The protective effect of vitamin E, idebenone and reduced glutathione on free radical mediated injury in rat brain synaptosomes

In the present study the effect of ascorbate (0.8 mM)/iron (2.5 microM) on lipid and protein oxidation, in Synaptosomes isolated from rat brain cortex, was evaluated. Vitamin E, idebenone and reduced glutathione were used as free radicals scavengers, in order to analyze the mechanism involved in ascorbate/iron-induced oxidative stress. An increased formation of reactive oxygen species (ROS) in the cytosol and in the mitochondria was observed, in ascorbate/iron treated synaptosomes. Idebenone (50 microM) prevented the increased formation of ROS in both synaptosomal compartments, vitamin E (150 microM) protected partially this formation in mitochondria, whereas reduced glutathione (250 microM) (GSH) was ineffective. After ascorbate/iron treatment an increase in lipid peroxidation occurred as compared to control, which was completely inhibited by idebenone. A decrease in protein-SH content was also observed, and it was prevented by Vitamin E, idebenone and GSH. When synaptosomes were treated with ascorbate/iron the levels of GSH decreased, and the levels of oxidized glutathione (GSSG) increased as compared to controls under these conditions. Glutathione peroxidase activity was unchanged, whereas an inhibition of glutathione reductase activity was observed. These data suggest that the increased formation of free radicals in synaptosomes leads to lipid and protein oxidation, the role of the endogenous GSH being essential to protect protein thiol-groups against oxidative damage in order to maintain enzyme activity.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p < 0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p < 0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

Junctional sequences of immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements are used as patient-specific PCR targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukaemias (ALLs). Clonal evolution of gene rearrangements is a major pitfall of this strategy. Using high-resolution PCR-based analyses (including denaturing gel electrophoresis and single-stranded conformation polymorphism (SSCP)) we have compared Ig/TCR gene rearrangements at presentation and relapse in a series of ALLs. These methods allow an unambigous comparison of rearrangements taking into account junctional size and nucleotide sequence information and allow a precise assessment of the clonal evolution. V gamma-J gamma and V delta 1-J delta 1 rearrangements were analysed in 12 T-ALLs. VH-JH, V gamma-J gamma, V delta 2-D delta 3 and, in selected cases, DH-JH rearrangements were studied in 14 B-lineage ALLs. Clonal evolution, regarding major rearrangements, occurs for at least one of these loci in 2/12 T-ALLs and in 5/14 B-lineage ALLs. Clonal evolution is more marked for minor rearrangements than for major ones. As shown using SSCP analysis, rearrangements observed at relapse are sometimes found in minor clones at presentation which are therefore selected in vivo by a proliferative advantage. These data, as well as those from the available literature, suggest the use of at least two patient-specific probes to detect MRD in ALLs. A general strategy including selected Ig/TCR rearrangements and chromosomal abnormalities as PCR targets is proposed.     

Effect of propofol and thiopentone on free radical mediated oxidative stress of the erythrocyte

Propofol has free radical scavenging properties similar to those of recognized phenol-based antioxidants. We have examined these properties in an in vitro model of radical-induced cellular injury, comparing its activity with that of thiopentone (which has also been shown to have radical scavenging activity). Haemolysis of human erythrocytes was induced using the azo compound 2,2'-azo-bis(2-amidinopropane) dihydrochloride (ABAP). This was achieved by incubating a 10% suspension of erythrocytes with ABAP 100 mmol litre-1 at 37 degrees C. For propofol, at concentrations of 12.5, 25 and 50 mumol litre-1, the times to achieve 50% haemolysis were mean 126 (SEM 7) min (95% confidence interval 108-144 min), 150 (8) (129-170) min and 182 (12) (160-180) min, respectively (Intralipid control 107 (7) (90-125) min, ANOVA P < 0.0001). For thiopentone, at concentrations of 62.5, 125 and 250 mumol litre-1, the values were 117 (2) (112-121) min, 126 (3) (119-133) min and 138 (2) (132-144) min, respectively (saline control 109 (2) (104-113) min, ANOVA P < 0.0001). Spectroscopic analysis in the visible and ultraviolet spectra demonstrated a steady increase in the proportion of methaemoglobin during haemolysis, with the highest concentrations in the propofol-containing flasks. The formation of methaemoglobin was preceded by the generation of ferrylhaemoglobin (a Fe4+ haemoglobin species). Further experiments examining oxidation of purified methaemoglobin to ferrylhaemoglobin by hydrogen peroxide suggested that propofol, but not Intralipid or thiopentone, reduced ferrylhaemoglobin back to the met- state, and thereby explained the higher concentrations of methaemoglobin in the propofol-containing erythrocyte suspensions. We conclude that propofol is a more potent free radical scavenger in this model of oxidant stress than thiopentone, and that reduction of high oxidation states of haemoglobin may contribute to such activity.     

