Reduction in infarct size by the prostacyclin analogue iloprost (ZK 36374) after experimental coronary artery occlusion-reperfusion

In this study we attempted to determine whether administration of iloprost (ZK 36374), a chemically stable prostacyclin analogue, would reduce infarct size after experimental coronary artery occlusion and reperfusion. One hour of coronary artery occlusion was performed in 28 open-chest, anesthetized rabbits++, followed by 5 hours of reperfusion. Two minutes after occlusion, 99mTc-labeled albumin microspheres were injected into the left atrium for later assessment of the area at risk of infarction. Fifteen minutes after occlusion animals were randomly assigned to either the treatment group (iloprost, 1.2 micrograms/kg/min intravenously for 6 hours; n = 14) or the control group (n = 14). In vitro platelet aggregation was inhibited in rabbits receiving iloprost. In 10 rabbits (five treated and five control) regional myocardial blood flow was also measured by means of differentially labeled radioactive microspheres. Infarct size was significantly smaller in treated rabbits (53.6 +/- 4.1% of the risk zone vs 89.4 +/- 3.8% in control rabbits; p less than 0.001). Flow to the nonischemic myocardium was higher in treated animals, that is, 1.87 +/- 0.20 ml/min/gm of tissue 50 minutes after occlusion and 1.90 +/- 0.20 ml/min/gm of tissue 4 hours after reperfusion, compared with 1.54 +/- 0.20 and 1.64 +/- 0.30 ml/min/gm of tissue, respectively, in control rabbits (p less than 0.01). Collateral flow to the ischemic region was not affected by the drug. Mean arterial blood pressure, heart rate, and pressure-rate product in treated rabbits were not significantly different from values in control rabbits. In conclusion, administration of iloprost reduced myocardial infarct size in this model of myocardial ischemia and reperfusion in absence of major hemodynamic effects.     

Sustained limitation of myocardial reperfusion injury by a monoclonal antibody that alters leukocyte function

Pentobarbital anesthetized dogs were subjected to 90 minutes of left circumflex coronary artery (LCCA) occlusion followed by 72 hours of reperfusion. Control or anti-Mo1 (904) F(ab')2 fragments of monoclonal antibodies were administered intravenously at a dose of 1 mg/kg beginning 45 minutes after occlusion and at a dose of 0.5 mg/kg at 12, 24, 36, and 48 hours after reperfusion. Myocardial infarct size expressed as a percentage of the area at risk (IN/AR) measured postmortem after 72 hours of reperfusion was significantly reduced by 904 F(ab')2 (21.6 +/- 2.8%, n = 8) compared with control F(ab')2 (37.4 +/- 5.8%, n = 8; p less than 0.025). There were no significant differences between groups in heart rate, mean arterial blood pressure, rate-pressure product, or LCCA blood flow that could account for a reduced infarct size. Regional myocardial blood flow (RMBF) was determined with 15-microns radiolabeled microspheres. Transmural blood flows (ml/min/g) within the region of myocardium at risk were not statistically different between treatment groups. Infarct size in both groups was related to regional myocardial blood flow, and the relation was shifted downward in the group treated with the anti-Mo1 F(ab')2 antibody (analysis of covariance, p = 0.01). Thus, anti-Mo1 F(ab')2 produces a sustained limitation of myocardial infarct size compared with controls under similar hemodynamic conditions and a similar degree of myocardial ischemia as determined by RMBF. These data suggest that inhibition of neutrophil adhesive interactions (as suggested by the inhibitory effect of anti-Mo1 on canine neutrophil aggregation) may be an effective mechanism for protection against myocardial injury secondary to myocardial ischemia and reperfusion.     

Identification of a time window for therapy to reduce experimental canine myocardial injury: suppression of neutrophil activation during 72 hours of reperfusion

The cardio-protective effects of neutrophil depletion or inhibition of neutrophil activation early in the course of myocardial reperfusion has been established. Whether these treatments would be effective during extended periods of reperfusion has not been ascertained. Open-chest anesthetized dogs were subjected to left circumflex artery (LCX) occlusion for 90 minutes followed by 72 hours of reperfusion. Dogs were randomized into one of four groups: 1) control; 2) Ilo-2 (iloprost 100 ng/kg/min administered via the left atrium beginning 10 minutes after LCX occlusion and continuing 2 hours into reperfusion); 3) Ilo-48 (iloprost 100 ng/kg/min administered as above until 1 hour after reperfusion then 25 ng/kg/min for 48 hours of reperfusion; or 4) antibody (neutrophil antibody administered before occlusion and 1/2 hourly for 2 hours of reperfusion and then every 24 hours). Myocardial infarct size, as a percentage of the area at risk assessed after 72 hours of reperfusion, was significantly smaller in the antibody-treated group (32.1 +/- 5.0% mean +/- SEM) or Ilo-48 (22.6 +/- 4.0%) treatment group compared with control (48.7 +/- 5.6%) or Ilo-2 (57.6 +/- 5.2%) groups. Regional myocardial blood flow studies demonstrated that all groups developed similar degrees of ischemia. The iloprost-treated groups had lower mean arterial blood pressures during occlusion and reperfusion than groups 1 and 4 (p less than 0.05). Circulating neutrophil counts were increased in groups 1 and 2 at 24 and 48 hours after reperfusion compared to groups 3 and 4 (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beneficial effects of iloprost in the stunned canine myocardium

