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1 Division of Cardiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21224; 2 Federico II School of Medicine, 80131 Naples; and 3 University of Perugia School of Medicine, 06122 Perugia, Italy
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Although many studies document oxygen radical formation during ischemia-reperfusion, few address the sources of radicals in vivo or examine radical generation in the context of prolonged ischemia. In particular, the contribution of activated neutrophils remains unclear. To investigate this issue, we developed a methodology to detect radicals without interfering with blood-borne mechanisms of radical generation. Dogs underwent aorta and coronary sinus catheterization. No chemicals were infused; instead, blood was drawn into syringes prefilled with a spin trap and analyzed by electron paramagnetic resonance spectroscopy. After 90 min of coronary artery occlusion, transcardiac concentration of oxygen radicals rose severalfold 10 min after reflow and remained significantly elevated for at least 1 h. Radicals were mostly derived from neutrophils, as shown by marked reduction after the administration of 1) neutrophil NADPH oxidase inhibitors and 2) a monoclonal antibody (R15.7) against neutrophil CD18 adhesion molecule. Reduction of radical generation by R15.7 was also associated with a significantly smaller infarct size and no-reflow areas. Thus our data demonstrate that neutrophils are a major source of oxidants in hearts reperfused in vivo after prolonged ischemia and that antineutrophil interventions can effectively prevent the increase in oxygen radical concentration during reperfusion.
reperfusion injury; electron paramagnetic resonance; myocardial infarction
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IT IS WELL ESTABLISHED that postischemic reflow is associated with generation of oxygen radicals (5, 7, 38) and that the oxidant stress occurring during reperfusion may produce deleterious effects in reperfused hearts (20). However, important aspects remain incompletely defined, such as the role of neutrophils as a source of oxygen radicals. Documentation that activated neutrophils can contribute to oxygen radical formation during reperfusion comes from in vitro experiments in isolated, neutrophil-perfused hearts (22, 31). Yet, despite the widely held notion that neutrophil activation plays a major role in reperfusion injury (12, 30), the role of neutrophil-derived oxygen radicals has never been directly investigated in vivo. Moreover, most studies of oxygen radical formation have utilized relatively brief episodes of ischemia resulting in myocardial stunning (5-7, 37), whereas neutrophil-mediated damage is thought to become prominent after prolonged ischemia associated with myocardial infarction (1, 12, 29, 30).
Another factor limiting our understanding of the role of neutrophils is the lack of suitable methodology to investigate the sources of oxygen radical formation in vivo. Typically, radicals are detected by electron paramagnetic resonance (EPR) spectroscopy, either of tissue specimens quickly frozen in liquid nitrogen (2, 3, 38) or of cardiac effluent collected after intracoronary administration of chemicals, which react to form stable radical adducts ("spin traps") (3, 5-9, 17). However, both methodologies have significant limitations. Biopsies cannot be performed often enough to provide a sufficiently detailed time course of oxygen radical production, whereas the administration of spin traps interferes with the very process of radical formation and its consequences because, in reacting with radicals, spin traps behave like scavengers (7, 17). This characteristic obviously interferes with precise evaluation of the role of blood-borne elements such as neutrophils. Infusion of salicylate or phenylalanine, which form stable derivatives with hydroxyl radicals (15), has also been used (28, 36), but these chemicals share with spin traps the characteristic of acting like scavengers (36).
To address these issues, we took advantage of previous work by Mergner et al. (24), who showed the feasibility of measuring oxygen radicals in vivo in the cardiac effluent. However, their technique required surgical exposure and cannulation of a coronary vein and infusion of a spin trap. We employed instead a minimally invasive approach based on coronary sinus catheterization and subsequent withdrawing of blood in a syringe-containing spin trap. After successful accomplishment and extensive validation of the technique, we used it to investigate the role of neutrophils in cardiac production of oxygen radicals after prolonged ischemia and reperfusion.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Adult mongrel dogs of either sex (20-25 kg) were used. Experiments were performed in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" from the Declaration of Helsinki and the American Physiological Society.
