Pharmacologic perturbation of neutrophils by Fluosol results in a sustained reduction in infarct size in the canine model of reperfusion.

Previous studies have demonstrated that intravenous administration of large doses of Fluosol, a perfluorochemical preparation, reduced infarct size 24 h after reperfusion, an effect that was associated with reduced neutrophil infiltration. The effect of a clinically tolerable dose of Fluosol on infarct size after a prolonged period of reperfusion and its mechanism of action on neutrophils remain unknown. Twenty-one anesthesized closed chest dogs were subjected to 90 min of proximal left anterior descending coronary artery occlusion and 72 h of reperfusion. An additional five dogs that did not undergo regional myocardial ischemia were utilized to explore the mechanism of action of Fluosol on neutrophil function. In the infarct study, animals were randomized to receive either intravenous Fluosol (n = 10) or an equivalent volume of Ringer's lactate solution (control; n = 11) at 15 ml/kg body weight during the last 30 min of occlusion and for the 1st 30 min of reperfusion. Fluosol significantly reduced infarct size when expressed as percent area at risk 72 h after reperfusion (13.7 +/- 2.7% vs. 38.3 +/- 4.5%, respectively, p less than 0.001). This reduction was associated with significant improvement in regional wall motion (18.4 +/- 2.3% vs. 5.5 +/- 2%, p less than 0.001). Endocardial blood flow in the ischemic bed was significantly higher 3 h after reperfusion in Fluosol-treated dogs (0.63 +/- 0.08 vs. 0.34 +/- 0.07 ml/min per g, p = 0.01). Reduced capillary plugging by neutrophils with relative preservation of endothelial cell structure was observed in Fluosol-treated animals. Infusion of Fluosol produced a marked transient decrease in peripheral neutrophil and platelet counts in both ischemic and nonischemic dogs and was associated with a significant reduction in total hemolytic complement levels. Studies of neutrophil function ex vivo revealed a reduction in chemotaxis and lysozyme degranulation after infusion of Fluosol. In vitro experiments showed that Fluosol produced a rapid and sustained activation of neutrophils determined by superoxide anion production. These data demonstrate that low dose intravenous Fluosol produces a sustained reduction in infarct size in the canine model. The beneficial effect may be in part due to the suppression of various neutrophil functions in the reperfused myocardium subsequent to peripheral activation by Fluosol. Such interventions may offer a novel therapy to enhance myocardial salvage by sequestration of circulating neutrophils during the critical early reperfusion period.     

Intracoronary adenosine administered after reperfusion limits vascular injury after prolonged ischemia in the canine model

Myocardial salvage after reperfusion may be limited by deleterious vascular changes in the previously ischemic microcirculatory bed. This could result in a progressive decrease in blood flow in the capillary bed to potentially viable myocytes (no-reflow phenomenon). The effect of intracoronary adenosine on these changes was assessed in 15 closed-chest dogs subjected to 2 hours of proximal left anterior descending artery (LAD) occlusion followed by 3 hours of reperfusion. Animals randomly received adenosine (n = 8) 3.75 mg/min into the proximal LAD or an equivalent volume of saline (control) (n = 7) for 1 hour after reperfusion. Endothelial-dependent and independent coronary vasodilator reserve was determined using a chronically implanted volume-flowmeter on the mid-LAD at baseline and 1 and 3 hours after reperfusion with acetylcholine and papaverine infusions, respectively, into the proximal vessel. Regional myocardial blood flow was measured serially with radioactive microspheres and regional contractile function with contrast ventriculography. Both agonists produced a significant increase in LAD flow before occlusion. Endothelial-dependent and independent vasodilatory reserve was significantly reduced (p less than 0.05) at 1 and 3 hours after reperfusion in control animals compared with adenosine treatment. A progressive decrease in mid-LAD flow and increase in coronary vascular resistance after reperfusion was observed in control animals (p less than 0.05). The treated group manifested improved regional myocardial blood flow in endocardial regions from the central (0.73 +/- 0.15 versus 0.24 +/- 0.11 ml/g/min; p less than 0.02) and lateral ischemic zones (0.80 +/- 0.15 versus 0.34 +/- 0.12 ml/g/min; p less than 0.05) 3 hours after reperfusion. A significant reduction (p less than 0.05) in endocardial and midmyocardial flow compared with baseline was seen in control animals at 3 hours. Intravascular and interstitial neutrophil infiltration was reduced in adenosine animals and this was associated with relative ultrastructural preservation of endothelial cells. Regional ventricular function in the ischemic zone was improved in the adenosine group 3 hours after reperfusion (13.4 +/- 3.9% versus -5.3 +/- 1.6%; p less than 0.001). This study demonstrates that selective administration of adenosine after reperfusion significantly attenuates functional and structural abnormalities in the microvasculature after prolonged (2 hours) regional ischemia in the canine model. Prevention of microvascular injury and the non-reflow phenomenon by adenosine may preserve reversibly injured myocytes following restoration of blood flow to previously ischemic myocardium.     

