, blocks of tissue were cut from both the center of the previously ischemic LAD bed and remote, nonischemic areas. These tissue blocks were then subdivided into endo-, mid-, and epicardial segments and RMBF quantified by the method of Domenech et al.25 All measurements were made by one investigator (B.R.R.) who remained blinded with respect to the treatment groups of each animal until the study had been completed.
Statistics All dogs with high collateral blood flow during coronary occlusion and a small AR (that is, animals that were not sufficiently ischemic to develop myocyte necrosis) were excluded from the final analysis. Specifically, our exclusion criteria, established prior to the onset of the study, were: values of RMBF in the "ischemic" endocardium > 0.15 ml/min/g tissue and/ or AR < 10% of the LV. In addition, dogs which did not appear to reperfuse successfully (i.e. did not exhibit characteristic hyperemic distention of the LAD upon removal of the vascular clamps) were excluded from analysis. No attempt was made to revive dogs that developed ventricular fibrillation during occlusion or during reperfusion. Comparison of hemodynamic parameters, RMBF, infarct size data, and urinary iron concentrations among the three treatment groups were made using analysis of variance (ANOVA). If significant differences among the groups were detected by ANOVA, pairwise comparisons were made using Tukey's test. 26 All values are quoted as mean --- SEM, and values are considered to differ significantly if p < 0.05.
47 RESULTS
Of the 53 dogs entered into the study, 24 died of ventricular fibrillation* during LAD occlusion. Specifically, 11 of 24 dogs (or 46%) initially randomized to receive deferoxamine preocclusion, 6 of 13 animals (46%) assigned to receive deferoxamine at reperfusion, and 6 of 15 randomized to the saline control group (40%) fibrillated during the ischemic period. In addition, 2 dogs (both pretreated with deferoxamine) died during reperfusion. Thus, treatment with deferoxamine did not protect against ventricular fibrillation due to coronary artery occlusion or reperfusion in this canine model. Four dogs were excluded from the study because they were not " i s c h e m i c " as defined by the exclusion criteria: mean endocardial RMBF during coronary occlusion was 0.35 ml/min/g tissue, and AN averaged < 1% of the LV in these animals. In addition, 2 dogs were excluded from analysis because they did not appear to reperfuse successfully upon removal of the vascular clamps, probably due to thrombus formation at the site of the occlusion. No hyperemic distention of the LAD was observed in these 2 animals at the time the clamps were removed, infarcts in both animals were essentially transmural (suggesting a period of occlusion > 2 h), and values of endocardial RMBF measured after 2 h of " r e f l o w " in these 2 dogs remained ischemic by our exclusion criteria, averaging only 0.13 ml/min/g tissue. Thus, a total of 21 dogs (n = 7 in each treatment group) are included in the final analysis.
Hemodynamic parameters (Table 1) Baseline values of heart rate, mean arterial pressure and LV dP/dt were comparable among the three groups prior to randomization and treatment. Infusion of deferoxamine had no apparent effect on any of these hemodynamic parameters, as heart rate, arterial pressure and dP/dt did not differ significantly among the three groups either preocclusion, during LAD occlusion, or following reperfusion.
Regional myocardial blood flow (Table 2) Values of RMBF in the ischemic LAD bed did not differ significantly among the three treatment groups in any myocardial layer. For example, RMBF to the ischemic endocardium at 30 min postocclusion averaged 0.01 +- 0.01, 0.04 +_ 0.02 and 0.03 -+ 0.02 ml/
*The assigned treatment group of one dog that fibrillated during occlusion was not recorded.
