Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in canine heart

The prostacyclin analogue iloprost (ZK 36374) inhibits neutrophil activation in vitro, reduces neutrophil accumulation in inflammatory skin lesions, and reduces ultimate infarct size in an anesthetized open-chest canine model of regional ischemia and reperfusion. Iloprost (0.1-100 microM) inhibited the in vitro production of superoxide anion by canine neutrophils in a concentration-dependent manner. Iloprost (100 ng/kg/min i.v.) inhibited C5a-induced neutrophil migration into inflammatory skin lesions as assessed by the neutrophil-specific enzyme marker, myeloperoxidase. The myeloperoxidase activity determined 2 hours after the intradermal administration of C5a in each of the groups was control 13.3 +/- 1.8 units/g tissue (n = 12) and iloprost 6.5 +/- 0.9 units/g (n = 12), p less than 0.01. Iloprost was administered to anesthetized open-chest dogs (100 ng/kg/min) 10 minutes after left circumflex coronary artery (LCCA) occlusion and continued during the 90-minute occlusion period and the first 2 hours of reperfusion. Regional myocardial blood flow was similar between treatment groups at baseline, 5 minutes and 80 minutes after LCCA occlusion, and after 1 hour of reperfusion. Infarct size, assessed 6 hours after reperfusion, was reduced by iloprost treatment: 22.4 +/- 3.1% of the area at risk (n = 15) compared with 42.4 +/- 3.3% of control (n = 13), p less than 0.01. Iloprost treatment reduced the accumulation of neutrophils (measured by myeloperoxidase activity) in the ischemic myocardium at the interface between infarcted and noninfarcted tissue: control (n = 9) 9.0 +/- 1.8 units/g tissue, iloprost (n = 6) 2.0 +/- 0.4 units/g, p less than 0.01.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs.

Single or multiple brief periods of ischemia (preconditioning) have been shown to protect the myocardium from infarction after a subsequent more prolonged ischemic insult. To test the hypothesis that preconditioning is the result of opening ATP-sensitive potassium (KATP) channels, a selective KATP channel antagonist, glibenclamide, was administered before or immediately after preconditioning in barbital-anesthetized open-chest dogs subjected to 60 minutes of left circumflex coronary artery (LCX) occlusion followed by 5 hours of reperfusion. Preconditioning was elicited by 5 minutes of LCX occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. Glibenclamide (0.3 mg/kg i.v.) or vehicle was given 10 minutes before the initial ischemic insult in each of four groups. In a fifth group, glibenclamide was administered immediately after preconditioning. In a final series (group 6), a selective potassium channel opener, RP 52891 (10 micrograms/kg bolus and 0.1 micrograms/mg/min i.v.) was started 10 minutes before occlusion and continued throughout reperfusion. Transmural myocardial blood flow was measured at 30 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percent of the area at risk. There were no significant differences in hemodynamics, collateral blood flow, or area at risk between groups. The ratio of infarct size to area at risk in the control group (28 +/- 6%) was not different from the group pretreated with glibenclamide in the absence of preconditioning (31 +/- 6%). Preconditioning produced a marked reduction (p less than 0.002) in infarct size (28 +/- 6% to 6 +/- 2%), whereas glibenclamide administered before or immediately after preconditioning completely abolished the protective effect (28 +/- 6% and 30 +/- 8%, respectively). RP 52891 also produced a significant (p less than 0.03) reduction (28 +/- 6% to 13 +/- 3%) in infarct size. These results suggest that myocardial preconditioning in the canine heart is mediated by activation of KATP channels and that these channels may serve an endogenous myocardial protective role.     

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.     

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.     