Neutrophil accumulation in ischemic canine myocardium. Insights into time course, distribution, and mechanism of localization during early reperfusion

BACKGROUND. We have previously demonstrated that chemotactic factors released from the ischemic canine myocardium peak early during reperfusion and that they elicit neutrophil adherence reactions in vitro that are dependent on the CD18 glycoprotein family. In this study we investigated the hypothesis that neutrophil localization in ischemic canine myocardium in vivo occurs over a similar time course during early reperfusion and involves a CD18-dependent mechanism. METHODS AND RESULTS. We occluded the circumflex coronary artery for 1 hour in acute, open-chest dogs, followed by reperfusion for 1, 2, 3, or 4 hours. Regional myocardial blood flow was determined using radiolabeled microspheres, and localization was traced using technetium-99m-labeled autologous neutrophils. In the first hour of reperfusion, neutrophil localization occurred preferentially within the subendocardial region and was inversely related to flow. Neutrophil localization diminished across the ischemic myocardium from endocardium to epicardium but remained negatively related to flow in the midmyocardial region. Regardless of flow, little neutrophil localization occurred in the subepicardial region. Neutrophil localization was greatest in the first hour of reperfusion and diminished thereafter. By 4 hours of reperfusion, the rate of localization was markedly attenuated relative to 1 hour. Dogs given anti-CD18 monoclonal antibody R15.7 (1 mg/kg i.v.) before occlusion underwent 1 hour of occlusion followed by 1 hour of reperfusion. When compared with 1-hour reperfusion controls, the R15.7-treated dogs demonstrated significant attenuation of neutrophil localization in the subendocardial region. CONCLUSIONS. These data support the concepts that rapid neutrophil localization during reperfusion occurs within regions of previous myocardial ischemia and that neutrophils preferentially localize within the subendocardial region. The rate of neutrophil localization is greatest within the first hour after the initiation of reperfusion, and localization is, at least in part, CD18 dependent. Therapies directed against neutrophil-mediated reperfusion injury should be initiated with these considerations in mind.     

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.     

Reduction of myocardial infarct size by neutrophil depletion: effect of duration of occlusion

Experiments were performed in the dog to examine the effects of neutropenia on ultimate infarct size resulting from short (90 minutes) or prolonged (4 hours) circumflex coronary artery occlusion. Sheep antiserum to canine neutrophils was used to produce neutropenia. Control animals received nonimmune serum. Neutrophil infiltration into myocardial infarcts was examined using histopathologic techniques and a semiquantitative scoring system. In 90-minute occlusions with 24-hour reperfusion, neutropenia was associated with the development of significantly smaller infarcts: normopenic group, 43.2% +/- 3.3% (n = 7) vs. neutropenic group, 26.6% +/- 3.7% (n = 10) of the area at risk, means +/- SEM. However, in 4-hour occlusion with 6-hour reperfusion experiments, the tendency of neutrophil depletion to reduce infarct size did not reach statistical significance (46.4% +/- 7.2% vs. 31.5% +/- 6.0% of the area at risk, normopenic vs. neutropenic) despite differences in neutrophil infiltration into the reperfused region. The observed differences in ultimate infarct size could not be attributed to differences in myocardial oxygen consumption. The results suggest that a significant amount of myocardial infarction induced by a limited duration of coronary artery occlusion followed by reperfusion is neutrophil dependent and appears to be less important in determining the fate of myocardium subjected to more prolonged periods of ischemia followed by reperfusion.     

