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Role of oxygen radicals in postischemic myocardial dysfunction (“stunning”)
Andoxidant Therapy:Ischemia/Reperfusion, Bum/Trauma 16.9 ROLE O F OXYGEN RADICALS IN POSTISCHEMIC MYOCARDIAL DYSFUNCTION ("STUNNING*). Roberto Bolli, M.D., Baylor College of Medicine, Houston, TX 77030, USA.
A series of studies was conducted to test the hypothesis that oxygen radicals contribute to the genesis of postischemic myocardial dysfunction or myocardial "stunning". In open-chest dogs undergoing a 15-rain coronary occlusion followed by reperfusion, recovery of contractile function was enhanced by a variety of antioxidants, such as superoxide dismutase (SOD) plus catalase, N-2-mercaptopropionyl glycine, dimethylthiourea, allopurino], and desfem~oxamine. The benefid a l effects of SOD plus catalase were reproduced in conscious dogs undergoing a 15-rain coronary occlusion followed by 7 days of reperfusion, indicating that oxygen metabolites contribute to postischemic dysfunction under physiologic conditions. To provide direct evidence for a role of oxygen radicals, we administered the spin trap alpha-phenyl N-tert-butyl nitrone (PBN) in vivo (i.c. or 1."v.) in our open-chest canine model of myocardial stunning. Using electron paramagnetic resonance (EPR) spectroscopy, radical adducts of PBN were detected in the coronary venous effluent, with a burst of production occurring in the first minutes of repcrfusion. l h r e e antioxidant interventions (SOD plus catalase, N2-mercaptopropionyl glycine, desferrioxamine) inhibited productl~on of PBN adducts and, at the same time, enhanced recovery of contractility, suggesting that the radicals generated play a causal role in postischemic dysfunction. Taken together, these results provide strong evidence for an important role of reactive oxygen species in the genesis of the prolonged depression of contractility (stunning) observed after reversible myocardial ischemia.
16.11
PREVENTION OF CARDIAC REPERFUSION DAMAGE BY A NITROXlDE SUPEROXlDE DISMUTASE MIMIC. Dan Gelvan*, Paul Saltman* and Saul Powell+ Dept. of Biology, University of Califomia, San Diego (*) and North Shore - Comell University Hospital, Manhasset, N.Y.(+). Considerable evidence suggests that oxygen derived free radicals are involved in ischemia/repeflusion damage. Nitroxide sp~n labels disproportionate superoxide radicals and oxidize the reduced metal ions required for OH" formation. In cell cultures, nitroxides have provided full protection from free radical damage. The nitroxide spin label TEMPO (2,2,6,6-tetramethylpipeddine1-gxyl) was tested for its ability to prevent repedusion damage in the isolated rat heart. The hearts, equilibrated with buffer ± TEMPO or ethanol (the solvent for TEMPO), were made regionally ischemic for 10 min by reversible occlusion of the left anterior descending coronary artery. The duration of severe reperlusion arrhythmia (SRA) - ventricular fibrillation and sustained ventricular tachycardia - was significantly reduced by the presence of 0.4 or I mM TEMPO, and only slightly by 0.01% ethanol (the highest concentration added with TEMPO), as compared to control hearts. This correlated well with a strong protective effect of TEMPO on post-ischemic release of lactate dehydrogenase and near-complete repression of OH" formation in hearts subjected to 10 min of global ischemia. Ethanol was moderately protective, probably due to OH" scavenging. In nonischemic hearts, hemodynamic function was net affected by ethanol while TEMPO was negatively chronotropic. However, compensatory pacing of the hearts during equilibration and ischemia did not alter the dramatic effect of TEMPO on SRA. When TEMPO was introduced 1 min into repertusion, the patterns of arrhythnda were identical to control hearts. TEMPO, by preventing the formation of OH', strongly protected against reperfusion arrhythmia and cell damage. This activity was not related to the negative chronotropic effect or to direct suppressionof arrhythmia. We conclude that both cell damage and SRA are directly related to oxidative damage incurred during the critical first minute of the reperfusion event.
ISCHEMIA-REPERFUSION RETINA HISTOLOGICAL DAMAGE IN RAT : A PROTECTIVE EFFECT OF EGB 761 Marie T. Droy-Lefaix*, Martha E. Szabo*, Michel Doly**~ Christian Carr4* and Pierre Braquet* * IHB/IPSEN Research Laboratories, Le Plessis Robinson, ** Biophysics Laboratory, ClermontFerrand, FRANCE.