Surgical Preparation
Open-chest experiments.
Dogs were anesthetized with an intravenous injection of 7% thiopental
followed by the intramuscular administration of 
-chloralose (14 mg/kg) and urethan (140 mg/kg), intubated, and mechanically ventilated
with air. A catheter was advanced in the right iliac artery for
hemodynamic measurements and for withdrawal of aortic blood. After a
left thoracotomy, the heart was suspended in a pericardial cradle.
Catheters were placed in the left atrium (for microsphere injection)
and in the coronary sinus (for blood sampling). Heparinized saline was
used to flush the lines. The left anterior descending coronary artery
(LAD) was isolated distal to the first diagonal branch. After baseline
measurements, the LAD was occluded by a snare that was removed 90 min
later. Animals were reperfused for 1-3 h according to the specific
protocol (see Experimental Protocol).
Closed-chest experiments. Closed-chest dogs underwent coronary artery occlusion-reperfusion and coronary sinus sampling via right- and left-sided catheterization. Dogs were anesthetized with intravenous thiopental (7%) and ventilated with halothane (1-2%) in room air. Animals were anticoagulated with heparin (45 U/kg iv). Sheaths (8-Fr) were placed in both femoral arteries. Under fluoroscopic guidance, a 6-Fr pigtail catheter was passed through the left sheath into the left ventricle for microsphere injection. The sidearm was used for hemodynamic measurements and for withdrawal of arterial blood. A 7-Fr guiding catheter was introduced through the right sheath and advanced to the aortic root. A 6-Fr multipurpose catheter with side holes (B2, Mallinckrodt) was placed in the coronary sinus from the right jugular vein for blood sampling. After baseline measurements, an angioplasty catheter (balloon diameter 2.5-3.5 mm) was inserted through the guiding catheter into the proximal LAD. The LAD was then occluded by inflating the balloon to 3-5 atm. After 90 min, the balloon was deflated. Coronary angiography was performed before and shortly after coronary occlusion, just before balloon deflation, and after reperfusion to confirm that complete arterial occlusion and reperfusion were achieved.
Regional Myocardial Blood Flow
Myocardial blood flow was measured at various time points (see Experimental Protocols). For each determination, 2 × 106 radioactive microspheres (10-15 µm, NEN-Track, New England Nuclear), labeled with 46Sc, 103Ru, 113Sn, or 153Gd, were injected into the left atrium or ventricle, followed by a 10-ml saline flush. Starting just before injection and continuing for 2 min, a reference arterial blood sample was withdrawn by a Harvard pump at a constant rate of 2.6 ml/min. After euthanization of the animal, tissue sampling for regional myocardial blood flow was made transmurally in ischemic and nonischemic areas of the left ventricle. Samples were weighed and counted for radioactivity with reference blood samples in a scintillation counter at appropriate energy windows. Regional myocardial blood flow was calculated by standard methods (19).No-reflow Area, Risk Region, and Infarct Size
To visualize myocardium with impaired perfusion (no-reflow area), a fluorescent dye (1 ml/kg thioflavin-S) was injected into the left atrium at the end of the experiment (1). Thioflavin-S was prepared daily as a 4% solution in warm (37°C) saline, centrifuged to remove particulate matter, and kept shielded from light. Thirty seconds after thioflavin-S injection, the LAD was reoccluded, and a bolus of 1 ml/kg monastral blue was injected into the left atrium to delineate risk region. The heart was arrested by an overdose of KCl; the right ventricle and atria were then removed, and the left ventricle was cut into five to six slices parallel to the atrioventricular groove. The contour of each slice and of risk region was traced onto a transparent acetate sheet. Subsequently, tissue slices were put under ultraviolet light, and the area not perfused by thioflavin-S (no-reflow area) was visualized and traced. To determine the extent of the infarcted area, the slices were then incubated for 20 min at 37°C in 1% triphenyltetrazolium chloride (TTC) in phosphate-buffered saline. The TTC-negative (necrotic) area was traced. Extent of no-reflow area, risk region, and infarcted area was calculated by planimetry.Blood Sampling for Oxygen Radical Measurements
Blood samples (2 ml) were withdrawn simultaneously from the femoral artery and the coronary sinus (at baseline, 85 min into ischemia, and at various time points during reperfusion) into syringes prefilled with 1 ml of the spin trap N-tert-butyl--phenyl-nitrone (PBN, 50 mmol/l). Samples
were gently mixed by inverting the syringe several times over 10 s
and then immediately frozen by immersion in liquid nitrogen. For each
experiment, PBN was dissolved in the dark in cold NaCl solution (150 mmol/l) and stored shielded from light on ice.