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)     

Effects of transient coronary ischemia and reperfusion on myocardial edema formation and in vitro magnetic relaxation times

The effects of transient ischemia and reperfusion on regional myocardial function, salvage and swelling have been systematically analyzed in experimental canine preparations. The results of these interventions on myocardial in vitro measurements of magnetic relaxation times (T1 = magnetization recovery, T2 = spin echo) are of significant importance with respect to future nuclear magnetic resonance tomographic imaging. Thus, using a pulsed magnetic resonance spectrometer (10.7 MHz), myocardial tissue samples from two groups of dogs were evaluated. In group 1 (n = six dogs), the left anterior descending artery was occluded for 3 hours before sacrifice; in group 2 (six dogs), 3 hours of occlusion was followed by 1 hour of reperfusion. Multiple tissue samples from normal and ischemic (or ischemic and reperfused) myocardium were obtained for measurement of T1, T2 and % water content (wet weight--dry weight/wet weight). Water content increased with ischemia (78 +/- 4%) and reperfusion (81 +/- 4%) (both p less than 0.01 versus control values). Values for T1 increased with ischemia (598 +/- 39 versus 487 +/- 23 ms in normal tissue from the same heart, p less than 0.01). Even greater T1 changes occurred in the animals with reperfusion (654 +/- 52 ms, p less than 0.01 versus the intra-animal control values). Changes in T2 were similar but less marked (ischemic zone 43.9 +/- 1.0 versus 41.2 +/- 1.0 ms in nonischemic tissue in the corresponding heart, p less than 0.05; reperfusion zone 48.3 +/- 3.5 versus 41.9 +/- 2.3 ms in the normal zone, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influx of neutrophils into the walls of large epicardial coronary arteries in response to ischemia/reperfusion

BACKGROUND. There are several clinical situations in which large epicardial coronary arteries are deprived of blood flow, such as occurs when an obstructing thrombus or embolus lodges within a vessel or during coronary dissection. There is little information concerning the effect of flow deprivation on large epicardial coronary arteries. METHODS AND RESULTS. We studied a model in which a segment of a large epicardial coronary artery was deprived of blood flow using both proximal and distal clamps for 3 hours followed by reperfusion. On examination by light microscopy of cross sections of the arteries, 19 +/- 6 neutrophils were present in the intima of ischemic/reperfused vessels, whereas only a mean of 4 +/- 3 (SEM) were present in the intima of nonischemic vessels (p less than 0.02). On average, there were 17 +/- 9 neutrophils just under the elastic lamina in ischemic/reperfused vessels versus none in the nonischemic vessels (p less than 0.05); there were 16 +/- 10 neutrophils present within the media of ischemic/reperfused vessels, and none (p less than 0.05) in the nonischemic vessels. Electron microscopic analysis revealed that neutrophils in the ischemic/reperfused vessels were often "sandwiched" between the endothelial cells and the elastic lamina. Ultrastructural abnormalities within the myocardium also revealed damage to the microvasculature, including the presence of neutrophils within the vessels and erythrocyte stasis. To rule out the possibility that findings in the large epicardial arteries were due to toxic substances from static blood within the isolated arterial segment, a protocol was performed in which blood was removed from the isolated segment. Again, neutrophil infiltration into the vessel was observed. Resting mean epicardial coronary artery blood flow before coronary occlusion was 19 +/- 3 ml/min; mean coronary blood flow 2.5 hours after reperfusion was identical at 19 +/- 3 ml/min. Response to both endothelial-dependent vasodilation (acetylcholine) and endothelial-independent vasodilation (nitroglycerin) challenges was normal early after reperfusion but was depressed late after reperfusion, suggesting progressive vascular dysfunction and hence a form of vascular reperfusion injury in this model. CONCLUSIONS. When large epicardial coronary arteries are deprived of blood flow, followed by reperfusion in this model, neutrophils migrate into the vessel wall as well as into the microvasculature. These abnormalities are associated with reduced endothelial-dependent and endothelial-independent coronary vasodilator reserve.     