153 16.10
Histological alterations induced by ischemia/ reperfusion of retina depend on ischemia duration. On anesthetized male Sprague-Dawley rats, a suture was placed on the central retinal artery. Untreated eyes exhibit severe histological damage after reperfusion. To obtain a maximum lesion score, we tested different times of ischemia. Eyes (n = 6-12 in each group) were submitted to 30, 60 or 90 minutes ischemia followed by respectively 0.5, I, 2, 4 and 24 hours reperfusion. Thirty minutes ischemia, followed by reperfusion did not result in any histological changes, while 60 minutes ischemia induced a moderate retinal edema. Ninety minutes ischemia further increased retinal edema and migration of neutrophils. In our study, rats were treated with superoxide dismutase (SOD-PEG, 15 x 103 U/kg) or EGB 761 (i00 mg/kg) for i0 days (chronic treatment). In conclusion, SOD as well as EGB 761 significantly reduced the development of reperfusion-induced retinal edema and prevented neutrophil infiltration even if they were administered just before reperfusion.
FLUID RESUSCITATION WITH A DEFEROXAMINE-16.12 COLLOID CONJUGATE: APPLICATIONS TO BURN INJURY AND HEMORRHAGIC SHOCK Bo E. Hedlund and Philip E. Hallaway Biomedical Frontiers, Inc., Minneapolis, MN 55414, USA Attachment of the powerful iron chelator deferoxamine (DFO) to colloids, such as dextran and hydroxyethyl starch (HES), yields non-toxic conjugates with long vascular half-lives (Hallaway et al., Proc Nat. Acad. Sci. USA 86:10108, 1989). These compounds afford protection against tissue injury caused by oxygen and lipid radicals formed in the presence of free iron. In an ovine model of 40% total body surface area burn, lung and liver lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) in animals receiving lactated Ringer's (LR), 2% HES or HES-DFOin LR 30 min post burn. Huid therapy with the HES vehicle was associated with increases in lung and liver TBARS of 43% and 92%, respectively, compared to unburned controls. Treatment with HES-DFO attenuated these levels to those observed in the control group. Oxygen consumption increased 30% in HES-DFOtreated animals, despite identical oxygen delivery in the three groups. In a porcine model of hemorrhagic shock, animals were bled and the MAP maintained at 40 mmHg for 60 min. Resuscitation with LR (3 times volume of shed blood) was compared to one volume 5% HES or HES-DFO in LR. All LR-treated animals expired within 2.5 hr. All animals in the colloid groups survived until sacrifice at 24 hr. Levels of liver maiondialdehyde increased 70% in the HES group but only 10% in the HES-DFO group. Plasma aspartate aminotransferase levels at 24 hr post resuscitation were elevated 5-fold in the HES-treated animals, but only 50% in animals receiving HES-DFO. Conjugation of DFO to colloids permits bolus parenterai administration of high dose (100-500 mg/kg) chelator/anti-oxidant as an integral part of resuscitation. This therapeutic approach attenuates oxygen- and lipid radical-mediated reperfusion injury.
A series of studies was conducted to test the hypothesis that oxygen radicals contribute to the genesis of postischemic myocardial dysfunction or myocardial "stunning". In open-chest dogs undergoing a 15-rain coronary occlusion followed by reperfusion, recovery of contractile function was enhanced by a variety of antioxidants, such as superoxide dismutase (SOD) plus catalase, N-2-mercaptopropionyl glycine, dimethylthiourea, allopurino], and desfem~oxamine. The benefid a l effects of SOD plus catalase were reproduced in conscious dogs undergoing a 15-rain coronary occlusion followed by 7 days of reperfusion, indicating that oxygen metabolites contribute to postischemic dysfunction under physiologic conditions. To provide direct evidence for a role of oxygen radicals, we administered the spin trap alpha-phenyl N-tert-butyl nitrone (PBN) in vivo (i.c. or 1."v.) in our open-chest canine model of myocardial stunning. Using electron paramagnetic resonance (EPR) spectroscopy, radical adducts of PBN were detected in the coronary venous effluent, with a burst of production occurring in the first minutes of repcrfusion. l h r e e antioxidant interventions (SOD plus catalase, N2-mercaptopropionyl glycine, desferrioxamine) inhibited productl~on of PBN adducts and, at the same time, enhanced recovery of contractility, suggesting that the radicals generated play a causal role in postischemic dysfunction. Taken together, these results provide strong evidence for an important role of reactive oxygen species in the genesis of the prolonged depression of contractility (stunning) observed after reversible myocardial ischemia.
