Nobuo Hatori

Beneficial effects of coronary venous retroinfusion of superoxide dismutase and catalase on reperfusion arrhythmias, myocardial function, and infarct size in dogs.

The efficacy of coronary venous retroinfusion of superoxide dismutase and catalase was studied in anesthetized closed chest dogs with 90-min left anterior descending coronary artery (LAD) occlusion followed by 3-h reperfusion. In group A, superoxide dismutase (2.5 mg/kg) and catalase (2.5 mg/kg) were administered by a 30-min continuous right atrial infusion beginning 15 min before reperfusion and supplemented by a bolus injection of superoxide dismutase (2.5 mg/kg) and catalase (2.5 mg/kg) through the great cardiac vein immediately before reperfusion. The treatment in group B was similar to that in group A, except that the bolus injection was into the right atrium. In the control group (group C), saline was administered in the same manner as in group A. Infarct size, expressed as a percentage of the risk area, was significantly smaller in group A (11.3 ± 8.9%) than in groups B (31.3 ± 21.1%) and C (43.0 ± 16.9%; p < 0.05). Regional function of the ischemic zone measured by two-dimensional echocardiography exhibited significantly (p < 0.05) greater recovery after 3-h reperfusion in group A (30.3 ± 8.4%) versus groups B (12.5 ± 13.7%) and C (12.1 ± 11.7%). Moreover, there were significantly fewer postreperfusion ventricular arrhythmias in group A as compared with groups B and C. The results of this study indicate that coronary venous retroinfusion is an effective method for delivery of superoxide dismutase and catalase.

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial infarct size in pigs.

The efficacy of human extracellular-superoxide dismutase type C (EC-SOD C) to limit infarct size after ischemia and reperfusion was explored and compared to that of EC-SOD C combined with catalase (CAT) and to that of CAT alone. EC-SOD C binds to heparan sulphate proteoglycan on the cell surfaces. Thirty-two pigs were subjected to 45 min of myocardial ischemia followed by 4 h of reperfusion. Control pigs (group A; n = 8) received 300 mL of saline into the great cardiac vein during a 30-min period started 5 min prior to reperfusion; pigs in group B (EC-SOD C; n = 8) got 16.6 mg of EC-SOD C; pigs in group C (EC-SOD C + CAT; n = 8) got 16.6 mg of EC-SOD C together with 150 mg of CAT. Pigs in group D (CAT; n = 8) received 150 mg of CAT. In groups B, C, and D, the drug was dissolved in saline and infused into the great cardiac. Infarct size expressed as percent of area at risk was smaller in groups B (14.5 +/- 16.7%) and C (40.8 +/- 13.3%) than in groups A (78.8 +/- 8.6%) and D (67.2 +/- 18.6%; p less than .05). Creatine kinase (CK) activity in ischemic myocardium was higher in groups B (1740 +/- 548 U/g) and C (1729 +/- 358 U/g) than in groups A (1184 +/- 237 U/g) and D (1251 +/- 434 U/g; p less than .05). There was an inverse relation (r = -.83) between infarct size and CK content. The EC-SOD C infusions resulted in only minimal increases in plasma SOD activities. In conclusion, the presence of SOD on the cell surfaces is of importance in the prevention of reperfusion injury rather than circulating SOD.

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.

Differences in infarct size with lidocaine as compared with bretylium tosylate in acute myocardial ischemia and reperfusion in pigs

Summary: The effects of the antiarrhythmic drugs lidocaine and bretylium tosylate on myocardial necrosis were studied in anesthetized pigs subjected to 60-min coronary occlusion followed by 3-h reperfusion. Group A (n = 7) received lidocaine (average dose × SD = 1,828 × 515 mg) before and during coronary occlusion and after reperfusion; the other series (group B, n = 7) received bretylium tosylate (3,457 × 1,323 mg). Infarct size was 16.3 × 14.7% in the lidocaine group as compared with 68.6 × 12.6% (p < 0.01) in the bretylium group. In vitro release of superoxide anion from porcine granulocytes was studied using the lucigenin-dependent chemiluminescence technique. Lidocaine significantly reduced the peak chemiluminescence response to zymosan from 3.34 × 0.44 without lidocaine to 2.23 × 0.46 (p < 0.01) and 1.06 × 0.17 mV (p < 0.001), with lidocaine concentrations of 20 and 200 μg/ml, respectively. Bretylium had no effect on the chemiluminescence response. Adherence of porcine granulocytes to plastic was also reduced from 332 × 32 cells/mm2 (no lidocaine) to 247 × 35 and 206 × 26 cells/mm2 with lidocaine concentrations of 20 and 200 μg/ml, respectively (p < 0.001). Bretylium had no significant effect. Eight additional bretylium-treated pigs received either rabbit antiporcine granulocyte serum (group C, n = 4) to reduce circulating granulocytes or nonimmune serum (group D, n = 4). Infarct size in the granulocyte-depleted pigs was 26.6 × 5.6% as compared with 51.4 × 5.5% in pigs that received nonimmune serum (p < 0.01). Smaller infarct size with lidocaine as compared with bretylium tosylate therapy is associated with decreased adherence of granulocytes to vascular endothelium in the ischemic myocardium and inhibition of superoxide anion release.