Reduction of reperfusion injury in the canine preparation by intracoronary adenosine: importance of the endothelium and the no-reflow phenomenon

We hypothesized that the endogenous coronary vasodilator adenosine may reduce infarct size by progressively increasing reflow in a preparation of coronary occlusion-reperfusion. After 90 min of proximal left anterior descending artery occlusion, 20 dogs were randomized to blood reperfusion with (n = 10) or without (n = 10) adenosine into the proximal left anterior descending vessel at 3.75 mg/min for 60 min after reperfusion. Regional myocardial blood flow was determined serially with microspheres and regional ventricular function was assessed by a computerized radial shortening method. At 24 hr, the area at risk was defined in vivo with monastral blue dye and area of necrosis was determined after incubation of left ventricular slices in triphenyltetrazolium chloride. Hemodynamic variables were similar in the two groups during the experimental protocol. Infarct size was significantly reduced in treated animals, both when expressed as a percentage of the area at risk (9.9 +/- 2.8% vs 40.9 +/- 6.6%, p less than .001) and as a percentage of the left ventricle (4.6 +/- 1.3% vs 18.0 +/- 3.4%, p = .002). This was associated with significant improvement in radial shortening in the ischemic zone 24 hr after reperfusion (10.1 +/- 2.5 vs -2.8 +/- 2.2%, p less than .01). Regional myocardial blood flow was significantly increased in endocardial and epicardial regions from the lateral ischemic zone 1 hr after reperfusion in adenosine-treated animals. Light microscopy demonstrated decreased neutrophil infiltration in the ischemic zone and electron microscopy showed relative preservation of endothelial structure in the subendocardium with reduced neutrophil and red cell stagnation of capillaries in the treated group. These findings suggest that intracoronary administration of adenosine after reperfusion significantly reduces infarct size and improves regional ventricular function in the ischemic zone in the canine preparation.     

Paralysis of phagocyte migration due to an artificial blood substitute

We investigated the effect of a candidate artificial blood substitute, Fluosol-DA (FDA), on human neutrophil function in a serum-free medium. In a 50% (vol/vol) mixture with polymorphonuclear cells (PMN), FDA had no effect on PMN viability, phagocytosis, superoxide anion generation, degranulation, or bactericidal activity. In striking contrast, the random migration and chemotaxis of PMN to both f-Met-Leu-Phe (fMLP) and activated serum were inhibited by 98% +/- 2%, 95% +/- 2%, and 88% +/- 6%, respectively. Inhibition of chemotaxis by FDA required no preincubation, was dose-dependent (50% inhibition [ID50] with a 14% vol/vol mixture with FDA), and was fully reversible by washing PMN free of FDA after one hour but not after 18 hours of incubation (32% +/- 11% inhibition of chemotaxis). FDA itself was not chemotactic and did not impair either the chemotactic activity or binding of fMLP to PMN. FDA also inhibited PMN adhesion (ID50, 9 +/- 1 vol/vol%). The inhibitory component of FDA was found to be its detergent additive, Pluronic F-68, which inhibited random migration, chemotaxis, and adhesion with ID50s of 1.4, 2.4, and 2.9 mg/mL, respectively (equivalent to FDA concentrations of 5, 9, and 11 vol/vol%, respectively). All the other components of FDA were noninhibitory. Plasma samples from humans injected with 8 mL/kg FDA and plasma samples from rabbits injected with 16 mL/kg FDA or an equivalent concentration of Pluronic F-68, when mixed with autologous PMN, also severely inhibited PMN chemotaxis. We conclude that exposure of PMN to clinically relevant concentrations of FDA inhibits PMN migration, presumably due to inhibition of adhesion. The inhibitory effect is entirely due to the detergent, Pluronic F-68. Artificial blood substitutes containing Pluronic F-68 may compromise the ability of PMN to prevent or effectively control microbial infections.     

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.     