The effect of the prostacyclin-mimetic, iloprost, on the reversibly damaged ("stunned") myocardium was studied in barbital-anesthetized, open-chest dogs subjected to 15 minutes of coronary artery occlusion and 3 hours of reperfusion. Regional myocardial segment shortening (%SS) was measured in the subendocardium of nonischemic and ischemic-reperfused areas by sonomicrometry. Iloprost was infused for 30 minutes beginning 15 minutes prior to occlusion (0.05 microgram/kg/min, ILO-LOW, or 0.1 microgram/kg/min, ILO-HIGH) or immediately prior to reperfusion (0.1 microgram/kg/min, ILO-REP). %SS in the ischemic-reperfused region recovered to 3% of pretreatment values in the control (saline-treated) group by 3 hours of reperfusion. In contrast, %SS in the iloprost-treated groups was significantly enhanced versus the control group at all times of reperfusion. At 3 hours of reperfusion, %SS recovered to 43% (ILO-LOW), 58% (ILO-HIGH), and 35% (ILO-REP) of pretreatment values. The beneficial effect on functional recovery was significantly greater when iloprost was administered before occlusion versus immediately prior to reperfusion. Thus, part of the salutory effects of iloprost appear to occur prior to and/or during ischemia. Iloprost did not improve collateral blood flow to the ischemic region or myocardial high energy phosphate content at 3 hours of reperfusion. While iloprost significantly decreased mean arterial pressure during ischemia and early reperfusion, the hypotensive action did not appear to play a role in the amelioration of postischemic dysfunction, as preocclusion treatment with an equihypotensive dose of sodium nitroprusside produced no significant effect on postischemic recovery beyond 5 minutes of reperfusion. Results of in vitro experiments indicated that iloprost had no effect on the xanthine oxidase free-radical generating system including lipid peroxidation. However, iloprost decreased the neutrophil-derived superoxide burst after chemotactic stimulation. This beneficial action may, in part, explain the efficacy of iloprost in enhancing postischemic function of the stunned myocardium.     

Role of neutrophil adherence receptors (CD 18) in lung permeability following lower torso ischemia.

Ischemia and reperfusion of the lower torso lead to leukotriene- and neutrophil (PMN)-dependent lung injury characterized by lung PMN sequestration, increased permeability, and noncardiogenic edema. It is thought that PMNs require adhesion to endothelium to alter barrier function. This study tests the role of CD 18, the PMN adherence receptor, in mediating lung permeability after lower torso ischemia and reperfusion. Anesthetized rabbits (n = 9) underwent 3 hours of bilateral hind limb ischemia. Ten minutes after the release of the tourniquets, plasma leukotriene B4 levels increased to 395 +/- 85 pg/ml, higher than 129 +/- 35 pg/ml in controls (n = 9, p less than 0.01). At this time there was a reduction in circulating white blood cells (x 10(3)), 3.56 +/- 0.49/mm3 relative to 6.07 +/- 0.61/mm3 in controls (p less than 0.01). PMNs were sequestered in the hind limbs, indicated by increased myeloperoxidase activity of 1.06 +/- 0.19 units/g compared with 0.56 +/- 0.09 units/g in controls (p less than 0.05). Four hours after tourniquet release, PMNs were sequestered in the lungs, 52 +/- 4 PMNs per 10 high-power fields, a value higher than 31.5 +/- 3 PMNs per 10 high-power fields in controls; bronchoalveolar lavage fluid protein content increased to 554 +/- 90 micrograms/ml relative to 277 +/- 46 micrograms/ml in controls; and there was lung edema, measured by increased wet weight-to-dry weight ratios of 5.19 +/- 0.10, higher than 4.29 +/- 0.21 in controls (all p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostacyclin protects ischemic reperfused myocardium in the dog by inhibition of neutrophil activation