48
B. R. REDDY et al. Table 1. H e m o d y n a m i c P a ra me t e rs Occlusion
I. H e a r t Rate (beats/min): Control (n = 7) DF-Pre (n = 7) DF-at Re p (n = 7) II. Mean Arterial Pressure (ram Hg): Control (n = 7) DF-Pre (n = 7) DF-at R e p (n = 7) III. + LV d P / d t (mm Hg/sec): Control (n = 7) DF-Pre (n = 7) D F - a t R e p ( n = 7)
Reperfusion
Baseline
Pre-Occlusion
30'
120'
60"
120'
141 -+ 5 150 -+ 11 150 -+ 8
149 _+ 6 146 --- 11 150 -+ 8
142 -+ 5 147 _+ 4 151 _+ 7
136 _+ 4 143 -+- 10 155 -+ 6
141 -+ 5 150 -+ 10 152 + 7
144 + 5 147 -+ 7 157 +- 7
147_+ 5 150 + 9 157 +- 9
115 -+ 4 121 -+ 9 120 --- 8
120 -+ 5 123 -+ 10 119 -+ 7
117 -+ 6 121 -+ 10 120 -+ 5
111 _+ 9 117 _+ 9 121 _+ 5
112-8 112- 7 115 + 6
118-+6 112-+ 8 117 -+ 6
119-+6 108-+ 5 114 -+ 6
1443 -+ 142 1364 -+ 133 1467-+ 130
1607 +-- 151 1479 -+ 150 1492-+ 173
1400 _+ 144 1436 -+ 160 1483 _+ 123
1386 + 142 1307-+ 122 1325 - 89
1471 -+ 154 1300-+ 107 1450 + 135
1464 -+ 126 1279-+ 136 1442 + 111
1408 _+ 181 1421 _+ 158 1475-+ 79
240'
DF-Pre = Deferoxamine pretreatment. DF-at Rep = Deferoxamine given at reperfusion.
min/g tissue in the control, deferoxamine pretreatment and deferoxamine at reperfusion groups respectively (p = NS). Values of RMBF in the LAD bed during reperfusion were also comparable among the three treatment groups: in the previously ischemic endocardium, mean RMBF was 0.54 - 0.10, 0.64 + 0.11 and 0.58 -+ 0.09 m l / m i n / g tissue in the 3 groups respectively (p = NS). RMBF was also comparable among all groups in the remote, nonischemic tissue perfused by the circumflex bed, both during LAD occlusion and following reperfusion. Thus, deferoxamine had no significant effect on myocardial blood flow in this canine model. Urinary iron content (Fig. 1)
As anticipated 24, only small amounts of iron (42 --16 micrograms) were detected in the urine of control animals. In contrast, total urine iron content averaged 662 --- 177" micrograms in dogs pretreated with de-
feroxamine and 803 --- 205* micrograms in animals that received deferoxamine at the time of reperfusion (*p < 0.05 vs. controls). Thus, deferoxamine was equally effective in chelating free iron in both drugtreated groups. Infarct size and area at risk (Fig. 2)
Treatment with deferoxamine did not have a significant effect on area at risk in this canine model: AR/ LV was 17.2 +-_ 1.4%, 19.2 + 2.1% and 19.6 -+ 2.6% in the control, deferoxamine pretreatment and deferoxamine at reperfusion groups respectively (p = NS; Fig. 2). Area of necrosis averaged 55.4 + 4.7% of the AR in the saline control group. In contrast, A N / A R was reduced significantly in animals that had been pretreated with deferoxamine, averaging 34.6 + 3.7%* (*p < 0.05 vs. controls by ANOVA; Fig. 2). Deferoxamine given at the time of reperfusion, however,
Table 2. Regional Myocardial Blood Flow ( m l / m i n / g tissue) 30" Postocclusion
I. Ischemic L A D Bed: Control (n = 6) DF-Pre (n = 7) DF-at R e p (n = 7) II. R e m o t e Nonischemic Circumflex Bed: Control (n = 6) DF-Pre (n = 7) DF-at Rep (n = 7)
2 H ours Postreperfusion
ENDO
MID
EP I
ENDO
MID
EP I
0.01 +_ .01 0.04 --+ .02 0.03 --_ .02
0.04 4- .02 0.07 -+ .02 0.04 _+ .02
0.12 +- .05 0.24 _+ .06 0.15 -+ .07
0.54 --- .10 0.64 _+ .11 0.58 + .09
0.44 +- .02 0.64 - .06 0.49 -+ .09
0.59 - .09 0.81 -+ .12 0.89 -+ .12
1.34 _+ .21 1.38 _+ .22 1.62 _+ .23
1.22 _+ .28 1.34 _+ .24 1.68 -+ .24
1.04 -+ .15 1.18 +- .24 1.44 --- .23
1.15 _+ .13 1.32 _+ .18 1.60 -+- .33
1.09 -+ .13 1.26 --- .18 1.69 -+ .32
1.00 _+ .12 1.17 _+ .21 1.70 -+ .34
DF-Pre = Deferoxamine pretreatment. DF-at Rep = Deferoxamine given at reperfusion. ENDO = endocardial; MID = midmyocardial: EPI = epicardial.
Deferoxamine and infarct size URINE IRON LEVELS
E
1200
£
1ooo
,o
E
"-" ~E
£
400
",U-.