Synaptic remodeling and free radical formation after brain contusion injury in the rat

The purpose of this study was to explore whether bilateral frontal cortex contusion in rats would demonstrate changes relevant for understanding the pathology of frontal lobe injury in humans. Rats were allowed to survive for 3, 7, or 18 days postinjury (dpi). In the contused rats, albumin was trapped in frontal cortices, as well as in other brain areas, showing that neurons were exposed to plasma components. In the sham-operated rats, which had only craniotomy but no penetration of dura, the level of trapped albumin was also increased compared to intact controls, suggesting a partial lesion-like condition. Choline acetyltransferase activity was severely decreased in the frontal cortices of contused rats, compared to the sham-operated controls. The decrease was most pronounced at 3 dpi and less pronounced 18 dpi, suggesting that after the initial damage, regeneration of the cholinergic terminals occurred. The concentration of the mature presynaptic membrane protein D3(SNAP-25) was also decreased in the frontal cortices of contused rats at 3 and 7 dpi, whereas it was normalized at 18 dpi. Previously, we have evaluated changes in the rate of synaptic remodeling in brain injury by calculating the ratio of the neural cell adhesion molecule (NCAM) to D3(SNAP-25). The NCAM/D3(SNAP-25) ratio at 3 dpi was elevated by more than 60% in the frontal cortices of contused rats, suggesting a high initial rate of synaptic remodeling. The ratios were smaller at 7 and 18 dpi, suggesting that after the initial burst, the rate of remodeling leveled off. In contrast, astrocyte activation was less pronounced at 3 dpi than at 7 and 18 dpi, as measured by the levels of glial fibrillary acidic protein and glutamine synthetase immunoactivities. The immunoreactivity of glutamine synthetase more than doubled in the contused brains but its enzymatic activity increased less than 50%, suggesting that many enzymatic centers had been inactivated by free radicals. Calculated as the difference between the relative immunoreactivity and the relative enzymatic activity the "lost glutamine synthetase activity" increased continuously in frontal cortex and striatum from 3 to 18 dpi, indicating the production of free radicals long after the initial contusion event. In conclusion, following frontal cortical contusions the early synaptic damage was partly compensated by synaptic remodeling. We suggest that the continuous production of free radicals may have contributed to the declining remodeling rate and impair functional recovery.     

Indicators of oxidative stress in aging rat brain. The effect of nerve growth factor

INTRODUCTION AND OBJECTIVE: Increased oxidative stress during ageing and the neurodegenerative disorders associated with this has been described. The central nervous system is particularly vulnerable to oxidative damage because of its high energy requirements, high oxygen consumption, high tissue concentration of iron and relatively low levels of some antioxidant systems. Treatment with neurotrophic factors may reverse neurone deterioration and stimulate cholinergic activity in aged rats. It may have a similar neuroprotector effect against damage due to ischaemic reperfusion, hypoglycaemia, inflammation and other pathological conditions involving oxidative stress. In this study we determined some indicators of oxidative stress in rat brains during ageing and evaluated this in response to a plan of treatment with murine nerve growth factor (FCN) for 38 days. MATERIAL AND METHODS: Biochemical techniques were used for determination of oxidative stress indicators. RESULTS AND CONCLUSIONS: We found that with age there was a significant increase in phospholipase A2 and superoxide dysmutase activity and concentration of hipoperoxidases, whilst the concentration of reduced glutathion fell. Catalase activity increased in the hippocampal and striate regions and decreased in the cortex and septal area. There was less oxidative stress in rats treated with FCN. In view of our results, we conclude that the level of oxidative stress increases with ageing, with significant differences between areas of the brain. The region most vulnerable to damage from species reactive to oxygen was the hippocampus, and the protective effect of FCN may be related to potentiation of antioxidant defenses.     