16.11
PREVENTION OF CARDIAC REPERFUSION DAMAGE BY A NITROXlDE SUPEROXlDE DISMUTASE MIMIC. Dan Gelvan*, Paul Saltman* and Saul Powell+ Dept. of Biology, University of Califomia, San Diego (*) and North Shore - Comell University Hospital, Manhasset, N.Y.(+). Considerable evidence suggests that oxygen derived free radicals are involved in ischemia/repeflusion damage. Nitroxide sp~n labels disproportionate superoxide radicals and oxidize the reduced metal ions required for OH" formation. In cell cultures, nitroxides have provided full protection from free radical damage. The nitroxide spin label TEMPO (2,2,6,6-tetramethylpipeddine1-gxyl) was tested for its ability to prevent repedusion damage in the isolated rat heart. The hearts, equilibrated with buffer ± TEMPO or ethanol (the solvent for TEMPO), were made regionally ischemic for 10 min by reversible occlusion of the left anterior descending coronary artery. The duration of severe reperlusion arrhythmia (SRA) - ventricular fibrillation and sustained ventricular tachycardia - was significantly reduced by the presence of 0.4 or I mM TEMPO, and only slightly by 0.01% ethanol (the highest concentration added with TEMPO), as compared to control hearts. This correlated well with a strong protective effect of TEMPO on post-ischemic release of lactate dehydrogenase and near-complete repression of OH" formation in hearts subjected to 10 min of global ischemia. Ethanol was moderately protective, probably due to OH" scavenging. In nonischemic hearts, hemodynamic function was net affected by ethanol while TEMPO was negatively chronotropic. However, compensatory pacing of the hearts during equilibration and ischemia did not alter the dramatic effect of TEMPO on SRA. When TEMPO was introduced 1 min into repertusion, the patterns of arrhythnda were identical to control hearts. TEMPO, by preventing the formation of OH', strongly protected against reperfusion arrhythmia and cell damage. This activity was not related to the negative chronotropic effect or to direct suppressionof arrhythmia. We conclude that both cell damage and SRA are directly related to oxidative damage incurred during the critical first minute of the reperfusion event.
ISCHEMIA-REPERFUSION RETINA HISTOLOGICAL DAMAGE IN RAT : A PROTECTIVE EFFECT OF EGB 761 Marie T. Droy-Lefaix*, Martha E. Szabo*, Michel Doly**~ Christian Carr4* and Pierre Braquet* * IHB/IPSEN Research Laboratories, Le Plessis Robinson, ** Biophysics Laboratory, ClermontFerrand, FRANCE.
153 16.10
Histological alterations induced by ischemia/ reperfusion of retina depend on ischemia duration. On anesthetized male Sprague-Dawley rats, a suture was placed on the central retinal artery. Untreated eyes exhibit severe histological damage after reperfusion. To obtain a maximum lesion score, we tested different times of ischemia. Eyes (n = 6-12 in each group) were submitted to 30, 60 or 90 minutes ischemia followed by respectively 0.5, I, 2, 4 and 24 hours reperfusion. Thirty minutes ischemia, followed by reperfusion did not result in any histological changes, while 60 minutes ischemia induced a moderate retinal edema. Ninety minutes ischemia further increased retinal edema and migration of neutrophils. In our study, rats were treated with superoxide dismutase (SOD-PEG, 15 x 103 U/kg) or EGB 761 (i00 mg/kg) for i0 days (chronic treatment). In conclusion, SOD as well as EGB 761 significantly reduced the development of reperfusion-induced retinal edema and prevented neutrophil infiltration even if they were administered just before reperfusion.
FLUID RESUSCITATION WITH A DEFEROXAMINE-16.12 COLLOID CONJUGATE: APPLICATIONS TO BURN INJURY AND HEMORRHAGIC SHOCK Bo E. Hedlund and Philip E. Hallaway Biomedical Frontiers, Inc., Minneapolis, MN 55414, USA Attachment of the powerful iron chelator deferoxamine (DFO) to colloids, such as dextran and hydroxyethyl starch (HES), yields non-toxic conjugates with long vascular half-lives (Hallaway et al., Proc Nat. Acad. Sci. USA 86:10108, 1989). These compounds afford protection against tissue injury caused by oxygen and lipid radicals formed in the presence of free iron. In an ovine model of 40% total body surface area burn, lung and liver lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) in animals receiving lactated Ringer's (LR), 2% HES or HES-DFOin LR 30 min post burn. Huid therapy with the HES vehicle was associated with increases in lung and liver TBARS of 43% and 92%, respectively, compared to unburned controls. Treatment with HES-DFO attenuated these levels to those observed in the control group. Oxygen consumption increased 30% in HES-DFOtreated animals, despite identical oxygen delivery in the three groups. In a porcine model of hemorrhagic shock, animals were bled and the MAP maintained at 40 mmHg for 60 min. Resuscitation with LR (3 times volume of shed blood) was compared to one volume 5% HES or HES-DFO in LR. All LR-treated animals expired within 2.5 hr. All animals in the colloid groups survived until sacrifice at 24 hr. Levels of liver maiondialdehyde increased 70% in the HES group but only 10% in the HES-DFO group. Plasma aspartate aminotransferase levels at 24 hr post resuscitation were elevated 5-fold in the HES-treated animals, but only 50% in animals receiving HES-DFO. Conjugation of DFO to colloids permits bolus parenterai administration of high dose (100-500 mg/kg) chelator/anti-oxidant as an integral part of resuscitation. This therapeutic approach attenuates oxygen- and lipid radical-mediated reperfusion injury.