Dose-dependent reduction of myocardial infarct size with the perfluorochemical Fluosol-DA

The perfluorochemical Fluosol-DA has been shown to reduce infarct size. However, the dose-response relationship of the agent is unknown. Because perfluorochemicals (PFC) can potentially saturate the reticuloendothelial system and decrease carbon clearance as well as cause a transient elevation in liver enzymes, the present study was conducted to determine the lowest effective dose. New Zealand White rabbits (n = 73) were randomly selected prior to infarction to receive 10, 15, 20, 25, or 30 ml/kg PFC or an equivalent volume of 5% dextran (control) intravenously. Animals underwent 30 minutes of coronary artery occlusion with PFC or dextran infused over a 30-minute period starting at 20 minutes into the occlusion. Animals were put to death at 24 hours and infarct size was determined histologically and quantitated by computerized planimetry. Neutrophil infiltration into the ischemic myocardium was evaluated semiquantitatively. No hemodynamic differences were noted within groups. Infarct size was similar to that of controls in animals treated with 10 or 15 ml/kg PFC. Significant infarct size reduction, however, was noted in animals treated with 20, 25, and 30 ml/kg PFC versus controls; (p = 0.05, 0.04, and 0.02, respectively). Maximal infarct size reduction was seen with 30 ml/kg PFC (35%). Neutrophil infiltration was significantly decreased in all groups treated with PFC. These results show that intravenous Fluosol-DA significantly reduces infarct size at a minimal dose of 20 ml/kg.     

Protective effects of ginsenosides in myocardial ischemia and reperfusion injury

To verify whether ginsenosides will attenuate the myocardial ischemia and reperfusion injury, the left anterior descending coronary artery (LAD) was snared for 2 hours in 23 dogs and then the ischemic myocardium was reperfused. 45 minutes after ischemia, the animals were randomly divided into a ginsenosides group (n = 11, receiving a slow IV bolus of ginsenosides 10 mg/kg and then a continuous infusion of 80 micrograms/kg/min) and a saline solution group (n = 12 receiving equal amount of glucose in saline). The treatment was started 45 minutes after coronary occlusion and stopped one hour after reperfusion. 24 hours later, the dogs were killed and the extent of myocardial necrosis was determined histologically. The LVEDP, arterial pressure and heart rate were markedly lower in the ginsenosides group. Electrocardiographic findings of myocardial ischemia were significantly improved in the ginsenosides group. 8 controls developed malignant arrhythmia after reperfusion, but none in ginsenosides group. The myocardial ultrastructure can be protected by ginsenosides during the period of ischemia and reperfusion. The infarct size in saline group was 22.7 +/- 3.2% while in the ginsenosides group it was 5.2 +/- 1.3% (P less than 0.05). These results show that ginsenosides can protect the ischemic myocardium and reperfusion injury of myocardium.     

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)     

Limitation of no reflow injury by blood-free reperfusion with oxygenated perfluorochemical (Fluosol-DA 20%)

This study was designed to assess the effects of blood-free reperfusion with oxygenated or unoxygenated intracoronary perfluorochemical (Fluosol-DA 20%) on myocardial perfusion and to determine its mechanism or mechanisms of limiting no reflow. Twenty-four dogs underwent 90 min of coronary occlusion followed by 210 min of reperfusion and were randomized to either: 1) blood-free reperfusion with intracoronary oxygenated perfluorochemical (20 ml/kg per min) for 20 min followed by blood reperfusion (n = 8); 2) intracoronary unoxygenated perfluorochemical administered as in those treated with oxygenated perfluorochemical (n = 8); and 3) blood reperfusion alone (control) (n = 8). Regional myocardial blood flow was serially determined and global myocardial perfusion was assessed by an intravenous injection of the fluorescent dye (thioflavin-S). Quantitative studies were performed to determine neutrophil infiltration and extent of endothelial injury. Hemodynamic variables were similar in all groups. The zone of impaired perfusion (thioflavin negative), expressed as a percent of the left ventricle, averaged 10 +/- 2%, 6 +/- 2% and 3 +/- 1%, in control and unoxygenated and oxygenated perfluorochemical groups, respectively (control versus oxygenated perfluorochemical p less than 0.004). The reduction in thioflavin-negative area with oxygenated perfluorochemical was associated with a notable recovery of endocardial blood flow (0.97 +/- 0.22 vs. control 0.39 +/- 0.08 ml/min per g; p less than 0.04) at 210 min of reperfusion. The number of capillaries plugged by neutrophils (per 200 capillaries) in thioflavin-negative areas was similar with both oxygenated (5.9 +/- 1.4) and unoxygenated perfluorochemical (5.4 +/- 0.8) treatment and was significantly less than that with the control group (18.9 +/- 3.2, p less than 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced myocardial neutrophil accumulation and infarct size following thromboxane synthetase inhibitor or receptor antagonist