200
"6
dence suggests that the formation of the .OH radical and subsequent production of lipid peroxides, rather than O2- per se, may be the primary mediators of tissue injury in the myocardium. 29'3° One major pathway of •OH production is the two-step metal catalyzed reaction: 2
800 02
600 c-
49
0
+ Fe+++/Cu ÷÷
Fe++/Cu+ + HzO2
T
p.-
r---I CONTROLS
DF: PREOCCLUSION
1771 DF: AT REPERFUSION
Fig. 1. Totalurine ironcontent in controlanimals, dogsthat received deferoxamine preocclusion, and those treated with deferoxamine at the time of reperfusion. DF = deferoxamine;*p < 0.05 vs. controls.
had no significant effect on infarct size: mean A N / A R was 54.3 +-- 8.7% (p = NS vs. controls; Fig. 2). In summary, the iron chelator deferoxamine administered prior to two hours of coronary artery occlusion acutely reduced infarct size, assessed at 4 h postreperfusion, in the absence of any apparent effect on hemodynamic parameters, the degree of collateral blood flow during occlusion, or RMBF following reperfusion. Although deferoxamine given at the time of reperfusion also chelated free iron, this " d e l a y e d " treatment had no significant beneficial effect on myocyte necrosis in this model.
) Fe++/Cu ÷ + 02 >
Fe+++/Cu ++ + OH- + .OH
There are no specific physiologic or enzymatic defenses that act to scavenge the excess quantities of .OH thought to be produced in pathologic conditions such as ischemia-reperfusion. 2 The highly reactive .OH radicals may then attack unsaturated fatty acid side chains of cellular membranes and undergo numerous chain reactions, resulting in the formation of lipid radicals, organic oxygen radicals, and lipid peroxides. Lipid peroxides can, in the presence of transition metals such as iron, further decompose to form alkoxy- and peroxyradicals. 2 These free radical mediated alterations in the polyunsaturated side chains of the lipid components of cell membranes result in an increase in membrane fluidity and permeability and decrease in membrane integrity. 2 Iron and/or copper clearly play an important theoretical role in the mechanisms of tissue injury by free radicals: in the absence of transition metals, the rate constant for the metal catalyzed formation of .OH is essentially zero. 14 Deferoxamine, a trihydroxamic INFARCT SIZE AND AREA AT RISK
DISCUSSION
70
Considerable recent attention has been focused on the possible role of O2- (the superoxide anion), H202 (hydrogen peroxide) and .OH (the hydroxyl radical) in the pathogenesis of myocardial ischemia-reperfusion injury. To address this issue, several investigators have attempted to reduce the extent of myocyte necrosis by administration of free radical scavenging agents and antioxidants. 4-~3 While results have been controversial, 4-v early administration of superoxide dismutase (SOD) + catalase, 8 SOD alone, 9-~ allopurinol 1°42 and oxypurinop3--either prior to or during the period of ischemia--have been shown to acutely reduce infarct size produced by 6 0 - 9 0 min of ischemia and 4 - 2 4 h of reflow. Although treatment with the specific O2- scavenger SOD has been shown to have beneficial effects in both in v i v o 9-11 and in vitro models 27,28of ischemia/hypoxia followed by reperfusion/reoxygenation, in vitro evi-
60
T
50 40
2O 10 0
AN/AR (g)
F"-I CONTROLS
AN/LV (~)
AR/LV (~)
DF: PREOCCLUSION
17"-21 DF: AT REPERFUSION
Fig. 2. Area of necrosis (AN) expressed as a percentage of the area at risk (AR), AN expressed as a percentage of the total LV weight, and AR expressed as a percentage of the LV for control animals, dogs that receiveddeferoxaminepreocclusion, and those treated with deferoxamine at the time of reperfusion. DF = deferoxamine; *p < 0.05 vs. controls.
50
B . R . REDDY et al.