Prostacyclin (PGI2) and the stable PGI2 analogue SC39902 (6,9 alpha-epoxy,5S-fluoro-11 alpha, 15S-dehydroxyprosta-6,13E-dien-1-oic acid, sodium salt) were studied in anesthetized open-chest dogs subjected to 90 minutes of left circumflex coronary artery (LCCA) occlusion and 6 hours of reperfusion. PGI2 (50 ng/kg/min, infused into the left atrium) reduced infarct mass by 59% compared to control, but SC39902 (1.5 micrograms/kg/min) failed to produce a significant reduction in infarct size. Both PGI2 and SC39902 reduced mean arterial blood pressure, heart rate, and rate-pressure product to the same extent. Regional myocardial blood flow measured with radiolabelled tracer microspheres did not demonstrate an increase in regional blood flow to the ischemic myocardium during the 90 minutes of LCCA occlusion in the PGI2 and control treatment groups. Canine neutrophils were isolated from whole blood and activated with opsonized zymosan. PGI2 produced a concentration-dependent inhibition of neutrophil activation as measured by superoxide production in vitro, whereas SC39902 failed to effectively inhibit neutrophil activation. Neutrophil migration into inflammatory skin lesions was effectively attenuated when dogs were pretreated with PGI2 (50 ng/kg/min, intravenously). Therefore, it is suggested that the cytoprotective effect of PGI2 during myocardial ischemia and reperfusion is related to an inhibition of neutrophil migration and the production of cytotoxic activated oxygen species.     

Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog

Accumulation of polymorphonuclear neutrophils during the acute inflammatory response may exacerbate tissue injury through the release of activated oxygen products or proteolytic enzymes or both. To assess the role of neutrophils in acute myocardial infarction, circulating neutrophil levels in dogs were reduced by 77 +/- 2% (mean +/- SEM) by administering rabbit antiserum to dog neutrophils. Acute myocardial infarction was induced in open-chest anesthetized dogs by 90 minutes of left circumflex coronary artery occlusion followed by 6 hours of reperfusion. Dogs treated with neutrophil antiserum (n = 8) developed myocardial infarcts that were an average of 43% smaller than infarcts in dogs treated with nonimmune rabbit serum (n = 7) (27.0 +/- 4.5% vs 47.1% +/- 7.5% of the area at risk, p less than 0.05). In a saline-treated control group (n = 8), infarct size was 48.0 +/- 4.7% of the area at risk, a value not significantly different from that of the nonimmune serum group but significantly greater than that in the neutrophil antiserum dogs (p less than 0.05). There were no major hemodynamic differences between groups. Histopathologic examination revealed that infarcted myocardium from dogs given saline or treated with nonimmune serum had a substantial neutrophilic infiltrate, which was virtually absent in infarcted tissue from dogs treated with neutrophil antiserum. These observations suggest that neutrophil accumulation in response to myocardial ischemia may be responsible for a substantial portion of the irreversible myocardial injury resulting from temporary coronary artery occlusion.     

Immediate rebound followed by deterioration of regional left ventricular function with coronary reperfusion

The immediate and early effects of coronary artery reperfusion initiated 1 and 3 hours after coronary artery occlusion were evaluated by two-dimensional echocardiographic measurements of overall and regional left ventricular function. A total of 29 anesthetized open chest dogs underwent one of the following: 1 hour occlusion followed by reperfusion (Group I, n = 9), 3 hour occlusion followed by reperfusion (Group II, n = 12) or 5 hour occlusion without reperfusion (Group III, n = 8). Serial two-dimensional echocardiography was performed at baseline; at 1, 3 and 5 hours of coronary occlusion; within 5 minutes of reperfusion; and at 2 hours of reperfusion. After occlusion, all groups manifested significant (p less than 0.01) increases in left ventricular diastolic and systolic area and decreases in left ventricular area ejection fraction. With coronary reperfusion, there was no improvement in these global variables in Groups I and II. However, immediately after reperfusion, there was improvement in the regional extent of dysfunction (Group I, 138 +/- 35 to 66 +/- 62 degrees, p less than 0.05; Group II, 156 +/- 51 to 85 +/- 77 degrees, p less than 0.05) as well as improvement in the regional degree of dyskinesia (p less than 0.05). These regional improvements were transient and resolved by 2 hours of coronary reperfusion. This immediate rebound of function was not associated with the duration of coronary occlusion, hemodynamic variables or ultimate infarct size. Thus, in the anesthetized open chest dog model, coronary artery reperfusion at 1 or 3 hours produces an immediate but transient improvement in regional systolic myocardial function.     

Effect of superoxide dismutase on myocardial infarct size in the canine heart after 6 hours of regional ischemia and reperfusion: a demonstration of myocardial salvage