Reduction of oxidative tissue injury and endothelial dysfunction by graduated reperfusion after cardioplegic arrest in isolated rat hearts

The aim of this study was to investigate whether or not a graduated resumption of the perfusion pressure after cardioplegic ischaemic arrest will reduce the impact of oxygen free radicals on myocardium and the cardiovasculature. Langendorff-perfused rat hearts were subjected to cardioplegia and subsequent 40 min of global ischaemia at 25 degrees C. Reperfusion was carried out either abruptly (AR) or gradually (i.e., perfusion pressure stepwise increased from 40 to 75 mmHg within 30 min -GR). GR resulted in a significant improvement of percentage recovery of left ventricular systolic pressure as compared to AR. A marked increase of thiobarbituric acid reactive substances (TBARS) was detected in the effluent during AR, accompanied by an impaired release of the endothelial vasodilator NO and diminished coronary flow rates compared to the baseline values. GR resulted in a significant reduction of TBARS in the effluent and promoted a better recovery of coronary flow as well as endothelial release of NO during the later phase of reperfusion. It is concluded that graduated reperfusion is beneficial in reducing free radical mediated peroxidative tissue injury and endothelial dysfunction upon reoxygenation.     

Marchantinquinone, isolated from Reboulia hemisphaerica, as inhibitor of lipid peroxidation and as free radical scavenger

Recent years have witnessed a renewed interest in plants as pharmaceuticals in the Western world. This interest is channeled into the discovery of new biologically-active molecules by the pharmaceutical industry and into the adoption of crude extracts of plants for self-medication by the general public. In both of these areas some attention is being paid to the investigation and use of ethnopharmacology, the traditional use of plants for medicinal purposes by particular cultural groups. Ethnopharmacologic leads have resulted in the introduction of new single molecule drugs but have a greater role to play if crude extracts are accepted for clinical use in the West. The problems confronting such usage are discussed. Considerable benefits for developing countries are possible when the local medicinal plants are subjected to scientific methods of validation of traditional use and quality control. This approach has met with success in some parts of the world but is not always appreciated by national governments and international agencies. Related areas of concern such as conservation of ecology and culture must be integrated with any such program. Plants used in traditional medicine therefore have an important role to play in the maintenance of health in all parts of the world and in the introduction of new treatments.     

Inhibitory effect of estrogens on the oxidative hemolysis induced by 2-amidinopropane hydrochloride, a free radical generator

Verapamil was shown to improve the glucose absorption whereas perfusion of the rat small intestine with high-calcium solution decreased it. The transport system of glucose following the membrane hydrolysis proved rather stable under the effect of calcium. Stress seems to depress somewhat the mechanisms of glucose transport and to increase the calcium level in enterocytes against the background of occurring hypercalcemia.     

The effect of metal ions on free radical formation in lung and brain of rats

The influence of 14 metal ions on free radical (FR) formation in subcellular systems of lung mitochondria and brain synaptosomes (cortex and cerebellum) of rats was investigated. Iron and manganese showed a highly significant increase in FR formation in lung, whereas other investigated metal ions showed relatively slight, insignificant decreases in FR. The significant enhancement effects of iron and manganese increased with their content in the media. Scandium exhibited an insignificant enhancement effect at a low concentration of 0.01 microM, but also an insignificant decrease in FR formation in lung at higher concentrations. In brain cerebellum only iron significantly increased FR formation but manganese, as well as trivalent metals (Al, La, Sc), showed insignificant changes in FR formation. The possible causes of differences induced by metal ions at various concentrations, as well as the biological functions of the investigated tissues, were considered in the explanation of obtained results.     