Regional differences in postischemic recovery in the stunned canine myocardium

To determine if differences exist in the degree of ischemic damage and in postischemic recovery when different coronary arteries are occluded and reperfused, 40 barbital-anesthetized dogs were subjected to brief 15-minute periods of coronary artery occlusion followed by 3 hours of reperfusion ("stunned" myocardium) of the left anterior descending (LAD) or the left circumflex (LCX) coronary arteries. Myocardial segment shortening (%SS) in the subendocardium of nonischemic and ischemic reperfused areas was measured by sonomicrometry, and regional myocardial blood flow was measured by radioactive microspheres. Transmural tissue biopsies were taken at the end of reperfusion for the measurement of adenine nucleotides and total tissue water content. Arterial and local coronary venous blood samples were collected during preocclusion, during occlusion, and at 30 and 180 minutes of reperfusion for determination of blood oxygen content and oxygen consumption in the ischemic area. During occlusion, subendocardial blood flow (LAD flow = 0.11 +/- 0.02; LCX flow = 0.15 +/- 0.04 ml/min/gm), myocardial oxygen consumption (LAD = 2.4 +/- 0.7; LCX = 2.7 +/- 0.7 ml/min/100 gm), and areas of the left ventricle at risk (LAD = 27.4 +/- 2.3%; LCX = 32.4 +/- 2.4) were similar in both groups, thus indicating equivalent degrees of ischemia. There were no differences between groups in hemodynamics throughout the experiment or in the loss of myocardial high-energy phosphates or increase in total tissue water in the ischemic reperfused area at 3 hours of reperfusion. There was a significantly greater loss (p less than 0.05) of systolic wall function during LAD versus LCX occlusion and a greater recovery of segment function from 5 minutes throughout 1 hour of reperfusion after LCX occlusion (p less than 0.05), with no difference in %SS at 2 and 3 hours following reperfusion. Thus, although similar changes occurred in blood flow, metabolite parameters, tissue edema, wall function, and overall hemodynamics when either the LAD or LCX perfusion territories were occluded and reperfused, the loss of systolic wall function and recovery of segment shortening were more variable after regional stunning of the LCX perfusion bed. These data suggest that evaluation of pharmacologic or surgical interventions to improve postischemic functional recovery may be more reliably performed when the LAD coronary artery is the vessel occluded.     

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.     

Prostacyclin analogue iloprost decreases thrombolytic potential of tissue-type plasminogen activator in canine coronary thrombosis.

Platelets play an important role in the formation of a coronary thrombus and reocclusion after thrombolysis. Therefore, we examined the thrombolytic potential of concomitant intravenous administration of potent platelet inhibitor iloprost, a prostacyclin analogue, with tissue-type plasminogen activator (t-PA; n = 8) and t-PA alone (n = 9) in dogs with an electrically induced occlusive coronary artery thrombus. t-PA (0.75 mg/kg) was given over 20 minutes, and iloprost (4 micrograms/kg) was given over 40 minutes. Reperfusion rate was 63% (five of eight dogs) in the t-PA plus iloprost group and 67% (six of nine dogs) in the t-PA alone group (p = NS). The time to thrombolysis (or reperfusion) in the t-PA plus iloprost group was almost twice as great as in the t-PA alone group (33.0 +/- 13.3 vs. 18.5 +/- 6.7 minutes, mean +/- SD, p less than 0.02), and the duration of reperfusion was much shorter (3.4 +/- 1.8 vs. 39.3 +/- 17.4 minutes, p less than 0.005). Peak coronary artery blood flow after reperfusion in the t-PA plus iloprost group was also less (20 +/- 17 ml/min) than in the t-PA alone group (58 +/- 21 ml/min, p less than 0.005). Reocclusion occurred in all dogs given t-PA with iloprost despite potent synergistic platelet inhibitory effects of t-PA and iloprost, whereas four of six dogs given t-PA alone reoccluded. Neither regimen exerted a significant beneficial effect on regional myocardial shortening during coronary reperfusion. Plasma levels of t-PA were lower when iloprost was given with t-PA (1,022 +/- 360 vs. 1,459 +/- 270 ng/ml in t-PA alone group, p less than 0.05). The detrimental effects of iloprost identified in this study may relate to the reduction in plasma t-PA concentrations by its degradation in the liver caused by the prostacyclin analogue iloprost.     