EPR Spectroscopy
Samples were thawed on ice and transferred into glass tubes, combined with 1.5 ml spectroscopic-grade toluene, gently shaken 10 times, and centrifuged for 5 min at 4,000 rpm at 4°C. The upper phase was collected on ice and used for EPR analysis. EPR spectra were recorded in flat cells at room temperature with a Bruker-IBM ER300 spectrometer operating at X-band with a TM110 cavity. The instrument was set at modulation frequency 100 kHz, modulation amplitude 0.5 G, time constant 0.3 s, scan time 60 s, and microwave power 20 mW. Radical signals were quantitated by comparing the double integral of the first derivative of the signal with that of known concentrations of 2,2,6,6-tetramethyl-piperidinoxy (TEMPO) radical (21). EPR spectroscopy was typically performed within 48 h of sample collection. However, in eight parallel experiments, we determined that samples maintained 1 mo at
80°C under N2 retained 95 ± 6% of the signal measured immediately after collection.
Characterization of EPR Signals
To investigate the reaction of a known source of radicals with PBN under the conditions of our study, dog blood samples containing PBN were exposed to hydroxyl radicals generated in the reaction of H2O2 (500 µmol/l) with ammonium-iron(II)-sulfate (100 µmol/l). After 60-s incubation, samples were either immediately processed or extracted with toluene as described above and analyzed by EPR.In additional experiments, we investigated which oxidant species was responsible for the signal observed in coronary sinus blood. In control dogs from the neutrophil arm of the study (see Role of neutrophils in oxygen radical formation and tissue injury), blood was withdrawn from the coronary sinus 15 min after reperfusion (at the time near peak radical generation) into syringes containing, in addition to PBN, one of the following: 1) the superoxide-radical scavenger superoxide dismutase (SOD; 630 units); 2) the iron-chelator deferoxamine (900 nM); 3) the H2O2 scavenger catalase (1,650 units); or 4) the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 55 µM in DMSO 0.5%) (16, 26).
Experimental Protocol
Oxygen radical measurements in open-chest dogs. Six dogs were used for this protocol. At baseline, blood was collected from the coronary sinus and aorta. The LAD was then occluded, and blood samples were again taken 85 min into ischemia. After 90 min, the LAD was reopened, and blood samples were taken at 1, 5, 10, 20, 30, and 60 min of reperfusion. Myocardial blood flow was measured at baseline and 80 min into ischemia.
Oxygen radical measurements in closed-chest dogs. Seven dogs were used for this protocol. After baseline collection of blood from the coronary sinus and aorta, the LAD was occluded by inflating the percutaneous transluminal coronary angioplasty (PTCA) balloon. Blood samples were taken again 85 min into ischemia. After 90 min, the balloon was deflated, the catheter withdrawn, and blood samples were taken at 1, 5, 10, 20, 30, and 60 min of reperfusion. Myocardial blood flow was measured at baseline and 80 min into ischemia.