Carvedilol, a new beta adrenoreceptor blocker and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits

Oxygen-derived free radicals play a critical role in atherogenesis and reperfusion injury. The present experiment evaluated the effects of carvedilol, a new beta adrenoreceptor blocker with potent free radical-scavenging activity, on myocardial ischemia and reperfusion injury in a hypercholesterolemic rabbit model. New Zealand rabbits were fed a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with 1200 ppm carvedilol or propranolol. Eight weeks later, the rabbits were subjected to 60 min of myocardial ischemia followed by 60 min of reperfusion. The nontreated cholesterol-fed animals experienced greater cardiac damage after ischemia and reperfusion than rabbits fed a normal diet (necrosis 51% +/- 4% vs. 28% +/- 3% in the normal-diet group, P < .01). In addition, nontreated cholesterol-fed rabbits showed a significantly decreased vasorelaxant response to ACh in U-46619-precontracted aortic rings (56% +/- 5% vs 90% +/- 3% in the control group, P < .001). Treatment with propranolol neither preserved endothelial function after cholesterol feeding nor reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. Propranolol treatment did significantly decrease HR, pressure-rate index and infarct size (necrosis 33% +/- 4%). Despite their having essentially identical effects on HR and pressure-rate index, carvedilol exerted more profound cardiac protective effects than propranolol (necrosis 19% +/- 3%). Moreover, carvedilol treatment significantly preserved aortic endothelial function and markedly reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. These results indicate that in addition to its beta blocking activity, the antioxidant and endothelial protective activities of carvedilol contributed significantly to its cardiac protective effects after ischemia and reperfusion.     

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

Selective ruminants, which prefer easily digestible plants, cannot digest fibrous forage as well as grass eaters. Low enzyme activity or short retention time of ingesta particles in fermentation chambers appeared to be responsible for reduced cellulose breakdown. Seasonal activity of cellulolytic enzymes, cellulose concentration and protozoa population in reticulorumen (RR) and caecocolon (CC) of roe deer as a typical concentrate selector were investigated. Cellulase activities were lowest in winter when cellulose concentration in RR contents were highest. Highest enzyme activities and lowest cellulose concentration were measured in early spring. Cellulolytic activities were significantly correlated with the number of protozoa in RR. Only one entodinomorphic genus was identified in the RR. The enzyme activities in CC were far lower compared with those in RR. Low cellulose digestion in the RR cannot be compensated for by cellulose breakdown in the CC. The reduced cellulose digestion of roe deer may be attributed to the short retention time of food particles in spring and summer, whereas decreased colonisation of microorganisms in the rumen may be the main reason for low cellulose breakdown in winter.     

Increased oxidative stress in rat liver and pancreas during progression of streptozotocin-induced diabetes

1. Oxygen free radicals have been suggested to be a contributory factor in complications of diabetes mellitus. There are many reports indicating the changes in parameters of oxidative stress in diabetes mellitus. In this study we aimed to identify whether oxidative stress occurs in the liver and pancreas in the initial stages of development of diabetes. 2. We therefore investigated the lipid peroxide level (thiobarbituric acid-reactive substances, TBARS) and activities of antioxidant enzymes [superoxide dismutase (SOD), catalase and glutathione peroxidase] in liver and pancreas of control and streptozotocin-induced diabetic rats at various stages of development of diabetes. 3. Male Sprague-Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups consisting of six rats each. Rats in these subgroups were studied at weekly intervals (0 to 6 weeks). Plasma glucose levels, TBARS levels and activities of antioxidant enzymes were measured in liver and pancreas at various time intervals. 4. There was a significant (P < 0.05) and progressive increase in TBARS levels of liver and pancreas in the diabetic group. Total SOD and Cu-Zn-SOD activity increased (P < 0.05) with progression of diabetes while Mn-SOD activity showed no significant change in either tissue. Catalase and glutathione peroxidase activities increased significantly (P < 0.05) in liver and pancreas. 5. Immunohistochemical study of pancreatic islet revealed a decrease in the expression of insulin with progression of diabetes. However, glucagon and somatostatin showed an increase in immunoreactivity and a difference in their distribution pattern. 6. The findings of the present study suggest that oxidative stress starts at early onset of diabetes mellitus and increases progressively. In conclusion, the structural damage to these tissues or complications of diabetes mellitus may be due to oxidative stress.     