Coronary venous retroinfusion of felodipine reduces myocardial necrosis after coronary occlusion and reperfusion

The effect of the vasoselective calcium antagonist felodipine on myocardial necrosis was studied in 14 anesthetized pigs subjected to 45-min occlusion of the left anterior descending coronary artery (LAD) followed by 24-h reperfusion. Felodipine (7 nmol/kg, n = 7), or the corresponding amount of vehicle diluted in 300 ml saline (n = 7) was infused in the great cardiac vein for 30 min beginning 5 min before onset of reperfusion. Regional myocardial function was measured as percentage of systolic segment shortening (%SS) by sonomicrometry. The recovery in ischemic myocardium was significantly already better among felodipine-treated animals after 30-min reperfusion (mean ± SD = 9.1 ± 6.1 vs. 0.1 ± 3.2%, p < 0.05). The improved recovery in the felodipine group persisted throughout the observation period; %SS was 8.5 ± 5.9% after 24-h reperfusion. No significant improvement was observed in the vehicle group after 24 h (%SS = 2.7 + 2.0%, p < 0.05). The area of the infarct was measured by triphenyl tetrazolium chloride staining. When expressed as percentage of the left ventricle, the infarct was smaller in the felodipine group (8.2 ± 4.0%) than in the vehicle group (14.6 ± 3.5%; p < 0.01). The corresponding values for infarct size expressed as percentage of the area at risk was 38.6 ± 18.9% in the felodipine group and 69.8 ± 6.7% in the vehicle group (p < 0.01). Peak plasma felodipine concentration was reached 30 min after onset of reperfusion (4.9 ± 1.2 nM). The vascular selective calcium antagonist felodipine reduced myocardial infarct size and improved myocardial functional recovery in pigs subjected to myocardial ischemia and reperfusion.

Acute cobalt exposure and oxygen radical scavengers in the rat myocardium.

Excessive amounts of cobalt are cardiotoxic, although the mechanism for this toxicity remains unclear. We studied the effects of acute cobalt exposure on the activities of free radical scavengers in the myocardium in 5 groups of rats. Six rats served as a control group and were given a daily subcutaneous injection of 1 ml saline for 8 days. The other 4 groups of rats received a daily injection subcutaneously of cobalt chloride in doses of 1 mg/kg bw, 5 mg/kg bw, 20 mg/kg bw and 50 mg/kg bw, respectively for 8 days. There was a marked and dose-dependent accumulation of cobalt in the myocardium of the cobalt exposed rats. Creatine kinase, copper-zinc superoxide dismutase (CuZn-SOD) and alpha-tocopherol content did not differ between the control and the cobalt exposed groups. The activity of glutathione peroxidase increased, while the activity of manganese-superoxide dismutase (Mn-SOD) was significantly reduced in the cobalt exposed groups. There was an inverse relationship (r = 0.60, P < 0.0001) between the cobalt content and Mn-SOD activity in the myocardium. These results suggest that acute cobalt cardiotoxicity may involve a reduction of intrinsic scavengers resulting in an increased vulnerability to oxygen free radical toxicity.

Purification and properties of porcine polymorphonuclear cells

A method for the rapid separation of highly purified populations of porcine polymorphonuclear cells from whole blood is described. Porcine blood, anti-coagulated with EDTA, was layered over a discontinuous Percoll gradient (62.5% and 75%) and then centrifuged at 400 X g for 25 min. This results in the formation of a band of cells at the interface of the two Percoll layers which is greater than 99% granulocytes (93.8 +/- 1.8% neutrophils and 5.3 +/- 1.8% eosinophils) with a 77% recovery. The mononuclear cells remain above the 62.5% Percoll layer, and most erythrocytes pellet to the bottom of the tube. The isolated porcine granulocytes were found to respond to opsonized zymosan, phorbol myristate acetate (20 ng/ml), and the calcium ionophore A23187 (10(-5) M) in chemiluminescence assays with kinetics similar to those of human granulocytes. The porcine cells did not respond to the chemotactic peptide N-formyl-methionyl-leucyl-phenalanine (FMLP; 10(-6) M) unlike the human granulocytes which display a very rapid response to FMLP. Both porcine and human granulocytes readily changed shape by elongating and developing pseudopods when exposed to zymosan-activated serum, but only human granulocytes changed on exposure to FMLP. Thus, porcine granulocytes may be rapidly isolated on discontinuous Percoll gradients with little mononuclear cell contamination. Porcine and human PMN have similar oxidative and chemotactic responses, but porcine PMN differ from human granulocytes in the inability of porcine granulocytes to respond to FMLP.