Role of neutrophils in oxygen radical formation and tissue injury. effect of nadph oxidase inhibition. In this set of experiments, we tested whether in vivo inhibition of radical formation by neutrophils would reduce radical concentration in the coronary sinus. Twelve open-chest dogs undergoing 90 min of LAD occlusion followed by reperfusion were randomly allocated to receive saline (controls) or ethyl-gallimidate (VF244), an inhibitor of NADPH oxidase (25, 40). VF244 was provided by Dr. Howard Elford (Molecules For Health). The drug was dissolved in saline and administered as a slow bolus (30 mg/kg iv) 10 min before reperfusion, followed by an additional 15 mg/kg over 1 h. Myocardial blood flow was measured at baseline and 80 min into ischemia. Samples for oxygen radical measurements were collected from the aorta and coronary sinus at baseline, 85 min into ischemia, and at 1, 5, 10, 20, 30, and 60 min of reperfusion.
EFFECT OF ANTINEUTROPHIL ANTIBODY. Open-chest dogs undergoing 90 min of LAD occlusion followed by 180 min of reperfusion were randomly allocated to receive saline (controls, n = 9 dogs) or R15.7, a monoclonal antibody against the CD18 neutrophil adhesion complex (n = 8 dogs) (4, 13, 32). The antibody was administered (1 mg/kg iv bolus) 10 min before reperfusion. Myocardial blood flow was measured at baseline, 80 min into ischemia, and 10 and 180 min after reperfusion. Samples for oxygen radical measurements were collected from the aorta and the coronary sinus at baseline, 85 min into ischemia, and at 1, 5, 10, 20, 30, 60, and 120 min of reperfusion. At the end of the experiment, no-reflow, risk region, and infarct size were measured. The murine monoclonal antibody R15.7, an immunoglobulin G1 that recognizes a functional epitope on CD18 on dog neutrophils (4, 13, 32), was provided by Dr. Robert Rothlein (Boehringer Ingelheim Pharmaceuticals). Antibody concentration was 12.17 mg/ml; the solution contained <1 U/mg of endotoxin. In this experimental model, this dose of R15.7 results in ~90% saturation of neutrophil binding sites in vivo, which persists for at least 24 h, with no effect on platelet count (4). Blockade of CD11/CD18 complex on neutrophils by R15.17 results in almost complete inhibition of integrin-mediated adherence and oxidant production (4, 13, 32).Chemicals
The following were purchased from Sigma: copper/zinc SOD (bovine erythrocytes, specific activity 4,000 U/mg protein), catalase (bovine liver, specific activity 5,000 U/mg protein), deferoxamine, DMSO, H2O2 (30% wt/wt solution), TEMPO, thioflavin-S, TTC, and DPI. Ammonium-iron(II)-sulfate (99.997% purity) and PBN were from Aldrich Chemical, and Monastral blue was from Heubach.Statistical Analysis
Data are expressed as means ± SE. Repeated-measures ANOVA was used to test the time course of radical production. Comparison between controls and R15.7-treated dogs was performed by repeated-measures ANOVA with a design for orthogonal comparisons. When the overall trend was significant, specific time points were tested by post hoc analysis. Infarct size data were compared by Student's t-test for unpaired samples.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Oxygen Radical Measurements in Open-chest Dogs
A trace EPR signal was detected in coronary sinus blood at baseline (Fig. 1A), and a small increase was observed during coronary artery occlusion (collateral blood flow 7.8 ± 3.3% of baseline) (Fig. 1B). In contrast, a prominent signal was observed in the coronary sinus blood collected during postischemic reperfusion (Fig. 1C). Radical generation was negligible immediately on reflow, but then it rose markedly, reaching a peak 10 min after reperfusion (Fig. 2). Oxygen radical concentration remained significantly higher than baseline throughout 60 min of reperfusion (Fig. 2).
