David M. Ansley

The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion

Myocardial ischemia-reperfusion injury is an important cause of impaired heart function in the early postoperative period subsequent to cardiac surgery. Reactive oxygen species (ROS) generation increases during both ischemia and reperfusion and it plays a central role in the pathophysiology of intraoperative myocardial injury. Unfortunately, the cellular source of these ROS during ischemia and reperfusion is often poorly defined. Similarly, individual ROS members tend to be grouped together as free radicals with a uniform reactivity towards biomolecules and with deleterious effects collectively ascribed under the vague umbrella of oxidative stress. This review aims to clarify the identity, origin, and progression of ROS during myocardial ischemia and reperfusion. Additionally, this review aims to describe the biochemical reactions and cellular processes that are initiated by specific ROS that work in concert to ultimately yield the clinical manifestations of myocardial ischemia-reperfusion. Lastly, this review provides an overview of several key cardioprotective strategies that target myocardial ischemia-reperfusion injury from the perspective of ROS generation. This overview is illustrated with example clinical studies that have attempted to translate these strategies to reduce the severity of ischemia-reperfusion injury during coronary artery bypass grafting surgery.

Oxidative stress and myocardial injury in the diabetic heart.

Reactive oxygen or nitrogen species play an integral role in both myocardial injury and repair. This dichotomy is differentiated at the level of species type, amount and duration of free radical generated. Homeostatic mechanisms designed to prevent free radical generation in the first instance, scavenge, or enzymatically convert them to less toxic forms and water, playing crucial roles in the maintenance of cellular structure and function. The outcome between functional recovery and dysfunction is dependent upon the inherent ability of these homeostatic antioxidant defences to withstand acute free radical generation, in the order of seconds to minutes. Alternatively, pre‐existent antioxidant capacity (from intracellular and extracellular sources) may regulate the degree of free radical generation. This converts reactive oxygen and nitrogen species to the role of second messenger involved in cell signalling. The adaptive capacity of the cell is altered by the balance between death or survival signal converging at the level of the mitochondria, with distinct pathophysiological consequences that extends the period of injury from hours to days and weeks. Hyperglycaemia, hyperlipidaemia and insulin resistance enhance oxidative stress in the diabetic myocardium that cannot adapt to ischaemia–reperfusion. Altered glucose flux, mitochondrial derangements and nitric oxide synthase uncoupling in the presence of decreased antioxidant defence and impaired prosurvival cell signalling may render the diabetic myocardium more vulnerable to injury, remodelling and heart failure.

Large-dose propofol during cardiopulmonary bypass decreases biochemical markers of myocardial injury in coronary surgery patients: a comparison with isoflurane.

We investigated if increasing propofols dosage to augment its antioxidant capacity during cardiopulmonary bypass (CPB) could confer cardiac protection. Fifty-four coronary artery bypass graft surgery patients were randomly assigned to small-dose propofol (Group P; n = 18), large-dose propofol (Group HiP; n = 18), or isoflurane Group (Group I; n = 18). After the induction, anesthesia was maintained with an inspired concentration of isoflurane 1%–3.5% (Group I) or a continuous infusion of propofol 60 &mgr;g · kg−1 · min−1 (Group P) throughout the surgery. In Group HiP, this dose of propofol was increased to 120 &mgr;g · kg−1 · min−1 for 10 min before the onset of CPB until 15 min after aortic unclamping and then decreased to 60 &mgr;g · kg−1 · min−1 until the end of surgery. The duration of aortic cross-clamping was 83 ± 24, 88 ± 22, and 81 ± 20 min in Group P, Group HiP, and Group I, respectively (P > 0.1). Plasma malondialdehyde, a marker of oxidative stress, was significantly lower at 8 h after CPB, and Troponin I was lower at 24 h after CPB in Group HiP compared with Group P and Group I (P < 0.05). There was a significant reduction in inotropic requirements for separation from CPB in Group HiP compared with Group I. Postoperative systemic vascular resistance was significantly reduced in Group HiP as compared with Group I. Mean cardiac index was significantly higher at 24 h after CPB in Group HiP compared with Group P and Group I (P < 0.05) (Group I, 2.2 ± 0.1; Group P, 2.3 ± 0.2; and Group HiP, 2.8 ± 0.3 L · min−1 · m−2, respectively). The duration of intensive care unit stay was significantly shorter in Group Hi-P compared with Group I. We conclude that administration of a large dose of propofol during CPB attenuates postoperative myocardial cellular damage as compared with isoflurane or small-dose propofol anesthesia.