Ischemic and nonischemic tissue concentrations of felodipine after coronary venous retroinfusion during myocardial ischemia and reperfusion: an experimental study in pigs.

Tissue and plasma concentrations of felodipine, a dihydropyridine (DHP) calcium antagonist, retroinfused through the coronary venous system were studied in 27 pigs. The animals underwent 45-min myocardial ischemia followed by 4-h reperfusion. Felodipine (7 nmol/kg body weight) was administered in the coronary vein for 30 min, 5 min before reperfusion. Concentrations of felodipine in the ischemic and nonischemic myocardium and in plasma were determined by gas chromatography. In the ischemic area, felodipine concentration at start of reperfusion was 304 +/- 285, 171 +/- 160, and 52 +/- 47 nmol/kg (mean +/- SD) in the subepicardial, midmyocardial, and subendocardial layer, respectively. Corresponding concentrations in the nonischemic area were 15 +/- 13, 17 +/- 14, and 16 +/- 15 nmol/kg (p < 0.05 vs. ischemic area). Subepicardial concentration was highest at start of reperfusion, whereas concentrations in other layers peaked at the end of retroinfusion. The transmural concentration gradient of felodipine in the ischemic area decreased progressively during the reperfusion period. The nonischemic tissue concentration increased slightly during the reperfusion period. The plasma concentration was very low throughout the study (peak = 3.2 +/- 1.4 nM at 30 min). Coronary venous retroinfusion of felodipine resulted in profound accumulation of the drug, specifically in ischemic myocardium. The plasma concentration was low and did not affect systemic hemodynamics. Coronary venous retroinfusion is considered an advantageous technique for selective drug delivery.

The effect of nitric oxide synthase inhibitor on reperfusion injury of the brain under hypothermic circulatory arrest

The effect of nitric oxide synthase inhibitor-NG-nitro-L-argine methyl ester (L-NAME)-on reperfusion injury of the brain under deep hypothermic circulatory arrest was experimentally investigated in sixteen piglets weighing about 30 kg. Cardiopulmonary bypass was established and animals were cooled to a brain temperature of 20 degrees C and circulatory arrest was performed for 60 minutes followed by reperfusion of 120 minutes. The value of nitric oxide (NO) within the brain was measured with a needle electrode which was inserted into the brain. In the treatment group L-NAME was administered with a intravenous injection of 1.5 mg/kg at the beginning of the reperfusion followed by a 60-minute continuous venous infusion of 1.5 mg/kg/hour. In the control group the value of NO after 120 minutes reperfusion increased significantly compared with pre-circulatory arrest, but in the treatment group it did not. There was significant difference between the groups regarding the NO value after 120 minutes reperfusion. Blood pressure after 120 minutes reperfusion in the treatment group was a little higher than the control group, but there was no significant difference between the groups regarding cerebral blood flow, excess lactate and cerebral tissue water content. However, recovery of SEP after 120 minutes reperfusion was detected in all 8 cases of the treatment group, but only in one of the control group (p < 0.001). This data suggests that L-NAME protects the brain against the reperfusion injury under deep hypothermic circulatory arrest.

Neutralization of Prostaglandin E1 Intravenous Solution Reduces Infusion Phlebitis

Previous studies have shown moderate or severe phlebitis at the site of venipuncture in some patients who receiving prostaglandin E1 (PGE1) infusion therapy. Such phlebitis is sometimes severe enough to necessitate the cessation of PGE1 therapy. This study investigated how to continue PGE1 infusion therapy for 3 weeks with tolerable phlebitis. Although a 60 μg dose of PGE1 is usually dissolved in 500 mL of fluid to avoid phlebitis, we used 200 mL to prevent volume overload. This PGE1 solution was neutralized to pH 7.4 with 4 mL of 7% sodium bicarbonate. We examined the frequency and severity of phlebitis among patients who received a 2-h PGE1 infusion twice daily. Eighteen patients who were hospitalized for peripheral vascular disease between June 1998 and May 1999 were studied. All of them were men and their mean age was 63.3 ±8.9 years (range: 47-78 years). Fourteen patients had arteriosclerosis obliterans and four had Buergers disease. When the severity of phlebitis was determined according to Dinleys criteria, two patients (11%) had grade 0, four patients (22%) had grade 1, eleven patients (61%) had grade 2, and one patient (6%) had grade 3 phlebitis. Usually, PGE1 infusion therapy is stopped when phlebitis reaches grade 4 or more, but there were no such cases in this study. We also found that aging was significantly correlated with a decrease in the severity of phlebitis (Spearmans rank correlation test: r = -0.545, p = 0.0193).