Streptokinase improves reperfusion blood flow after coronary artery occlusion

Streptokinase is an effective thrombolytic agent which, with early restoration of coronary blood flow, has the potential for limiting infarct size. Distinct from thrombolysis, we studied the effects of streptokinase on reperfusion coronary blood flow and infarct size. Open-chest anesthetized canines underwent a 90 minute snare occlusion of the left circumflex coronary artery followed by release and reperfusion through a critical stenosis for 6 hours. The animals were assigned randomly to two groups. Intracoronary streptokinase [group 1 (n = 8): 6000 IU/kg in 3 ml of saline] or saline [group 2 (n = 8): 3 ml of saline] was infused at 0.05 ml/min for 60 minutes beginning 30 minutes before reperfusion. Coronary blood flow was stable in group 1 during reperfusion, while in group 2 it fell during 6 hours of reperfusion (30 +/- 4 ml/min to 18 +/- 2 ml/min, P = 0.05). The ST-segment elevation on the limb lead II electrocardiogram 15 minutes after coronary artery occlusion was similar in both groups (group 1: 3.9 +/- 0.6 mV, group 2: 2.3 +/- 0.5 mV), suggesting the extent of myocardial ischemia was also similar in both groups. The infarct sizes were similar when expressed both as a percent of the total left ventricular mass [(IZ/LV) group 1: 17 +/- 2.5%, group 2: 17.5 +/- 2.5%] or as a percent of the area at risk of infarction [(IZ/AR) group 1: 39 +/- 6%, group 2: 39 +/- 5%]. In both groups, the mass of left ventricle dependent on the blood flow distribution of the left circumflex coronary artery was similar when compared to total left ventricular mass [(AR/LV) group 1: 41 +/- 3%, group 2: 44 +/- 4%]. These results demonstrate that streptokinase maintains reperfusion coronary blood flow through a critical stenosis at a rate similar to baseline levels. Despite the fact that coronary blood flow remained stable with streptokinase during reperfusion, infarct size was not limited after 90 minutes of fixed coronary artery occlusion in this canine model of myocardial injury.     

Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion

Superoxide dismutase (SOD) and catalase (CAT), enzymes that degrade superoxide anion and hydrogen peroxide, respectively, reduce size of infarction in anesthetized, open-chest dogs subjected to coronary occlusion followed by reperfusion. To evaluate potential protective effects of these enzymes in conscious animals, three groups of dogs were instrumented at sterile surgery with a hydraulic occluder on the left circumflex (LCX) coronary artery, sonomicrometers to measure regional wall thickness, and catheters to monitor arterial and left ventricular pressures. Ten to 14 days after surgery, the animals were sedated with morphine sulfate (0.5 mg/kg). The LCX artery was occluded for 3 hr by inflation of the hydraulic cuff. Infusions of SOD (n = 7), CAT (n = 6), or saline (control group, n = 7) were begun 15 min before reperfusion and lasted for 45 min of reperfusion. The doses of SOD and CAT were 5 mg/kg, dissolved in 60 ml of saline, and infused at a rate of 1 ml/min. Myocardial blood flow was measured with tracer-labeled microspheres (15 micron diameter) before occlusion, after 5 to 10 min of occlusion, after 150 min of occlusion, and 5 to 10 min after reperfusion. Size of infarction was measured 24 hr later by dual-perfusion staining with Evans blue and triphenyl tetrazolium. Size of infarction (expressed as a percentage of area at risk) did not differ significantly among the three groups: control, 32 +/- 17% (mean +/- SD); SOD, 38 +/- 17%; CAT, 27 +/- 17%. Hemodynamic parameters and myocardial blood flows (measured before infusion of any agents) were not significantly different among the three groups. Serum SOD levels in SOD-treated dogs were 19 +/- 2 micrograms/ml at the onset of reperfusion and 29 +/- 3 micrograms/ml at the end of the infusion. Blood assays collected after infusion showed a monoexponential decay of SOD levels with a half-life of 22 +/- 6 min. We conclude that myocardial protection by SOD or CAT is model dependent. In conscious dogs subjected to 3 hr of coronary occlusion followed by reperfusion, SOD and CAT failed to alter size of infarction.     