Available data demonstrate that oxygen free radicals and derived reactive species of oxygen are produced during myocardial ischemia as well as upon reperfusion of the ischemic tissue. The present study was designed to determine if polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), with its extended plasma half-life in excess of 30 hours in contrast to the native form of the enzyme (Native-SOD), could provide protection to the ischemic myocardium subjected to a 6-hour regional ischemia followed by reperfusion for 24 hours. We hypothesized that myocardial injury due to an ischemic interval is a dynamic process involving the sustained production of cytotoxic oxygen radicals that may continue beyond the ischemic interval. The ability to demonstrate a protective effect of the free radical scavenger enzyme superoxide dismutase would require the continued presence of the antioxidant during the ischemic interval and especially during reperfusion. To test this hypothesis, 22 anesthetized, open-chest dogs underwent 6 hours of circumflex coronary artery occlusion followed by reperfusion for 24 hours. Rapid administration of either Native-SOD (1,000 U/kg), PEG-SOD (1,000 U/kg), PEG-albumin (PEG-ALB), or 0.9% sodium chloride solution for injection (saline) was administered via the left atrium 15 minutes before occlusion of the vessel. A continuous infusion of an additional 1,000 U/kg of the respective enzyme interventions or an equivalent volume of PEG-ALB or saline was given during the 6-hour coronary artery occlusion and terminated 15 minutes after reperfusion. The animals were euthanized 24 hours after reperfusion, and the myocardial region at risk and the infarct region were quantitated by the tetrazolium method. The area of myocardium at risk of infarction, expressed as a percent of the left ventricle, did not differ among the groups: Native-SOD (n = 8), 46.2 +/- 1.8%; PEG-SOD (n = 6), 45.7 +/- 2.1%; PEG-ALB, 38.4 +/- 2.3% (n = 4); and saline 46.0 +/- 2.1% (n = 4). Hemodynamic parameters, the calculated rate-pressure-product, as well as regional myocardial blood flow (radiolabeled microsphere method) in the endocardial, midmyocardial, and epicardial segments of the risk and the nonrisk regions were comparable for all groups. Mean infarct size, determined 24 hours after reperfusion, in the group treated with PEG-SOD was 47.1 +/- 2.9% of the area at risk (n = 6), significantly smaller than that observed in each of the other treatment groups: Native-SOD, 63.5 +/- 2.2% (n = 8); PEG-ALB, 64.6 +/- 2.4% (n = 4); saline, 70.8 +/- 2.2% (n = 4).(ABSTRACT TRUNCATED AT 400 WORDS)     

Limitation of myocardial reperfusion injury by intravenous perfluorochemicals. Role of neutrophil activation

Neutrophil activation and infiltration into the ischemic myocardium after reperfusion may limit the amount of salvageable myocardium (reperfusion injury). The effects of intravenous perfluorochemicals (Fluosol-DA) on infarct size, ventricular contractility, and neutrophil function were assessed in an occlusion-reperfusion canine model. Closed-chest dogs were subjected to 90 minutes of left anterior descending artery occlusion followed by 24 hours of reperfusion. Animals were randomized to receive either Fluosol-DA (FDA, n = 8) or Ringer's lactate (CONT, n = 10) intravenously over 30 minutes just before left anterior descending artery reperfusion. Neutrophil demargination and infiltration into the myocardium were assessed in vivo with In111. Neutrophil chemotaxis, superoxide radical production, and lysozyme degranulation were evaluated ex vivo at baseline, 1 hour after occlusion, and 1 hour after reperfusion. Perfluorochemicals significantly reduced infarct size expressed as percent of area at risk (FDA, 7 +/- 4%; CONT, 24 +/- 6%; p less than 0.01). This was associated with positive wall motion in the jeopardized zone of Fluosol-DA animals compared with dyskinesis in control animals (FDA, +4.4 +/- 2.1%; CONT, -1.1 +/- 1.5%; p less than 0.05). Electron microscopy showed reduced neutrophil and erythrocyte plugging of capillaries with relative preservation of endothelial cells in the Fluosol-DA animals. Myocardial blood flow was greater in the ischemic endocardium of Fluosol-DA animals 1 hour after reperfusion (FDA, 1.23 +/- 0.21; CONT, 0.62 +/- 0.08 ml/g/min; p less than 0.01). Neutrophil demargination and infiltration into the ischemic myocardium was reduced in the animals treated with Fluosol-DA. (FDA, 2.5 +/- 0.7 x 10(3); CONT, 14.1 +/- 2.7 x 10(3) neutrophils/g; p less than 0.01). Neutrophil chemotaxis and lysozyme release were also markedly suppressed in the Fluosol-DA groups ex vivo. These results show that intravenous Fluosol-DA significantly reduces reperfusion injury with greater salvage of myocardium and improved left ventricular function. The chief mechanism of action of Fluosol-DA appears to be the suppression of neutrophil function.     

Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion

OBJECTIVE: We previously showed that brief intermittent ischemia applied during the onset of reperfusion (i.e., postconditioning) is cardioprotective in a canine model of ischemia-reperfusion. This study tested the hypothesis that the early minutes of reperfusion (R) during which postconditioning (Post-con) is applied are critical to its cardioprotection. METHODS: In anesthetized open-chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 h. All rats were randomly divided into six groups: Control (n=8): no intervention at R; Ischemic preconditioning (IPC) (n=8): the LCA was occluded for 5 min followed by 10 min of R before the index occlusion; Post-con 1 (n=8): after LCA occlusion, three cycles of 10 s R followed by 10 s LCA re-occlusion were applied during the first minute of R; Post-con 2 (n=8): Six cycles of 10 s R and 10 s re-occlusion were applied during the first 2 min of R; Delayed Post-con (n=8): the ligature was loosened for full reflow for the first minute of R, after which the three-cycle Post-con algorithm was applied; Sham (n=6): the surgical procedure was identical to other groups, but the LCA ligature was not ligated. RESULTS: Infarct size (TTC staining) was 23% smaller in Post-con 1 (40+/-2%*) than in Control (52+/-3%), confirmed by plasma creatine kinase activity (18+/-2* vs. 46+/-6 IU/g protein). There was no further reduction in infarct size with 6 cycles of Post-con (40+/-2.9%, p>0.05 vs. Post-con 1). Meanwhile, infarct size reduction was significantly greater in the IPC group (17+/-3%) than in Post-con1 (p<0.01). The plasma lipid peroxidation product malondialdehyde (MDA, microM/ml) was less after R in IPC and Post-con 1 (0.8+/-0.07* and 0.8+/-0.06*) vs. Control (1.21+/-0.08), consistent with a visual decrease in superoxide anion generation (dihydroethidium staining) in the AAR myocardium after 3 h of reperfusion. Neutrophil accumulation (myeloperoxidase activity, MPO, U/100 g tissue) in the AAR was less in IPC (1.4+/-0.3*) and Post-con 1 (2.5+/-0.3*) vs. Control (5.5+/-0.6). The reductions in infarct size, creatine kinase, MDA and DHE staining were lost with delayed Post-con, while MPO activity remained lower than in Control (3.2+/-0.4*). CONCLUSIONS: (1) Post-con at onset of R reduces myocardial injury; (2) cardioprotection may be mediated, in part, by inhibiting oxidant generation and oxidant mediated injury; (3) the first minute of R in the rat model is critical to cardioprotection by Post-con; and (4) cardioprotection by Post-con may be independent of neutrophil accumulation in AAR. *p<0.05 Post-con vs. Control.     

Prostaglandin E1 attenuates postischemic contractile dysfunction after brief coronary occlusion and reperfusion

We have previously demonstrated that administration of the prostacyclin analogue iloprost improved postischemic functional recovery in reversibly injured ischemic-reperfused myocardium. The present study investigated the effects of administering an endogenous vasodilator prostanoid, prostaglandin E1 (PGE1), in the stunned myocardium (15 minutes of coronary artery occlusion and 3 hours of reperfusion) of anesthetized dogs. The percentage of regional myocardial segment shortening (%SS) after administration of PGE1 by two routes, intravenously (1 microgram/kg/min) or intraatrially (0.1 microgram/kg/min), to avoid pulmonary metabolism, 15 minutes before and throughout the period of occlusion, was compared to %SS in a control group treated with saline solution. Nearly equivalent reductions in mean arterial pressure during occlusion compared to pretreatment control (PTC) values were produced by intravenous (33%) or intraatrial (25%) PGE1. There was no difference in transmural myocardial blood flow (radioactive microsphere technique) in the ischemic region between the PGE1-treated and control groups at any time. Although there were no differences in %SS in the nonischemic region between groups throughout the experiment, postischemic recovery of segment function in the ischemic-reperfused area was significantly improved (p less than 0.05) at all times during reperfusion by intravenous PGE1 (%SS of PTC: 30 minutes = 65 +/- 8; 3 hours = 58 +/- 7) or intraatrial PGE1 (%SS of PTC: 30 minutes = 57 +/- 12; 3 hours = 50 +/- 4) compared to the control group (%SS of PTC: 30 minutes = 25 +/- 13; 3 hours = 10 +/- 13). Thus treatment with PGE1 attenuates postischemic contractile dysfunction in the stunned myocardium.2+ both.     

Sustained beneficial effects of gallopamil (D600) on size of myocardial infarction 24 hours after coronary artery occlusion in dogs

To examine whether gallopamil (D600), a methoxy derivative of verapamil, has sustained beneficial effects on the ischemic myocardium, its effects on the size of myocardial infarction determined 6 hours (protocol 1) and 24 hours (protocol 2) after left anterior descending coronary artery occlusion were compared in anesthetized, open-chest dogs. To quantify the extent of the hypoperfused zone, Tc-99m- or In-111-albumin microspheres were injected into the left atrium 1 minute after occlusion. Fifteen minutes after occlusion, dogs were randomly assigned to a control group or a gallopamil-treated group that received immediately after assignment 0.08 mg/kg of gallopamil followed by a continuous infusion of 0.2 mg/kg/hr for 6 hours. Six or 24 hours after occlusion, the left ventricle was cut into 3 mm thick slices for triphenyltetrazolium chloride staining and autoradiography. There were no differences in the extent of the hypoperfused zone among the four groups. In both protocols 1 and 2 the ratio of the extent of myocardial necrosis to the extent of the hypoperfused zone was significantly smaller in the treated groups (56.7 +/- 6.7% [n = 8], p less than 0.01 and 72.3 +/- 5.3% [n = 6], p less than 0.05 for protocols 1 and 2, respectively) than in the control groups (100.7 +/- 6.0% [n = 7] and 95.2 +/- 4.3% [n = 5] for protocols I and II, respectively). Thus gallopamil administered early after coronary artery occlusion had beneficial effects on the ischemic myocardium, which were sustained for at least 24 hours after the onset of infarction.     