Since thromboxane A2 (TXA2) release may relate to the extension of myocardial injury following coronary ligation, the authors examined the effects of pretreatment with a selective TXA2 synthetase inhibitor U-63,557A, or a TXA2 receptor antagonist SQ-29,548, on myocardial infarct size forty-eight hours following left coronary ligation in rats. Myocardial infarct size (as percent of left ventricle, LV) was decreased from 44 +/- 3% in saline-treated control animals to 34 +/- 4% (P less than 0.05) in U-63,557A-treated animals and to 32 +/- 4% (P less than 0.05) in SQ-29,548 treated animals (U-63,557A-treated vs SQ-29,548-treated, P = NS). LV creatine kinase (CK) was 5.08 +/- 0.42 IU/mg protein in noninfarcted untreated rats and 1.79 +/- 0.21 IU/mg protein in saline-treated infarcted rats. LV CK was 2.86 +/- 0.40 IU/mg protein in U-63,557A-treated rats and 3.11 +/- 0.51 IU/mg protein in SQ-29,548-treated infarcted rats (both P less than 0.05 compared with saline-treated rats). The beneficial effects of U-63,557A and of SQ-29,548 were not accompanied by reduction in indices of myocardial oxygen demand (heart rate and arterial pressure). However, neutrophil accumulation in the infarcted myocardium was markedly decreased by U-63,557A and SQ-29,548 pretreatment. Myocardial myeloperoxidase activity, a specific marker of neutrophil infiltration, was also decreased (P less than 0.02) in U-63,557A- and SQ-29,548-treated animals (0.09 +/- 0.03 and 0.07 +/- 0.02 units/100 mg, respectively) compared with saline-treated infarcted rats (0.19 +/- 0.04 units/100 mg). In vitro incubation of U-63,557A and SQ-29,548 caused a significant and similar reduction in f-MLP-induced neutrophil chemotaxis, and U-63,557A increased prostacyclin formation in whole blood. These data suggest that reduction in the extent of myocardial injury by TXA2 synthetase or receptor inhibitors may, in part, relate to a decrease in neutrophil accumulation in the infarcted tissue. In spite of differences in mechanisms of action of U-63,557A and SQ-29,548, both agents exert a similar protective effect on the extent of myocardial injury following coronary ligation. Reduction in neutrophil accumulation in the infarcted zone, as well as in f-MLP-directed chemotaxis in vitro, suggests that TXA2 inhibition may modulate neutrophil migration.     

Assessment of myocardial salvage after ischemia and reperfusion using magnetic resonance imaging and spectroscopy

To test the hypothesis that contrast-enhanced magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) can differentiate reversible from irreversible myocardial injury, these modalities were used to study ischemia and reperfusion in a rat model. The presence of ischemia and reperfusion were confirmed with radiolabeled microspheres (n = 6). Groups of animals were subjected to either 16 (n = 17), 30 (n = 14), 60 (n = 11), or 90 (n = 14) minutes of left coronary artery (LCA) occlusion and 60 minutes reperfusion. After albumin-gadolinium (Gd)-DTPA injection, contrast-enhanced, T1-weighted, spin-echo proton images were acquired at baseline and every 16 minutes during LCA occlusion and reperfusion. In separate experiments, 31phosphorus (31P) spectra were acquired at similar time points during ischemia and reperfusion. After 16 minutes occlusion, normally perfused myocardium enhanced significantly compared with ischemic myocardium on MRI (104 +/- 7.9% vs. 61 +/- 11.0%, p less than 0.05, n = 5, mean +/- SEM, % of baseline value). MRS showed reduced phosphocreatine (PCr) and adenosine triphosphate (ATP) (58.8 +/- 2.4%, p less than or equal to 0.01; 81.4 +/- 2.4, p less than or equal to 0.01, n = 12). After 16 or 30 minutes ischemia, reflow resulted in uniform MRI signal intensity of the ischemic zone compared with normal myocardium (93.5 +/- 11.3 vs. 80.9 +/- 7.0, p = NS, n = 11, % of baseline value at 30 minutes reperfusion) and PCr recovery on MRS (94.3 +/- 4.0%, p = NS, n = 20, % baseline value at 30 minutes reflow). After 60 and 90 minutes ischemia, reflow resulted in marked enhancement of reperfused compared with normal myocardium on MRI (254.0 +/- 30.0 vs. 78.3 +/- 9.2, p less than or equal to 0.01, n = 10) and no recovery of PCr on MRS (64.1 +/- 3.0, p = NS, n = 14). Triphenyltetrazolium chloride (TTC) staining revealed transmural myocardial infarction (MI) in all hearts subjected to 60 or 90 minutes ischemia and reflow, and small nontransmural MIs in only 2/11 hearts subjected to 16 or 30 minutes ischemia and reperfusion. Thus, 1) MRI with albumin-Gd-DTPA is useful for identifying myocardial ischemia by enhancing the contrast between normally perfused and ischemic myocardia; 2) MRI with albumin-Gd-DTPA is useful for identifying reperfusion after myocardial ischemia; and 3) after reperfusion, reversible can be distinguished from irreversible myocardial injury by characteristic findings on MRI and MRS.     