Antagonism of selectin function attenuates microvascular platelet deposition and platelet-mediated myocardial injury after transient ischemia

OBJECTIVES: The goal of this study was to assess whether selectin blockade reduces myocardial platelet deposition and platelet-mediated injury after transient ischemia. BACKGROUND: Selectins participate in platelet adhesion to reperfused endothelium. METHODS: Thiopental-anesthetized, open-chest pigs were subjected to mechanical injury of the left anterior descending coronary artery followed by a 48-min occlusion and 2 (n = 20) or 4 (n = 16) h of reperfusion. Fifteen minutes before occlusion, animals were blindly allocated to receive a continuous intravenous infusion of the selectin blocker fucoidan (30 microg/kg/min, plus a 1-mg/kg bolus in the latter group) or saline. In isolated rat hearts infused with thrombin-activated platelets, the effects of fucoidan (30 microg/ml) administered during reperfusion after 40 min of global ischemia were also analyzed. RESULTS: Fucoidan did not prevent the development of cyclic reductions in coronary flow, but reduced the content of (99m)Tc-labeled platelets in reperfused myocardium after 2 h of reperfusion (23.4 +/- 3.3 vs. 42.1 +/- 8.3 x 10(6) platelets/g in treated and untreated animals, p = 0.03) and attenuated the impairment in the coronary flow reserve and reduced infarct size after 4 h (53 +/- 2% vs. 73 +/- 5% of the ischemic region, respectively, p = 0.003). Treated animals showed a trend toward less neutrophil infiltration early after reperfusion, but not after 4 h. In isolated hearts, fucoidan improved functional recovery and reduced coronary resistance and lactate dehydrogenase release, lacking any beneficial effects if given in the absence of platelets. CONCLUSIONS: The results suggest that selectin-dependent adhesion is a prominent mechanism of platelet deposition in reperfused cardiac microvessels and highlight its potential as a therapeutic target in patients with acute myocardial infarction.     

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.     

Two-dimensional contrast echocardiographic assessment of the time course of regional ischemic myocardial function

The time course of percent fractional area change (%FAC) of the ischemic left ventricular wall as identified by myocardial contrast echocardiography was assessed. Two-dimensional echocardiograms of the left ventricular short axis at the level of the chordae tendineae were recorded in 16 anesthetized open-chest dogs. Myocardial ischemia was produced by occluding the left circumflex coronary artery (LCX) for 30 min, and identified by myocardial contrast echocardiography using aortic root contrast injection. The left ventricular wall in the short-axis view was divided into eight segments. The experiments were completed in nine dogs. The %FAC of the segment which includes the center of the ischemic area was normal before LCX occlusion (35 +/- 6%: mean +/- S.D.), markedly decreased during 30 min of LCX occlusion (-3 +/- 4%) and gradually recovered after coronary reperfusion. However, it was significantly decreased 150 min after reperfusion (8 +/- 9%) (p less than 0.001) compared to that before LCX occlusion. The %FAC of the segment which includes the center of the non-ischemic area was not significantly changed throughout the experiment. In conclusion, 1) the time course of regional ischemic myocardial function could be assessed by the analysis of the %FAC of the ischemic area determined by myocardial contrast echocardiography, 2) the %FAC is significantly decreased 150 min after coronary reperfusion following 30 min occlusion compared to that before coronary occlusion.     

Intracoronary adenosine administration during reperfusion following 3 hours of ischemia: effects on infarct size, ventricular function, and regional myocardial blood flow