Superoxide dismutase conjugated to polyethylene glycol provides sustained protection against myocardial ischemia/reperfusion injury in canine heart

Disagreement regarding the cardioprotective role of superoxide dismutase may relate to the use of different durations for induction of ischemic injury and reperfusion. The present study employed superoxide dismutase conjugated to polyethylene glycol (PEG-SOD), which has a half-life greater than 30 hours. Two protocols differing in the mode of administration and the duration of the reperfusion interval were used. Dogs were subjected to occlusion of the circumflex coronary artery for 90 minutes, then reperfused for 6 hours (Protocol A) or 4 days (Protocol B). The dogs received either polyethylene glycol conjugated to albumin (PEG-ALB) or PEG-SOD (1,000 U/kg). In Protocol A, treatment was administered starting 15 minutes before coronary occlusion and continued for 2 hours, terminating 15 minutes after reperfusion. Infarct size was determined 6 hours later. In Protocol B, the conjugated proteins were given 15 minutes before reperfusion and ended simultaneously with reperfusion. Infarct size was measured after 4 days. Infarct size (percentage of area at risk) in control (n = 9) and treated (n = 9) dogs in Protocol A differed between groups: 46.7 +/- 3.5% versus 28.3 +/- 2.9%, respectively (p less than or equal to 0.005); risk regions did not differ: 42.8 +/- 1.5% versus 43.8 +/- 2.1%, respectively. Myocardial salvage also was observed in Protocol B. Infarct size in control (n = 13) and treated (n = 13) groups was 44.2 +/- 2.6% versus 29.2 +/- 1.6%, respectively (p less than or equal to 0.005), with risk regions being 44.4 +/- 1.4% versus 46.0 +/- 1.6% (p = NS). Hemodynamic variables did not differ during the period of coronary artery occlusion. The respective collateral blood flows to the inner two thirds of the ischemic myocardium determined 60 minutes after occlusion were 0.05 +/- 0.01 ml/min/g and 0.06 +/- 0.04 ml/min/g (p = 0.806) for the PEG-ALB and PEG-SOD treated groups, respectively. Infarct size was related inversely to collateral blood flow in the PEG-ALB treated group. This relation shifted downward (analysis of covariance, p = 0.017). Plasma SOD activity in Protocols A sustained for 6 hours. Significant enzymatic activity was present after 4 days in Protocol B. Previous negative studies with native SOD may be related to the short half-life of its free-radical scavenging capacity, which compromises the chances of observing a protective effect after 4 days of reperfusion. The present results support our previous observations, as well as those of other investigators, demonstrating that superoxide dismutase can reduce that component of myocardial injury associated with reperfusion.     

Ischemia and reperfusion-induced arrhythmias in the rat. Effects of xanthine oxidase inhibition with allopurinol

We have investigated the possibility that xanthine oxidase-linked free radical production has a role in the genesis of arrhythmias during ischemia and reperfusion. In this study, rats were treated with allopurinol (20 mg/kg, orally, 24 hours before study, plus 20 mg/kg, iv, 15 minutes prior to study). Using an anesthetized open-chest preparation with either coronary artery occlusion for 30 minutes, or 5 minutes followed by 10 minutes reperfusion, we monitored and compared the rhythm disturbances in experimental vs. placebo-treated rats (n = 18 in each group). Allopurinol treatment reduced the incidence of ventricular tachycardia during ischemia from 88% to 50% (P less than 0.05) and the number of premature ventricular complexes from 471 +/- 120 to 116 +/- 46 (P less than 0.02), but the treatment had no effect upon the incidence or duration of ventricular fibrillation or upon mortality. In contrast, far more dramatic protection was observed during reperfusion after 5 minutes of ischemia. Allopurinol treatment reduced the incidence of ventricular fibrillation from 67% to 11% (P less than 0.01), reduced the mean duration of fibrillation from 230 +/- 70 to 14 +/- 1 seconds (P less than 0.05), and reduced mortality by half (10/18 to 4/18), although this did not reach a level of statistical significance. In addition, the mean duration of tachycardia was reduced from 83 +/- 26 to 38 +/- 8 seconds (P less than 0.05). Allopurinol pretreatment thus affords some protection against ischemia-induced arrhythmias, but a higher degree of protection against reperfusion-induced arrhythmias. Allopurinol inhibits xanthine oxidase activity, and, in turn, this inhibits superoxide radical production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alleviation of myocardial stunning by leukocyte and platelet depletion