Inotropic stimulation of reperfused myocardium with dopamine: effects on infarct size and myocardial function

Prolonged postischemic ventricular dysfunction ("stunned myocardium") may be responsible for heart failure after myocardial reperfusion. Although inotropic stimulation can enhance the contractility of stunned myocardium, it could potentially increase infarct size and thereby impair ultimate recovery of myocardial function. In 16 anesthetized dogs, the left anterior descending coronary artery was occluded for 2 hours, and then reperfused. At 45 minutes of reperfusion, the dogs were randomized to receive a 3 hour intravenous infusion of either saline solution or dopamine (10 micrograms/kg per min), and 1 hour after the infusion was discontinued the area of necrosis and an in vivo area at risk of necrosis were determined. All dogs had serial two-dimensional echocardiograms with computer-assisted analysis and in vivo biopsies for determination of adenosine triphosphate and creatine phosphate levels. Dopamine caused an increase in the contractility of the reperfused myocardium, with systolic wall thickening increasing from -4.1 +/- 2.6 (mean +/- SEM) to +24.0 +/- 3.7% (p less than 0.002) and short-axis cross-sectional ejection fraction increasing from 27.1 +/- 4.7 to 71.6 +/- 4.4% (p less than 0.002) after 15 minutes of infusion. Regional myocardial blood flow in the previously ischemic epicardium was increased from 1.18 +/- 0.11 ml/min per g with saline to 2.95 +/- 0.36 ml/min per g with dopamine (p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction in experimental infarct size by recombinant human superoxide dismutase: insights into the pathophysiology of reperfusion injury

To determine the importance of reperfusion injury and the ability of the free-radical scavenger recombinant human superoxide dismutase (h-SOD) to prevent it, open-chest dogs underwent 90 min of proximal circumflex coronary artery occlusion, and only at the moment of reperfusion received either h-SOD (400,000 IU bolus into the left atrium followed by a 300,000 IU iv infusion over 1 hr) or saline. After 48 hr the surviving animals were killed and measurements were made of the risk region (by postmortem angiography) and infarct size (by gross pathology). All measurements were made by investigators blinded to treatment given, and the code was broken only at the end of the study. Hemodynamic variables and collateral flow during ischemia were similar in the two groups. Infarct size in control animals (n = 8) averaged 22.4 +/- 3.1% of the left ventricle and 52.2 +/- 7.1% of the risk region, compared with 13.3 +/- 0.8% of the left ventricle and 33.6 +/- 2.1% of the risk region in h-SOD-treated dogs (n = 8) (p less than .05). Infarcts in treated animals were not only smaller, but also exhibited a distinctive "patchiness," suggesting protection along vascular distributions. Furthermore, analysis of the relationship between infarct size and collateral flow measured during ischemia in the two groups indicated that protection by h-SOD was greatest in animals with the lowest collateral flows. This study supports the concept that reperfusion of ischemic myocardium results in a separate component of cell damage, presumably linked to the generation of oxygen free radicals on reflow. Since the h-SOD preventable reperfusion component of injury was most pronounced in hearts with the most severe ischemia, scavenging of oxygen radicals at the time of reflow may offer a novel and particularly promising therapeutic approach for the protection of ischemic myocardium.     

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.