Tissue Antioxidant Capacity During Anesthesia: Propofol Enhances in Vivo Red Cell and Tissue Antioxidant Capacity in a Rat Model

The effects of anesthesia on ischemia-reperfusion injury are of considerable scientific and clinical interest. We examined the effects of propofol (known to possess antioxidant activity) and halothane (devoid of antioxidant activity in vitro) on tissue and red blood cell (RBC) antioxidant capacity. Adult male Wistar rats were anesthetized with halothane 0.5%–1.0% (n = 7), propofol 500 &mgr;g · kg−1 · min−1 with halothane 0.25%–0.5% (small-dose propofol;n = 9), or propofol 2000 &mgr;g · kg−1 · min−1 (large-dose propofol;n = 8) for 45 min. Blood and tissue samples of liver, kidney, heart, and lung were then harvested for in vitro exposure to a peroxidizing agent. Red cell malondialdehyde and tissue thiobarbituric acid reactive substances were determined spectrophotometrically. Antioxidant capacities of blood and tissues in the Large-Dose Propofol group, and of blood and all tissues except lung in the Small-Dose Propofol group, were increased significantly compared with halothane (P < 0.003). The increases in tissue antioxidant capacities varied in their magnitude: RBC > liver > kidney > heart > lung. There was a high correlation between changes in RBC susceptibility to oxidative damage and corresponding changes in tissues. These findings demonstrate that large-dose propofol significantly enhances tissue antioxidant capacity, and RBC antioxidant capacity can serve as a functional measure of tissue activity, in vivo.

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H(2)O(2))-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H(2)O(2)-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H(2)O(2)-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H(2)O(2)-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.

Continuous epidural ropivacaine 0.2% for analgesia after lower abdominal surgery

The purpose of this study was to determine whether a lumbar epidural infusion of ropivacaine 0.2% would provide effective analgesia with an acceptably low incidence of motor blockade and side effects after lower abdominal surgery. After combined general and epidural anesthesia and surgery, 125 patients were randomly assigned to receive either saline or ropivacaine 0.2% at a rate of 6, 8, 10, 12, or 14 mL/h (Groups R6, R8, R10, R12, and R14, respectively) for 21 h. Supplemental analgesia, if required, was provided with intravenous patient-controlled analgesia with morphine. Data were collected at 4, 8, and 21 h, and included morphine consumption, pain scores at rest and with coughing, motor and sensory block, and adverse events. Cumulative morphine consumption was less in Groups R10, R12, and R14 compared with the saline group. At 4 h analgesia was better among patients receiving ropivacaine, but at 21 h pain scores were identical. Sensory blockade at 8 and 21 h was greater in the ropivacaine groups compared with the saline group. Approximately 30% of R8, R10, and R12 patients, and 63% of R14 patients had demonstrable motor block of the lower limbs at 21 hours. We conclude that lumbar epidural ropivacaine 0.2% reduces parenteral morphine requirements but has little effect on pain scores and may be associated with motor blockade. (Anesth Analg 1997;84:784-90)