Neutrophils accumulate in myocardium rendered ischemic and reperfused. Activated neutrophils release mediators such as metabolites of oxygen that can compromise myocellular integrity and provoke cardiac dysfunction. Although it is established that leukopenia reduces infarct size, the role of leukocytes and the source of free radicals in postischemic contractile dysfunction is unresolved. A carotid left anterior descending coronary-artery extracorporeal circuit without (n = 8) or with a Leukopak filter (n = 6) to deplete the leukocytes and platelets from blood entering the left anterior descending artery was established in the anesthetized, open-chest dog 30 minutes before ischemia. Subendocardial segmental function was monitored by sonomicrometry, and ischemia was produced by stopping flow for 15 minutes followed by 3 hours of reperfusion. Depleting leukocytes by 90 +/- 3.2% and platelets by 100% improved segmental function (from 30.5 +/- 7% to 74.1 +/- 12.7% for control versus leukocyte-depleted dogs, respectively) at 15 minutes of reperfusion. In the leukopenic group, however, there was a progressive decline in contractility to 32.5 +/- 13.8% by 3 hours of reperfusion that was associated with a return of leukocytes and, to a lesser extent, a return of platelets in the extracorporeal blood to 70.2 +/- 21.9% and 15.5 +/- 4.3% of systemic values, respectively. Removal of leukocytes and platelets from blood perfusing the coronary vascular bed only at reperfusion improved contractile function to 67.7 +/- 6.9% at 15 minutes and 54.7 +/- 12.1% at 3 hours (n = 6). Scanning electron microscopy revealed adherent leukocytes in the epicardial coronary arteries of control animals after 3 hours of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between myocardial glutathione content and extent of ischemia-reperfusion injury

The relation between the extent of myocardial injury sustained during reperfusion and total glutathione (GSH) content in the ischemic myocardium was examined in anesthetized open-chest pigs subjected to coronary occlusion for 45 minutes and reperfusion for 2 hours. In pigs infused with saline during reperfusion (n = 6) there was a decrease in myocardial GSH content from 380 +/- 48 micrograms/g in normally perfused myocardium to 182 +/- 36 micrograms/g in the ischemic reperfused myocardium (p less than 0.02). Myocardial infarct size (expressed as a percentage of the ischemic area) was 12.5 +/- 0.8%. There was a delay of recovery of contractile function before returning to 60% of preocclusion value. In pigs pretreated with buthionine sulfoximine (BSO) (n = 5), an inhibitor of cellular GSH synthesis, there was reduction in GSH content to 215 +/- 25 micrograms/gm in normally perfused myocardium and to 77 +/- 8 micrograms/gm in the ischemic reperfused myocardium. The extent of injury was greater as evidenced by an increase in infarct size to 30.4 +/- 4.0% (p less than 0.001), severe destructive changes in subepicardial ultrastructure, which were absent in saline-infused pigs, and persistence of dyskinesia throughout reperfusion. In pigs infused with glutathione intravenously (0.8 gm/kg) at a rate of 6.5 mg/kg/min (n = 6), 5 minutes before and continuously during reperfusion, there was an increase in GSH content to 582 +/- 67 micrograms/g in normally perfused myocardium and to 312 +/- 80 micrograms/g in ischemic reperfused myocardium. The increase in myocardial GSH was associated with a reduction in infarct size to 7.5 +/- 1.3% (p less than 0.05, compared with saline-infused pigs) and an early recovery of contractile function of the ischemic myocardium. GSH infusion into pigs pretreated with BSO (n = 4) failed to increase myocardial GSH content and failed to reduce the extent of myocardial injury. Thus, the extent of myocardial injury sustained during reperfusion is very dependent on the effectiveness of its antioxidant defenses. Markedly increased susceptibility to injury occurs when the GSH content in the ischemic myocardium becomes depleted.     

Protection by superoxide dismutase from myocardial dysfunction and attenuation of vasodilator reserve after coronary occlusion and reperfusion in dog

Previous studies indicate impairment of coronary arterial ring relaxation and loss of coronary vasodilator reserve after coronary artery occlusion and reperfusion. These changes are mediated in part through loss of endothelium-derived relaxing factor (EDRF) and/or myocardial neutrophil accumulation. To examine if superoxide dismutase (SOD), a scavenger of superoxide radicals, would modify the diminished coronary vasodilator reserve after temporary coronary occlusion in the intact animal, open-chest mongrel dogs were subjected to 1 hour of circumflex (Cx) coronary artery occlusion followed by 1 hour of reperfusion and treated with saline or SOD. Before Cx occlusion, coronary blood flow increased, and vascular resistance decreased (both p less than 0.01) in response to EDRF-dependent vasodilator acetylcholine as well as EDRF-independent vasodilator nitroglycerin. After Cx reperfusion, resting Cx coronary blood flow and vascular resistance were similar to the preocclusion values. In the saline-treated animals, there was evidence of myocardial dysfunction, which was measured by segmental shortening (-6 +/- 2% vs. 10 +/- 2%). Furthermore, increase in Cx coronary blood flow and reduction in vascular resistance in response to both vasodilators were significantly (p less than 0.01) impaired; these occurrences suggested loss of coronary vasodilator reserve. Myocardial histology showed extensive capillary plugging by neutrophils in the Cx-supplied myocardium. Myocardial myeloperoxidase activity, an index of neutrophil infiltration, was also increased in the Cx compared with the left anterior descending coronary artery region (p less than 0.02). Treatment of dogs with SOD, started at the end of Cx occlusion and continued during reperfusion, exerted significant (p less than 0.01) protective effect against reperfusion-induced attenuation of coronary vasodilator reserve in response to both acetylcholine and nitroglycerin. Loss of myocardial function (segmental shortening 5 +/- 1% vs. 10 +/- 1%) was less than in the saline-treated animals (p less than 0.01). Cx region-myocardial neutrophil accumulation and myeloperoxidase activity were also less (p less than 0.02) in the SOD-treated than in the saline-treated dogs. These observations suggest that coronary reperfusion impairs coronary vasodilator reserve in intact dogs. This impairment can be modified by treatment of animals with SOD before reperfusion. Capillary plugging by neutrophils may contribute to the altered coronary vasodilator reserve observed in the immediate postreperfusion period, and SOD modifies this reperfusion-induced impairment.     