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Characterization of EPR Signal
The spectral characteristics observed in blood samples collected from the coronary sinus consisted of a broad triplet with nitrogen hyperfine coupling constant aN = 14.35 Gauss. To better characterize the signal obtained, in parallel experiments, blood samples containing PBN were exposed ex vivo to a source of oxygen radicals. This process yielded spectra with EPR characteristics similar to those observed in samples taken in vivo from the coronary sinus (Fig. 3A). Signal characteristics were largely unaffected by the extraction procedure with toluene (Fig. 3B). Computer simulation of the observed spectra demonstrated triplet hyperfine splitting from nitrogen, aN = 14.35 G, and a poorly resolved hydrogen splitting aH = 2.64 G (Fig. 3C).
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Further characterization of the EPR signal was accomplished in coronary
sinus blood collected in syringes containing PBN and treated ex vivo
with different antiradical agents (Table
1). Oxygen radical concentration was
~90% lower in samples incubated with the superoxide-radical
scavenger SOD (P < 0.05; Table 1). Marked reduction of
radical concentration was also obtained with either deferoxamine, which
prevents iron-catalyzed formation of hydroxyl radicals, or with DPI, an
inhibitor of NADPH oxidase, the enzyme responsible for oxidant
production in neutrophils (16, 26) (P < 0.05; Table 1). Incubation with catalase (a scavenger of
H2O2) was associated with ~60% reduction of
radical concentration, although this result did not reach statistical
significance (P = 0.07; Table 1).
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Oxygen Radical Measurements in Closed-Chest Dogs
To reproduce the clinical setting, closed-chest dogs underwent coronary artery occlusion-reperfusion by PTCA and blood sampling via insertion of a coronary sinus catheter. One dog was excluded from the study because of PTCA balloon displacement. In the remaining six animals, coronary artery occlusion resulted in marked reduction of myocardial blood flow (collateral blood flow 7.1 ± 0.7% of baseline) similar to values found in the open-chest group. The radical signal was low in blood samples taken at baseline or during 90 min of ischemia (Fig. 4). However, a marked increase in oxygen radical concentration was observed in coronary sinus blood during reperfusion (Fig. 4). Similar to the findings in open-chest animals, signal intensity peaked at 10 min of reflow and remained elevated throughout 60 min of reperfusion (Fig. 4).
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Role of Neutrophils
Effect of NADPH oxidase inhibition. One dog fibrillated during ischemia, and another dog developed marked hypotension after coronary occlusion; both were excluded from the study. The remaining dogs were randomly assigned to a control group (n = 5 dogs) or to receive the NADPH oxidase inhibitor VF244 (25, 40) (n = 5 dogs). In preliminary experiments, we established that with phorbol ester (200 ng/ml)-activated neutrophils, 5 µM VF244 inhibited superoxide radical generation by >50%. In contrast, at least 50 µM concentration (i.e., 10-fold higher) of VF244 was required to achieve 50% reduction of radical concentration by direct scavenging effect when superoxide radicals were formed by nonneutrophil sources (i.e, xanthine oxidase or light-stimulated riboflavin).
The two groups were similar with respect to hemodynamic parameters (not shown) and collateral flow during ischemia (8.6 ± 2.6% of baseline in controls vs. 8.8 ± 2.1% in VF244-treated dogs). Administration of VF244 resulted in almost complete ablation of transcardiac concentration of radicals to <10% of control dogs. This effect persisted throughout the observation period (Fig. 5; Table 2).
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Effect of antineutrophil antibodies.
Two dogs died of ventricular fibrillation during ischemia. Of
the remaining dogs, eight received saline, whereas seven dogs received
R15.7, a monoclonal antibody against CD18 adhesion molecules (4,
13, 32). The two groups were similar with respect to hemodynamic
parameters (not shown) and collateral flow during ischemia
(Table 3). The extent of risk region
after coronary artery occlusion was also similar in the two groups
(Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we showed that neutrophils are a major source of oxygen radicals during reperfusion after prolonged myocardial ischemia in vivo and that interventions interfering with neutrophil activation result in less radical production and better tissue preservation.