In vivo free radical production after cross-clamping and reperfusion of the renal artery in the rabbit

Postischaemic reperfusion injury is often attributed to the generation of oxygenated free radicals which may subsequently promote lipid peroxidation in cell membranes. Electron paramagnetic resonance spectroscopy in association with the spin trap molecule alpha-phenyl-N-tert-butyl-nitrone allowed direct confirmation of lipid free radical production after renal ischaemia-reperfusion in an in vivo rabbit model. A 60-min period of ischaemia followed by reperfusion caused free radical production twofold greater than after 15 min of ischaemia. Glutathione and alpha-tocopherol have been measured in renal tissue, as indirect markers of lipid peroxidation. After 15 min of ischaemia followed by 10 min of reperfusion, the mean(s.e.m.) glutathione content of the ischaemic kidney was slightly but significantly reduced by 11.9(2.5)% (P < 0.003). The content of alpha-tocopherol was unchanged. However, 10 min of reperfusion following 60 min of ischaemia led to significant decrease in mean(s.e.m.) content of both glutathione (30.4(3.7)%) (2.23(0.2) versus 3.14(0.18) mumol/g wet tissue, P < 0.001) and alpha-tocopherol (46.1(7.8)%) (0.57(0.10) versus 1.09(0.14) micrograms/g wet tissue, P < 0.001) when compared to the control kidney. Under these experimental conditions, desferrioxamine (15 mg/kg administered intravenously before inducing ischaemia), a drug known to limit free radical production, significantly limited the decrease of alpha-tocopherol to 20.8(6.4)% (0.83(0.08) versus 1.05(0.04) micrograms/g wet tissue, P < 0.05), but did not prevent glutathione consumption in the reperfused kidney.     

Changes in QOL in patients with brain tumors measured by mood changes during and after treatment

Patients with brain tumors are exposed to severe stress which may have an influence on their quality of life (QOL). To measure QOL in those patients, we measure their mood state during and after the treatments. MATERIALS: 16 patients who were admitted to our department for treatment of brain tumors, were included in the study. The tumors included 5 gliomas, 6 meningiomas and others. They were 7 males and 9 females, and age distributed from 19 to 74 years old. All patients presented more than 90% of the Karnofsky performance score (KPS) on discharge, so they were expected to return to their previous social life. METHOD: The self-answering tests were performed on admission (Pre), on discharge (Post 1), and at more than 5 months after the discharge (Post 2). The tests included an original questionnaire asking consent to admission and treatment, and concerning the feeling of disability to cope with conditions of living, Cornell Medical Index (CMI) which measured the neurosis, and Profile of mood state (POMS) which measured the 6 subscales of patients mood states including tension-anxiety (TA), depression (D), anger-hostility (AH), vigor (V), fatigue (F), and confusion (C). RESULTS: The questionnaire showed that the patients feel satisfied with having consented to the treatment. The feeling of disability to cope with living became stronger in Post 2 than in Pre. CMI showed a borderline of neurosis in three patients on admission, and in four patients in Post 2. Only the TA of POMS subscale improved significantly in Post 2. However, other subscales were unchanged. It is characteristic that all of the subscales of POMS showed less disturbance on discharge compared with that on admission, but, they returned to the Prelevel after they returned to social life. CONCLUSIONS: Patients with brain tumors have satisfactory consent to the treatment, however, they feel disability to cope with social life. On discharge, they showed a better mood state compared to that on admission, but the mood state turned for the worse again during the follow-up period. It is evident that patients with brain tumors are exposed to severe stress even after the completion of the treatment. The results necessitate our taking patients' mental health into consideration for our treatment protocol.     