acid, is a potent chelator of free iron. 31 It is commonly used in the clinical treatment of acute iron intoxication and chronic iron overload and, in spite of some instances of deferoxamine toxicity, 32 is generally well tolerated. 33,34In addition to its capacity to chelate free iron, deferoxamine also inhibits lipid peroxidation 32'35 and acts as a scavenger of free radicals? 2'36 Thus, timely administration of this potent chelating and free radical scavenging agent may attenuate tissue injury in pathologic conditions associated with increased free radical production such as ischemia followed by reperfusion. Deferoxamine given at the time of cardiac resuscitation has previously been shown to reduce ischemiareperfusion injury to the brain and improve survival in an anesthetized rat model of cardiac arrest. 37 Similar beneficial results (i.e., reduced levels of conjugated dienes and malondialdehyde, a degradation product of lipid peroxidation) were noted in brain tissue of dogs resuscitated from cardiac arrest? 8 More recently, pretreatment with deferoxamine has been shown to enhance recovery of regional contractile function following a brief (15 min) period of transient coronary artery occlusion in the canine model of the stunned myocardium, ~8"39 a phenomenon thought to be due at least in part to the action of free radicals. ~9--'~However, iron chelation and its possible beneficial effects in the setting of prolonged periods of myocardial ischemia/ hypoxia followed by reflow have, to date, only been studied using isolated perfused heart models. For example, Myers et al. J5 found that deferoxamine, given during a 60 min period of global normothermic hypoxia, significantly reduced the release of creatine kinase following reoxygenation. Similar in vitro studies have demonstrated that " d e l a y e d " treatment with deferoxamine (i.e., added to the perfusate at the time of reoxygenation) lowered vascular resistance,~6 blunted release of creatine kinase, ~6 increased myocardial phosphocreatine content j7 and enhanced recovery of left ventricular developed pressure 17 when compared with controls. In addition, Badylak et a1.16 noted that the ultrastructure of the mitochondria, endoplasmic reticulum, and cell membranes were in part preserved by " d e l a y e d " administration of deferoxamine. Our observation that pretreatment with deferoxamine acutely limited the extent of myocyte necrosis produced by 2 h of regional ischemia and 4 h of reflow is in good agreement with these previous in vitro studies, L5-17and is similar to the beneficial results obtained using early treatment with free radical scavengers and antioxidants. 8-~3 Furthermore, our data suggest that iron may play an important role in the pathogenesis of myocyte necrosis in this canine model. What is the source of free iron in the setting of
myocardial ischemia/reperfusion? Iron is released from ferritin under anaerobic conditions; 4° thus, free iron may also accumulate during prolonged periods of coronary artery occlusion. In fact, an increase in nonprotein bound low molecular weight iron has been documented in the myocardium of dogs subjected to 2 h of regional ischemia. 4~ In addition, superoxide anions release iron from ferritin. 42 As both direct 43'44 and indirect evidence 45~46suggests that oxygen free radicals are generated during coronary artery occlusion, this superoxide-mediated iron release may be an important factor in our model. The precise source of free iron in myocardia ischemia and reperfusion is, however, speculative. While pretreatment with deferoxamine reduced infarct size produced by 2 h of coronary occlusion and 4 h of reflow, deferoxamine given at the time of reperfusion had no significant effect on the extent of myocyte necrosis when compared with saline controls. These data differ from previous in vitro results, in which " d e l a y e d " administration of deferoxamine preserved myocardial function, high energy phosphate stores and morphology in isolated perfused hearts. 16,17 The relative efficacy of " e a r l y " treatment (prior to or during occlusion) versus " d e l a y e d " treatment (at the time of reflow) on myocardial ischemia-reperfusion injury is an issue of both experimental interest and potential clinical importance. While treatment with scavenging agents or antioxidants prior to or during 60-90 min of coronary artery occlusion has, in most studies, resulted in an acute reduction in infarct size measured 4 - 2 4 hours after reperfusion, 8-1°12'~3 treatment at the time of reflow has produced conflicting results, j~.47.48 How can the difference in efficacy of deferoxamine pretreatment vs. deferoxamine given at reperfusion be explained? Our data may suggest that: 1) oxygen free radicals formed during the ischemic episode contribute to myocyte necrosis, whereas iron-catalyzed production of .OH and lipid peroxides at the time of reperfusion are not a significant cause of myocyte death in this model. Deferoxamine pretreatment may have reduced infarct size by chelating free iron released during ischemia, 3~ scavenging free radicals produced during coronary occlusion, 3z-36 and blunting the superoxidemediated release of iron from ferritin. 