Propofol enhances red cell antioxidant capacity in swine and humans

PurposeTo determine the effect of an anaesthetic with antioxidant potential, propofol, on red blood cell (RBC) antioxidant enzyme activities and RBC susceptibility to peroxidative challenge.MethodsPropofol was administered by intravenous bolus (2.5 mg·kg−1) and continuous infusion (36 and 72 ml·hr−1 in nine swine; 216 ml·hr−1 in two swine), to achieve serum concentrations between 5 and 30μg·ml−1 for two hours at each rate. Arterial blood sampling was at 0,10, 30, 60, and 120 min for each rate of infusion, for measurement of plasma propofol concentration, activities of plasma and RBC Superoxide dismutase, glutathione peroxidase, gluthathione reductase, RBC catalase, and RBC malondialdehyde (MDA) formation in response to exvivo oxidative challenge with t-butyl hydrogen peroxide (tBHP; 1.5mM). Antioxidant mechanisms were determined byin vitro study of MDA formation, GSH depletion, and oxidation of haemoglobin to methaemoglobin in human erythrocytes exposed to propofol 0–75 μM. The antioxidant potential of propofol was compared with that of alpha-tocopherol utilising the reaction with 2,4,6-tripyridyl-s-triazine (TPTZ).ResultsPropofol had no effect on plasma or RBC antioxidant enzyme activities. It inhibited RBC MDA production over the range of 0–20 μg·ml−1 (y = −18.683x + 85.431 ; R2 = 0.8174). Effective propofol concentrations for 25% and 50% reductions in MDA levels were 7–12 and 12–20 μg·ml−1, respectively. Propofol has a similar effect on human erythrocytesin vitro (R2 = 0.98).ConclusionPropofol antagonises the effects of forced peroxidation of red cells at anaesthetic and sub-anaesthetic concentrations in swine. Its actions include scavenging of oxygen derived free radicals in a tocopherol-like manner.RésuméObjectifDéterminer l’effet d’un agent anesthésique possédant un potentiel antioxydant, le propofol, sur l’activité d’un enzyme antioxydant des globules rouges (GR) et sur la susceptibilité des GR à une provocation peroxydative.MéthodesLe propofol a été administré en bolus intraveineux (2,5 mg·kg−1) et en infusions continues (36 et 72 ml·h−1 chez 9 porcs; 216 ml·h−1 chez 2 porcs) pour obtenir des concentrations sériques entre 5 et 30 μg·ml−1 durant deux heures à chaque vitesse d’infusion. Des prélèvements sanguins par voie artérielle ont été réalisés à 0, 10, 30, 60 et 120 min. pour chaque vitesse d’infusion; on a mesuré la concentration de propofol, l’activité de la superoxyde dismutase du plasma et des GR, de la peroxydase du glutathion, de la réductase du glutathion, de la catalase du GR, ainsi que de la formation dans le GR de la malondialdehyde (MDA) en réponse à une provocation oxydative exvivo avec le peroxyde d’hydrogène t-butylique (tBHP, 1,5 mM). Les mécanismes antioxydants ont été déterminés par l’étudein vitro de la formation de MDA, de la déplétion de GSH ainsi que de l’oxydation de l’hémoglobine en methémoglobine dans des GR humains exposés au propofol 0–75 μM. Le potentiel antioxydant du propofol a été comparé à celui de l’alpha-tocophérol en utilisant la réaction avec le 2,4,6-tripyridyl-s-triazine (TPTZ).RésultatsLe propofol n’a pas eu d’effet sur l’activité de l’enzyme antioxydant du plasma ou des GR. Il a inhibé la production de MDA par les GR pour tout le spectre de 0–20 μg·ml−1 (y = −18.683x + 85.431 ; R2 = 0,8174). Les concentrations de propofol efficaces pour obtenir une réduction des taux de MDA de 25 et de 50% étaient respectivement de 7–12 et de 12–20 μg·ml−1. Le propofol a un effet analogue sur les globules rouges humainsin vitro (R2 = 0,98).ConclusionLe propofol, à des concentrations anesthésiques et subanesthésiques chez le porc, antagonise les effets d’une peroxydation forcée des globules rouges. Son mode d’action comporte l’épuration des radicaux libres provoqués par l’oxygène comme le fait le tocophérol.

Propofol enhances ischemic tolerance of middle-aged rat hearts: effects on 15-F2t-isoprostane formation and tissue antioxidant capacity