Formation of oxygen radicals on postischemic reperfusion has been demonstrated in numerous experiments (2, 3, 5-9, 17, 28, 38). However, those studies have mostly dealt with experimental models of reperfusion after relatively brief periods of ischemia, which is known to induce significant impairment of contractile function, but no or minimal irreversible cell injury (i.e., myocardial stunning). In contrast, the pathophysiology of oxygen radical production in hearts reperfused after prolonged ischemia (typically associated with myocardial infarction) has not been thoroughly investigated. Longer duration of ischemia may entail major differences in events taking place during reperfusion. One major difference is represented by the contribution of neutrophils to tissue injury. There is now substantial agreement that neutrophil activation plays little or no role in myocardial stunning (14, 18, 20, 27), whereas activation of circulating neutrophils enhanced by locally activated endothelium is considered a major player in cardiac injury associated with reperfusion after prolonged ischemia. In this context, neutrophils are recruited in large amounts within the previously ischemic tissue and may induce injury by local release of various mediators, chiefly oxygen radicals (1, 12, 23, 29, 30, 33). Release of oxygen radicals by the endothelium may also play a role in postischemic injury, potentially involving an interaction with circulating neutrophils (35). The neutrophil hypothesis is supported by a number of studies showing that interventions aimed at counteracting neutrophil activation confer tissue protection after prolonged ischemia and reperfusion (4, 22, 23, 29, 33). However, whether neutrophils are, in fact, a major source of radicals in hearts reperfused in vivo has never been directly investigated.
Role of Neutrophils
In the present study, we report that neutrophils contribute to oxidant load in reperfused hearts, as directly documented by several lines of evidence. First, addition of DPI to coronary sinus blood significantly reduced radical concentrations. DPI is a powerful inhibitor of NAD(P)H oxidase (16, 26), the enzyme responsible for radical generation in leukocytes, endothelial cells, and smooth muscle cells. Because we added DPI to the blood ex vivo, the effect of the drug can only be ascribed to inhibition of leukocyte production of oxidants. We then tested this issue in vivo. Because of possible toxic effects of DPI, we chose to administer VF244, which has also been shown to inhibit radical production by neutrophils (25, 40). Treatment with VF244 resulted in almost complete ablation of transcardiac production of radicals during reperfusion, further suggesting a major role for neutrophil activation in this phenomenon. Finally, in another arm of the study, we treated dogs with a monoclonal antibody against neutrophil CD18 adhesion complex. Administration of R15.7 in vivo was associated with a significantly lower concentration of oxygen radicals in the coronary sinus. This finding was accompanied by a significant reduction of infarct size and of the extent of no-reflow in treated dogs. We (4, 22) and others (23, 33-34) have previously shown that antineutrophil antibodies can significantly reduce myocardial injury in models of postischemic reperfusion. The results of the present study show for the first time that this protective effect of anti-CD18 antibodies occurs concomitantly with a reduction of oxygen radical generation in vivo. This finding is consistent with the concept derived from in vitro studies that an important trigger for oxidant production from neutrophils involves interaction with endothelial cells (4, 13, 32).Comparison with Previous Studies
In our study, radical concentration peaked 10 min into reperfusion, and it remained significantly elevated for at least 1 h afterward. This is apparently at variance with the widely held notion that radical generation peaks immediately on reflow and then subsides within minutes (2-3, 5-7, 17, 38). This discrepancy presumably reflects pathophysiological differences in experimental models, and it is consistent with a major role of neutrophils in generating radicals under our in vivo experimental conditions. Although tissue accumulation of neutrophils during reperfusion is a relatively slow process, adhesion to vascular endothelium and trapping of activated neutrophils within the microvasculature of reperfused myocardium may occur rapidly (1, 10-12, 14). However, brief periods of ischemia resulting in myocardial stunning are insufficient to recruit significant numbers of neutrophils, and neutrophil activation has been shown to play no major role in stunning (12, 18, 27). Lack of neutrophil contribution can similarly be invoked to explain the results from in vitro