Previous studies have demonstrated that adenosine significantly enhances myocardial salvage after 90 minutes of regional ischemia. To determine its effect after prolonged ischemia, closed-chest dogs underwent 3 hours of left anterior descending artery occlusion followed by 72 hours of reperfusion. Intracoronary adenosine (3.75 mg/min; at 1.5 ml/min:total volume = 90 ml; n = 10) or an equivalent volume of saline (1.5 ml/min: total volume = 90 ml; n = 9) was infused into the left main coronary artery during the first 60 minutes of reperfusion. Regional myocardial blood flow was assessed serially with microspheres and regional ventricular function was assessed by contrast ventriculography. Infarct size was determined histologically. Light and electron microscopy were utilized to assess neutrophil infiltration and microvascular injury. Adenosine failed to reduce infarct size expressed as a percentage of the area at risk (38.0 +/- 4.9% versus 34.8 +/- 4.6%; p = NS) or to improve regional ventricular function as measured by the radial shortening method (3.2 +/- 1.8% versus 2.2 +/- 3.1%; p = NS) at 72 hours after reperfusion. Vasodilatory effects were not observed in the endo- and midmyocardial regions of the ischemic zone during adenosine administration. This was associated with a similar extent of capillary endothelial changes and neutrophil infiltration in both adenosine-treated and saline control groups. These results suggest that severe functional abnormalities are present in the vasculature after 3 hours of ischemia and that adenosine therapy is ineffective in enhancing myocardial salvage.     

Failure of iloprost to protect the regionally ischemic, reperfused porcine heart.

The effect of iloprost (Schering AG, Berlin), a stable prostacyclin analogue, was investigated in ischemic, reperfused porcine hearts. The left anterior descending coronary artery (LAD) was distally occluded in 18 pigs for 45 min followed by 3-d of reperfusion. Nine pigs were continuously treated with iloprost at a dose of 25 ng/kg per min. Treatment was started as intracoronary infusion into the proximal LAD 10 min before occlusion. The intercoronary infusion was replaced by an intravenous infusion after 45 min of reperfusion, which was continued until the end of the experiment. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Myocardial concentrations of adenosine triphosphate were evaluated at the end of the experiment. Generation of free radicals by stimulated polymorphonuclear neutrophils was determined by luminol-enhanced chemiluminescence. Histologic and immunohistologic techniques were applied to characterize the myocardial inflammatory response. Global hemodynamics did not differ between the two groups. Neither infarct size (control group 68 +/- 18%, treated group 74 +/- 14%), recovery of systolic shortening (control group 3 +/- 6%, treated group 6 +/- 6%), nor myocardial adenosine triphosphate concentrations were improved by iloprost treatment. Myocardial inflammatory response remained unaffected by this treatment. The capacity of coronary venous, stimulated polymorphonuclear neutrophils to generate free radicals was slightly suppressed in the treated group before ischemia, at the end of ischemia and during early reperfusion. In this preparation, iloprost did not exhibit any beneficial effect on infarct size, recovery of systolic shortening and myocardial adenosine triphosphate concentrations.     

Limitation of myocardial infarct size by superoxide dismutase as an adjunct to reperfusion after different durations of coronary occlusion in the pig

Superoxide dismutase (SOD) has been documented to limit myocardial infarct size in the richly collateralized dog heart. This study was designed to explore this concept in a low-collateralized animal model. A blind, randomized, placebo-controlled protocol was used in 65 pentobarbital-anesthetized pigs subjected to closed-chest left anterior descending coronary artery occlusion for 30 (n = 22), 60 (n = 22), and 90 (n = 14) minutes followed by reperfusion up to 24 hours from the start of occlusion. Another seven control pigs were subjected to 24 hours of permanent occlusion. A total dose of 9 mg/kg bovine CuZn SOD was administered as a bolus injection immediately before reperfusion followed by a 1-hour infusion. Infarct size was assessed by tetrazolium staining. Myocardium at risk and collateral flow were determined by using cerium-141-labeled microspheres (15 microns) during the occlusion. After 30 minutes of occlusion, infarct sizes in placebo versus SOD-treated animals were 45.5 +/- 15.7% vs. 23.8 +/- 15.6% of myocardium at risk (p = 0.007). The corresponding values after 60 minutes of occlusion were 78.6 +/- 9.3% vs. 66.9 +/- 14.6% (p = 0.035). SOD administered after 90 minutes of occlusion did not limit infarct size (88.5 +/- 4.8% vs. 92.3 +/- 5.2%). Twenty-four hours of coronary occlusion resulted in infarction of 92.4 +/- 4.2% of myocardium at risk. (All values are mean +/- SD.) Ventricular fibrillation occurred in only nine pigs distributed equally between SOD and placebo. The results indicate that CuZn SOD has the potential to further improve the myocardial salvage established by reperfusion of an ischemic pig heart territory. However, the narrow time window for limiting infarct size in the pig by reperfusion is not much extended by SOD.     