Phagocyte-induced oxidative stress in patients with haemodialysis treatment and organ transplantation

This study was designed to investigate any seasonal (spring, summer, autumn and winter) changes in haematological parameters in the blood of Tinca tinca measuring the number of red blood cells (RBC), haematocrit, white blood cells, and total plasma proteins. The results show significant changes in RBC and haematocrit in males comparing spring and summer with autumn and winter, whereas in females the RBC remained constant for all 4 seasons but the haematocrit decreased in autumn and winter compared to spring and summer. The white blood cells of male and female animals were significantly lower in spring and winter compared to summer and autumn. In male fish total protein contents significantly decreased in autumn and winter compared to spring and summer, whereas in females protein output significantly decreased in winter compared to the other seasons. The results indicate marked seasonal variation in the blood of male and female Tinca tinca. This variation may play a important protective role for the survival of the animals.     

Free radical centers in isolated rat heart tissue in a normal state, in ischemia, and reperfusion

EPR spectroscopy was used to measure paramagnetic species in rat hearts freeze-clamped during control perfusion by the Neely procedure, after 25 min of normothermic global ischemia or 20 min of total reperfusion with oxygenated perfusate. The analysis of spectral and relaxation parameters measured at -40 degrees C showed that in all three cases free radicals in heart tissue were semiquinones of CoQ10 and flavins. Ischemia increased the amount of free radical species (mostly flavosemiquinones) in myocardium about two times, the beginning of reflow of perfusate resulted in decrease of the intensity of the EPR signal to an initial level. The saturation curves were different for control, ischemic and reoxygenated postischemic samples, and they demonstrated the heterogeneity of free radical centers in cardiac mitochondria.     

Enflurane and isoflurane, but not halothane, protect against myocardial reperfusion injury after cardioplegic arrest with HTK solution in the isolated rat heart

To investigate the effects of halothane, enflurane, and isoflurane on myocardial reperfusion injury after ischemic protection by cardioplegic arrest, isolated perfused rat hearts were arrested by infusion of cold HTK cardioplegic solution containing 0.015 mmol/L Ca2+ and underwent 30 min of ischemia and a subsequent 60 min of reperfusion. Left ventricular (LV) developed pressure and creatine kinase (CK) release were measured as variables of myocardial function and cellular injury, respectively. In the treatment groups (each n = 9), anesthetics were given during the first 30 min of reperfusion in a concentration equivalent to 1.5 minimum alveolar anesthetic concentration of the rat. Nine hearts underwent the protocol without anesthetics (controls). Seven hearts underwent ischemia and reperfusion without cardioplegia and anesthetics. In a second series of experiments, halothane was tested after cardioplegic arrest with a modified HTK solution containing 0.15 mmol/L Ca2+ to investigate the influence of calcium content on protective actions against reperfusion injury by halothane. LV developed pressure recovered to 59%+/-5% of baseline in controls. In the experiments with HTK solution, isoflurane and enflurane further improved functional recovery to 84% of baseline (P < 0.05), whereas halothane-treated hearts showed a functional recovery similar to that of controls. CK release was significantly reduced during early reperfusion by isoflurane and enflurane, but not by halothane. After cardioplegic arrest with the Ca2+-adjusted HTK solution, halothane significantly reduced CK release but did not further improve myocardial function. Isoflurane and enflurane given during the early reperfusion period after ischemic protection by cardioplegia offer additional protection against myocardial reperfusion injury. The protective actions of halothane depended on the calcium content of the cardioplegic solution. IMPLICATIONS: Enflurane and isoflurane administered in concentrations equivalent to 1.5 minimum alveolar anesthetic concentration in rats during early reperfusion offer additional protection against myocardial reperfusion injury even after prior cardioplegic protection. Protective effects of halothane solely against cellular injury were observed only when cardioplegia contained a higher calcium concentration.