42 " D e l a y e d " treatment with deferoxamine at the time of reflow clearly would not have affected free radical production during the period of coronary artery occlusion. Alternatively, 2) the burst of free radicals thought to occur within seconds of reperfusion 49'5° may cause lethal myocyte injury. Treatment prior to occlusion would ensure that deferoxamine was " o n board" at the time of reflow. However, deferoxamine given only 5 min prior
Deferoxamine and infarct size to reperfusion m a y not have entered the ischemic area in sufficient concentrations to e f f e c t i v e l y chelate iron and limit the burst o f iron c a t a l y z e d .OH production in the initial m o m e n t s f o l l o w i n g r e o x y g e n a t i o n . Finally, 3) recent e v i d e n c e indicates that d e f e r o x a m i n e m a y have a b i p h a s i c , a n t i o x i d a n t / p r o o x i d a n t effect. 5~ That is, d e f e r o x a m i n e chelates free iron and reduces free r a d i c a l - m e d i a t e d injury when given in low doses. H o w e v e r , d e f e r o x a m i n e appears to p a r a d o x i c a l l y amplify or i n c r e a s e o x i d a t i v e d a m a g e in the presence o f reducing agents 5~ ( i . e . , such as 0 2 - ) in a d o s e - d e p e n dent manner. 51 In the present study, the same dose o f d e f e r o x a m i n e was used in both treatment groups; in fact, the total c u m u l a t i v e dose given during the entire protocol was l o w e r in animals treated at the time o f reflow than in animals that r e c e i v e d d e f e r o x a m i n e prior to c o r o n a r y occlusion. H o w e v e r , Borg et al. 5~ have postulated that d e f e r o x a m i n e m a y exhibit a p r o o x i d a tive effect in instances o f p r o l o n g e d and intense ischemia. That is, the o b s e r v e d reduction in infarct size in animals pretreated with d e f e r o x a m i n e vs. the absence of a beneficial effect in dogs treated at the time o f reperfusion m a y reflect a biphasic antioxidative effect if given p r e o c c l u s i o n versus p r o o x i d a t i v e effect if given f o l l o w i n g 2 h of c o r o n a r y artery occlusion. This study has focused on the potential role of iron in the p a t h o g e n e s i s o f i s c h e m i c / r e p e r f u s i o n injury. It is important to note, h o w e v e r , that free c o p p e r can also act as the transition metal catalyst for the production o f .OH. A l t h o u g h the total p h y s i o l o g i c concentration o f iron in the human (and perhaps canine) b o d y is c o n s i d e r a b l y greater than that o f c o p p e r , 52 c o p p e r is both more labile and more soluble than iron. 52 Thus, higher levels o f c o p p e r m a y be available to participate in metal c a t a l y z e d production o f "OH. 52 In addition, the rate constant for the reaction between Fe ÷ ÷ and U 2 0 2 is higher than the c o r r e s p o n d i n g rate constant for Cu ÷.52 C o p p e r clearly m a y also be an important mediator o f free r a d i c a l - i n d u c e d m y o c y t e injury, yet the effect o f c o p p e r chelating agents in the setting of isc h e m i a and reperfusion remains to be evaluated. In c o n c l u s i o n , results o f our r a n d o m i z e d and blinded study indicate that the potent iron chelator deferoxamine, a d m i n i s t e r e d prior to c o r o n a r y artery o c c l u s i o n , acutely r e d u c e d the extent o f m y o c y t e necrosis produced by two hours o f transient coronary artery occlusion and 4 h o f reperfusion in the anesthetized openchest dog, p r e s u m a b l y by limiting the i r o n - c a t a l y z e d f o r m a t i o n o f c y t o t o x i c h y d r o x y l radicals and lipid peroxides during occlusion a n d / o r during reperfusion. W h e t h e r this acute salutary effect o f d e f e r o x a m i n e pretreatment represents an absolute reduction in infarct s i z e - - o r m e r e l y d e l a y s the d e v e l o p m e n t of tissue nec r o s i s - r e m a i n s to be investigated. In contrast, defer-
51