Short-term treatment with synchronized coronary venous retroperfusion before full reperfusion significantly reduces myocardial infarct size.

The efficacy of short-term synchronized coronary venous retroperfusion (SRP) before full arterial reperfusion was studied in a canine model. A control group (n = 6) was subjected to 90 minutes of occlusion of the left anterior descending coronary artery, which was followed by 6 hours of reperfusion. In another group (n = 6) the left anterior descending coronary artery was occluded for 2 hours followed by 5.5 hours of reperfusion. In this group SRP was applied for 30 minutes before full reperfusion. Myocardial regional blood flow was measured with the use of colored microspheres. During occlusion of the left anterior descending coronary artery, there was severe myocardial ischemia in both groups. Blood flow in the subendocardial area was, however, significantly better in the SRP group (0.51 +/- 0.17 ml/min/gm after 3.5 hours of reperfusion) than in the control group (0.29 +/- 0.16 ml/min/gm) after 4 hours of reperfusion (p less than 0.05). Left ventricular function was assessed as global ejection fraction from a left ventriculogram. Ejection fraction was reduced during ischemia in both groups (control = 38% +/- 3%, SRP = 32% +/- 8%). This dysfunction remained after 4 hours of reperfusion. Infarct size was assessed by means of triphenyltetrazolium chloride staining. The myocardial area at risk was similar in the two groups (control = 33.1% +/- 5.3%, SRP = 30.6% +/- 6.5%). Infarct size, which was expressed as the percent of the area at risk, was significantly smaller in the SRP group (17.2% +/- 14.6%) than in the control group (36.0% +/- 8.1%; p = 0.0197).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil accumulation in experimental myocardial infarcts: relation with extent of injury and effect of reperfusion

The effects of reperfusion on the myocardial accumulation of neutrophils and their role in the extent of injury were investigated in a canine preparation with a 3 hr coronary occlusion followed by 21 hr of reperfusion. The left anterior descending coronary artery (LAD) was permanently occluded in group 1 and reperfused after 3 hr in four others (groups 2 to 5). All but group 5 received lidocaine (1 mg/min over 8 hr). A critical stenosis was produced and left in place at reperfusion only in group 2. In groups 1 and 2, 111In-labeled autologous neutrophils were injected at the time of coronary occlusion. Group 4 animals were rendered leukopenic 2 hr before the coronary ligature and throughout the experiment by injection of an antineutrophil rabbit serum. Quantification of the radioactivity by digitized scintigraphy of the heart slices revealed an 80% (p less than .05) increase in neutrophil accumulation in the infarct region after reperfusion (group 2) as compared with permanent occlusion (group 1). Gamma counting of myocardial tissue samples showed that the neutrophil accumulation ratio in the subendocardial central zone of the infarct was increased five times (p less than .05) by reperfusion, whereas no difference was evident in the subepicardium. Infarct size and myocardial area at risk were not statistically different among the five groups. However LAD flow in the leukopenic group (group 4) was significantly higher (p less than .05) 30 min after reperfusion (40.0 +/- 5 ml/min) when compared with the preocclusion value (21.7 +/- 4 ml/min). In contrast, in a parallel experiment without leukopenia (group 3), LAD flow after reperfusion did not differ from the preocclusion value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined adenosine and lidocaine administration limits myocardial reperfusion injury