OBJECTIVE Experimental study has shown that myocardial ischemic tolerance is reduced during middle-age. We investigated the effect of propofol on ischemic tolerance of middle-aged rat hearts. METHODS Hearts of young adult (10 weeks old, Y) and middle-aged rats (20 weeks old, M) were assigned to propofol (P-Y, P-M) and control (C-Y, C-M) groups (n=6 each). Hearts were perfused using a Langendorff preparation with Krebs-Henseleit solution (KH) at constant flow rates. We applied propofol (P-Y, P-M) for 10 min at 12 microg/ml before inducing 40 min global ischemia. During ischemia, saline (C-Y, C-M) or propofol (P-Y, P-M) in saline was perfused through the aorta at 60 microl/min. Propofol in KH was perfused at 12 microg/ml for the first 15 min of reperfusion and subsequently reduced to 5 microg/ml in propofol treatment groups. Coronary effluent was assayed for 15-F(2t)-isoprostane after equilibration, during ischemia (T(1)) and at 0.5 (T(2)) and 5 (T(3)) min of reperfusion. After 90 min of reperfusion (T(4)), hearts were harvested to assess tissue antioxidant capacity. RESULTS In P-Y, we observed an increased latency to ischemic-contracture and a significantly reduced contracture after 35 min ischemia compared to control groups. No ischemic contracture was observed in P-M. There were significantly lower 15-F(2t)-isoprostane levels in P-M and P-Y than in C-M and C-Y at T(1). At T(4), the recovery of left ventricular developed pressure in P-M was greater than in P-Y (P<0.05); both were greater than in C-M and C-Y. CONCLUSION Propofol enhanced ischemic tolerance of middle-aged hearts, primarily by inhibiting lipid peroxidation.

Propofol Reduces Apoptosis and Up-regulates Endothelial Nitric Oxide Synthase Protein Expression in Hydrogen Peroxide-stimulated Human Umbilical Vein Endothelial Cells

BACKGROUND:Vascular endothelial cells play an important role in maintaining cardiovascular homeostasis. Oxidative stress is a critical pathogenic factor in endothelial cell damage and the development of cardiovascular diseases. In this study we evaluated the effects of propofol on oxidative stress-induced endothelial cell insults and the role of serine–threonine kinase Akt modulation of endothelial nitric oxide synthase (eNOS) as a mechanism of protection. METHODS:Human umbilical vein endothelial cells were used as the experimental model. Hydrogen peroxide (H2O2, 100 &mgr;M) was used as the stimulus of oxidative stress. Study groups included 1) control; 2) cells incubated with H2O2 alone; 3) cells incubated with propofol (50 &mgr;M) alone; or 4) cells pretreated with propofol 50 &mgr;M for 30 min then co-incubated with H2O2. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Trypan blue dye exclusion test. Cell apoptosis was evaluated by Hoechst 33258 staining. Caspase-3 activity was determined by the colorimetric CaspACE™ Assay System. Expressions of Akt, phospho-Akt, and eNOS were detected by Western blotting. RESULTS:H2O2 decreased cell viability, induced apoptosis, and increased caspase-3 activity in human umbilical vein endothelial cells. Propofol significantly protected cells from H2O2-induced cell damage, apoptosis and decreased H2O2-induced increase in caspase-3 activity. Propofol treatment significantly increased eNOS expression compared to control and H2O2-stimulated cells. There was no significant difference in phospho-Akt (Ser 473 or Thr 308) expression among the groups. CONCLUSIONS:Propofol 50 &mgr;M can reduce H2O2-induced damage and apoptosis in endothelial cells, by suppressing caspase-3 activity and by increasing eNOS expression via an Akt-independent mechanism.

Propofol Dose-Dependently Reduces Tumor Necrosis Factor--Induced Human Umbilical Vein Endothelial Cell Apoptosis: Effects on Bcl-2 and Bax Expression and Nitric Oxide Generation

We investigated whether propofol can inhibit tumor necrosis factor (TNF)-&agr;-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs). Isolated HUVECs were cultured in Dulbecco’s modified Eagle medium supplemented with 20% bovine calf serum. HUVECs in untreated and propofol control groups were cultured at 37°C for 24.5 h. HUVECs in the TNF treatment groups were initially cultured for 30 min in the presence of TNF or various concentrations of propofol, respectively, which were then cultured for 24 h with the addition of TNF at 40 ng/mL in the medium. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and confirmed by electron microscopy. The antiapoptotic Bcl-2 and proapoptotic Bax protein expressions were measured by immunocytochemical analysis. TNF stimulation resulted in a reduced Bcl-2/Bax ratio and increased apoptotic index (AI: percentage of apoptotic cells) in HUVECs. Propofol, at concentrations ≥12 &mgr;M, significantly (P < 0.001) and dose-dependently attenuated TNF-induced increase in AI and decrease in Bcl-2/Bax ratio. This was accompanied by increases in nitric oxide production. There is an inverse correlation between the ratio of Bcl-2/Bax expression and AI (P = 0.0009). These results suggest that propofol, at clinical relevant concentrations, can reduce TNF-induced HUVEC apoptosis.