o x a m i n e initiated at the time o f reperfusion had no significant beneficial effect on infarct size. A l t h o u g h d e f e r o x a m i n e given at the time o f reflow chelated free iron (as indicated by the total urine iron content in these animals), this " d e l a y e d " treatment may have been ineffective because 1) the i r o n - c a t a l y z e d production of .OH at the time of reperfusion m a y not be an important cause o f m y o c y t e necrosis in this canine model; 2) d e f e r o x a m i n e m a y not have entered the ischemic area in sufficient quantities to chelate the free iron prior to reflow; or 3) d e f e r o x a m i n e given at the time of reflow m a y have exerted a p a r a d o x i c a l prooxidant (rather than antioxidant) effect. The precise m e c h a n i s m o f action o f d e f e r o x a m i n e given preocclusion versus at the time o f reperfusion remains to be resolved. REFERENCES
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34. Propper, R.; Nathan, D. Clinical removal of iron. Ann. Rev. Med. 33:509-519; 1982. 35. Willis, E. D. Lipid peroxide formation of microsomes: the role of non-haem iron. Biochem. J. 113:325-332; 1969. 36. Halliwell, B. Use of desferrioxamine as a probe for iron-dependent formation of .OH. Biochem. Pharmacol. 34:229-233; 1985. 37. Babbs, C. E Role of iron ions in the genesis of reperfusion injury following successful cardiopulmonary resuscitation: preliminary data and a biochemical hypothesis. Ann. Emerg. Med. 14:777-783: 1985. 38. Komara, J. S.; Nayini, N. R.; Bialick, H. A.; Indrieri, R. J.; Evans, A. T.; Garritano, A. M.; Hoehner, T. J.: Jacobs, W. A.; Huang, R. R.; Krause, G. S.: White, B. C.; Aust, S. D. Brain iron delocalization and lipid peroxidation following cardiac arrest. Ann. Emerg. Med. 15:384-389; 1986. 39. Farber, N. E.: Vercelloti, G. M.; Pieper, G. M.: Gross, G. J. Enhancement of postischemic metabolic and functional recovery of the stunned myocardium by desferoxamine. Circulation 76(suppl IV):IV-230 abstr. (1987). 40. Sirivech, S.; Frieden, E.; Osaki, S. The release of iron from horse spleen by reduced flavins. Biochem. J. 143:311-315; 1974. 41. Holt, S.; Gunderson, M.; Joyce, K. M.; Nayini, N. R.; Eyster, G. F.; Garritano, A. M.; Zonia, C. L.; Krause, G. S.: Aust, S. D.; White, B. C. Myocardial tissue iron delocalization and evidence for lipid peroxidation after two hours of ischemia. Ann. Emerg. Med. 14:499 abstr. (1985). 42. Biemond, P.; Swaak, A. J. G.; van Eijk, H. G.; Koster, J. F. Superoxide dependent iron release from ferritin in inflammatory diseases. Free Radical Biol. Med. 4:185-198; 1988. 43. Arroyo, C. M.; Kramer, J.; Lieboff, R.; Mergner, G.; Dickens, B.; Weglicki, W. Detection by electron spin resonance (ESR) spectroscopy with spin trapping of oxygen- and carbon-centered free radicals in ischemic canine myocardium. Fed. Proc. 46:410 abstr. (1987). 44. Kramer, J. A.; Arroyo, C. M.; Weglicki, W. B. Direct observation of free radical generation in ischemic hearts. Circulation 76(suppl IV):IV-199 abstr. (1987). 45. Akizuki, S.; Yoshida, S.; Chambers, C. E.; Eddy, L. J.; ParmIcy, L. F.; Yellon, D. L.; Downey, J. M. Infarct size limitation by the xanthine oxidase inhibitor allopurinol in closed-chest dogs with small infarcts. Cardiovasc. Res. 19:686-692: 1985. 46. Przyklenk, K.; Kloner, R. A. Effect of oxygen-derived free radical scavengers on infarct size following six hours of permanent coronary artery occlusion: salvage or delay of myocyte necrosis? Basic. Res. Cardiol. 82:146-158; 1987. 47. Nejima, J.; Canfield, D. R.; Manders, W. T.; Knight, D. R.; Cohen, M. V. ; Fallon, J. T.; Vatner, S. F. Failure of superoxide dismutase and catalase to alter size of infarct and functional recovery in conscious dogs with reperfusion. Circulation 76(suppl IV):IV-198 abstr. (1987). 48. Przyklenk, K.; Kloner, R. A. "Reperfusion injury" by oxygenderived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature, and regional myocardial blood flow. Circ. Res. 64:86-96; 1989. 49. Kramer, J. A.; Arroyo, C. M.; Dickens, B. F.; Weglicki, W. B. Spin-trapping evidence that graded myocardial ischemia alters postischemic superoxide production. Free Radical Biol. Med. 3:153-159; 1987. 50. Garlick, P. B.; Davies, M. J.; Hearse, D. J.; Slater, T. F. Direct detection of free radicals in reperfused rat heart using electron spin resonance spectroscopy. Circ. Res. 61:757-760; 1987. 51. Borg, D. C.; Schaich, K. M. Prooxidant action of desferrioxamine: fenton-like production of hydroxyl radicals by reduced ferriosamine. J. Free Radical Biol. Med. 2:237-243; 1986. 52. Chevion, M. A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals. Free Radical Biol. Med. 5:27-37; 1988.