crystalloid-perfused models, which are obviously devoid of neutrophils. We have shown in buffer-perfused hearts that mitochondrial respiration is a major source of radicals (2). Radical production occurs immediately after reflow, on reintroduction of oxygen, and then quickly ceases once mitochondria have regained their normal redox status. Radicals are also generated from xanthine-oxidase of endothelial cells, triggered by oxygen reintroduction on reflow (39) and decline within minutes due to consumption of xanthine oxidase substrates (37). However, when neutrophils are added to crystalloid-perfused hearts, sustained radical formation is observed during reperfusion, which lasts throughout the period of neutrophil infusion (22, 31). Thus we believe that the source of oxidants generated during reperfusion may differ depending on whether ischemia has been brief or prolonged. An early, short-lasting radical production may occur via mitochondrial respiration and xanthine oxidase, whereas neutrophils may be the source of a slower-onset, longer-lasting production, more likely to be found with reperfusion after prolonged ischemia.Limitations of the Study
Our approach does not detect radicals formed in the interstitium or within myocytes. This is a problem common also to spin-trap techniques based on intracoronary infusion, and it can be overcome only by analysis of tissue biopsies. Another potential limitation is that the methodology allows detection only of oxidants with a relatively long half-life and/or of those being formed as a result of ongoing production in venous blood. Thus is it conceivable that a substantial fraction of total oxidant production [e.g., those produced by endothelial xanthine oxidase (35)] may escape detection, and, therefore, we may underestimate the total amount of radicals being formed. Also, occurrence of "no-reflow" would tend to reduce washout from the most severely ischemic areas.Implications. A major advantage of our approach is that it allows the investigation of radical production and of the effects of specific interventions simultaneously with measurements of other end points, such as contractile function, infarct size, or microvascular function. In contrast, infusion of spin traps or other agents that may act as scavengers requires that radical measurements and functional or morphologic evaluation be performed on separate groups of animals and the results extrapolated by analogy. The ability to perform repeated measurements in the same animal without interfering with the pathophysiology of oxygen radical-mediated injury should be of value in better understanding this phenomenon.
This technique also has potential clinical implications. Oxygen radical production has been shown in a variety of animal models of ischemia-reperfusion. However, demonstration of its occurrence in patients with acute myocardial infarction is still lacking. Here, we report on the feasibility of the procedure in closed-chest dogs undergoing cardiac catheterization. Thus we expect it to be applicable to the increasingly growing number of patients with acute myocardial infarction undergoing recanalization by means of percutaneous coronary artery angioplasty. In conclusion, by using a minimally invasive procedure, which does not interfere with the pathophysiology of blood-borne formation of oxygen radicals, our data demonstrate for the first time that activated neutrophils are a major source of oxidants in hearts reperfused in vivo after prolonged ischemia, that this phenomenon is long-lived, and that antineutrophil interventions can effectively prevent the increase in transcardiac concentration of oxygen radicals during reperfusion.| |
ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant P50HL-52315; by International Grant CRG972188 from the North Atlantic Treaty Organization, Bruxelles, Belgium; and by Grant CIP9806181192-002 from Ministero della Ricerca Scientifica, Rome, Italy.
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FOOTNOTES |
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The experiments reported in this paper were performed under a Framework Agreement between the Johns Hopkins School of Medicine, Baltimore, MD, and the University of Perugia School of Medicine, Perugia, Italy.
Address for reprint requests and other correspondence: G. Ambrosio, Cardiologia e Fisiopatologia Cardiovascolare, Università di Perugia, Policlinico Monteluce, Via Brunamonti, 06122 Perugia, Italy (E-mail: cardiopg{at}unipg.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 November 2000; accepted in final form 26 January 2001.
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TOP
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METHODS
RESULTS
DISCUSSION
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