The endogenous compound adenosine may play a role in limiting myocardial ischemia-reperfusion injury through its ability to cause vasodilation, modulate cardiac adrenergic responses, inhibit neutrophil function, or modulate energy supply and demand of the myocardium. The local anesthetic lidocaine has been shown to be protective against myocardial ischemia-reperfusion injury, although its mechanism of action remains unresolved. We hypothesized that administration of exogenous adenosine during reperfusion would limit the size of the infarct that results from a period of ischemia and reperfusion only when the animals are treated with lidocaine. Male, mongrel dogs (13.0-20.0 kg) were anesthetized (30 mg/kg i.v. sodium pentobarbital), and a left thoracotomy was performed. The left circumflex coronary artery (LCx) was isolated and instrumented with an electromagnetic flow probe, a 25-gauge nonobstructing intracoronary catheter, and a critical stenosis. The dogs were allocated randomly to one of four groups: 1) control, n = 13, (saline), 2) adenosine, n = 13, (0.15 mg/kg/ml/min i.c. for the first hour of reperfusion), 3) lidocaine, n = 9, (2.0 mg/kg i.v. given immediately before coronary artery occlusion and just before reperfusion), or 4) adenosine plus lidocaine, n = 11. The LCx was occluded for 90 minutes and reperfused for 6 hours. Regional myocardial blood flow (RMBF) was determined (n = 6 per group) at 80 minutes of occlusion and at 45 minutes of reperfusion with radiolabeled microspheres. RMBF determinations revealed an increase in blood flow to the inner two thirds of the myocardium at 45 minutes of reperfusion only in the presence of the combined treatment. Adenosine treatment alone or lidocaine treatment alone did not affect RMBF. Quantification of infarct size (triphenyltetrazolium method) expressed as a percent of the area at risk revealed a significant limitation of infarct size only in the group treated with both adenosine and lidocaine: control, 47.8 +/- 6.6%; adenosine, 45.0 +/- 3.2%; lidocaine, 46.9 +/- 6.0%; and adenosine and lidocaine, 20.8 +/- 5.6%. Statistical analyses were performed with two-way analysis of variance to account for the two individual drug treatments. The findings show that intracoronary administration of exogenous adenosine, at the dose used, is only effective at limiting myocardial infarct size when administered to lidocaine-treated animals.     

Pravastatin reduces myocardial infarct size via increasing protein kinase C-dependent nitric oxide, decreasing oxyradicals and opening the mitochondrial adenosine triphosphate-sensitive potassium channels in rabbits.

BACKGROUND: Statins reportedly protect against myocardial infarction, but the precise mechanism is unclear. METHODS AND RESULTS: Rabbits underwent 30 min of coronary occlusion followed by 48 h of reperfusion. Pravastatin (1 or 5 mg/kg) or saline was intravenously administered 10 min before ischemia. Pravastatin (5 mg/kg) was also administered 10 min before reperfusion. N(omega)-nitro-L-arginine methylester (L-NAME, 10 mg/kg), chelerythrine (5 mg/kg) or 5-hydroxydecanoic acid sodium salt (5-HD, 5 mg/kg) was intravenously administered 10 min before pravastatin injection. The infarct size was determined. The myocardial interstitial levels of 2,5-dihydroxybenzoic acid (DHBA) and nitrogen oxide (NOx), and the intensity of myocardial dihydroethidium staining were measured. Pre-ischemic treatment with pravastatin reduced the infarct size (34+/-5% and 24+/-4%, 1 and 5 mg/kg, respectively), but not pre-reperfusion treatment (42.1+/-3.7%), compared with the control (45+/-3%). This effect was blocked by L-NAME (42.6+/-4%), chelerythrine (50.9+/-3%) and 5-HD (52.7+/-2%). Pre-ischemic treatment with pravastatin increased myocardial NOx levels, and attenuated both the 2,5-DHBA level and the intensity of dihydroethidium staining during reperfusion. Chelerythrine abolished the increase in NOx levels by pravastatin. CONCLUSION: Pre-ischemic treatment with pravastatin reduces the myocardial infarct size via protein kinase C-dependent nitric oxide production, decreasing hydroxyl radicals and superoxide, and opening the mitochondrial adenosine triphosphate-sensitive potassium channels.