Ethanol does not exert myocardial preconditioning in an intact rabbit model of ischemia/reperfusion

Some epidemiologic analyses suggest that moderate drinking of alcohol may have beneficial effects on the heart. An experimental in vitro study also indicated that acute alcohol administration may precondition myocardium against ischemia, but only if it is washed out before ischemia. The goal of this study was to test whether alcohol + washout preconditions the myocardium to ischemia, reducing necrosis after ischemia/reperfusion in an in vivo model. Two groups of anesthetized open-chest rabbits received ethanol (0.5 mL/kg, intravenously) at 90 minutes before (Group 1, alcohol preconditioning, n = 8) or 5 minutes before (Group 2, acute, n = 8) coronary artery occlusion (CAO); control rabbits received saline (Group 3, n = 8). All rabbits received 30 minutes of CAO and 3 hours of reperfusion. Blood alcohol level at the time of CAO in Group 1 had returned to control level (< 10 mg/dL). Alcohol level in Group 2 was 162 +/- 9 mg/dL. There were no differences in hemodynamics, ischemic risk region size, or collateral blood flow among groups. Alcohol, with or without washout, failed to reduce necrosis. Infarct size (% risk region) was 45 +/- 5% in control rabbits, 48 +/- 6% with acute administration, and 53 +/- 7% with washout (P = 0.57). These data indicate that alcohol + washout did not precondition hearts against ischemia in this model, adding evidence to the theory that acute alcohol administration has no direct beneficial effect on ischemic myocardium.     

Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Evidence for neutrophil-mediated reperfusion injury

Reperfusion of ischemic myocardium may accelerate necrosis of injured myocytes. To determine the role of neutrophil leukocytes in this process, we examined whether neutrophil depletion during reperfusion could modify infarct size in anesthetized dogs. The proximal circumflex coronary artery was occluded for 90 minutes and then reperfused for 2 hours via an extracorporeal circuit with either whole blood (n = 11) or with blood depleted of neutrophils by leukocyte filters (n = 11). The leukocyte filters caused near-total neutropenia in blood reperfusing the ischemic myocardium (7 +/- 7 neutrophils/microliters compared with 2,551 +/- 317/microliters in controls, mean +/- SEM; p less than 0.001. Infarct size was measured by planimetry of myocardial slices stained with triphenyltetrazolium chloride (TTC), and the accuracy of TTC for identifying necrotic myocardium was verified by electron microscopy. The size of the ischemic risk region was the same in the control (41.6 +/- 1.0%) and neutropenic (41.8 +/- 2.1%) groups. Collateral blood flow to the risk region was the same in control (0.15 +/- 0.03 ml/min/g) and neutropenic (0.13 +/- 0.03 ml/min/g) groups. Among dogs with collateral flow less than 0.2 ml/min/g, infarct size was reduced in the neutropenic group (27.7 +/- 6.7% of risk region, n = 8), compared with control dogs (52.5 +/- 5.7%; n = 7; p = 0.02). Multiple linear regression described the relation between infarct size, risk region size, and collateral flow in the control group, and the same regression relation was used to predict infarct size for the neutropenic group. Mean predicted infarct size in the neutropenic group (n = 11) was 16.8 +/- 3.4% of left ventricle, whereas mean observed infarct size was 9.6 +/- 3.1% (p less than 0.01). The extent of the no-reflow zone (absence of thioflavin-S-fluorescence) was also less in the neutropenic than the control group (2.2 +/- 0.8% vs. 8.1 +/- 2.7% of the risk region, p less than 0.05). Neutropenia limited to the reperfusion period is associated with significant reductions in the extent of the infarct and no-reflow zones after 90 minutes of ischemia. These findings support the hypothesis that reperfusion necrosis occurs after prolonged myocardial ischemia and indicate that neutrophil leukocytes are important mediators of such reperfusion injury.