Infarct size limitation by the xanthine oxidase inhibitor, allopurinol, in closed-chest dogs with small infarcts.

The present study was designed to evaluate the ability of allopurinol to limit infarct size following permanent coronary occlusion in the greyhound. Coronary occlusion was produced by injecting 2.5 mm plastic beads into the coronary artery of the closed chest dog. Non-perfused myocardium, the area at risk, was visualised by autoradiography of 141Cerium labelled microspheres which were infused immediately following coronary embolization. The treated dogs (n = 12) received 400 mg of allopurinol orally one day before surgery. A 25 mg . kg-1 bolus was administered (iv) immediately before occlusion, and repeated every 8 h. 11 dogs served as controls. After 24 h, the dogs were killed and the hearts were sliced into 5.0 mm transverse sections. The infarcted myocardium was visualised by triphenyl tetrazolium chloride staining. The percentage of the risk zone which evolved to infarct was calculated. This percentage was 18.1 +/- 3.95% in the allopurinol group vs 58.4 +/- 2.81% in the control group (p less than 0.001). We conclude that allopurinol is a potent drug for the limitation of infarct size in the dog with permanent coronary occlusion.

Is the autoxidation of catecholamines involved in ischemia-reperfusion injury?

The autoxidation of catecholamines has been proposed to be a source of oxygen radicals in ischemia-reperfusion injury. However, this autoxidation per se is extremely slow at physiological pH and therefore is unlikely to be a primary source of oxygen radicals in ischemia-reperfusion injury. On the other hand, oxygen radicals from catecholamines are more likely to arise through catalyzed oxidations involving enzymatic systems and/or metal ions. It is these latter reactions that may be of interest with respect to damage associated with ischemia-reperfusion injury.

Classification of ischemic-induced damage to Na+, K+-ATPase in gerbil forebrain. Modification by therapeutic agents.

The activity of three forms of ATPase were examined in fractions of the brain of the gerbil treated with ethylene glycol-N-N-tetra-acetic acid (EGTA) under a variety of conditions of primary and secondary (reflow) ischemia. In animals which were unilateral ischemic (ligation of the right common carotid), damage to Na+, K+-ATPase alone was observed only after at least 6 hr of ischemia had elapsed. The phenomenon occurred in only symptomatic gerbils and was absent in animals which were either asymptomatic or only displayed partial neurological symptoms. Under conditions of bilateral cerebral ischemia, in which both carotid arteries were clamped, only irreversible ischemia (60 min) followed by reflow, was associated with highly significant damage to cerebral Na+, K+-ATPase. In regional studies of the forebrain involving ischemia for 60 min plus 30 min reflow, damage to Na+, K+-ATPase was evident in the cerebrum, hippocampus, striatum and thalamus, while the hypothalamus and olfactory bulb were spared. Pretreatment of gerbils with allopurinol, clonazepam or combinations of thiopental plus either indomethacin or methylprednisolone offered protection to cerebral Na+, K+-ATPase subsequent to secondary ischemia. With only minor exceptions (striatum) neither Ca2+, Mg2+- nor Mn2+-ATPase were altered by stroke or treatment with drugs.

Pharmacological protection of reoxygenation damage to in vitro brain slice tissue.

Pharmacological protection of central neural function against damage by hypoxia and reoxygenation was studied electrophysiologically and biochemically in hippocampal brain slices. Hypoxia causes a loss of both orthodromically and antidromically evoked potentials in CA1 pyramidal cell neurons. Damage due to hypoxia lasting more than 10 min cannot be restored by reoxygenation. Following pretreatment with methylprednisolone (10(-5) M), indomethacin (10(-5) M) or allopurinol (10(-5) M), reoxygenation after 10 min of hypoxia resulted in complete recovery of the evoked activity. Na+,K+-ATPase activity was not reduced by 10 min of hypoxia, but was reduced by 50% during the first 10 min of subsequent reoxygenation. Allopurinol (10(-5) M) protected against loss of this enzyme activity. The protective action by these drugs of both electrophysiological and biochemical aspects of neural function is consistent with the hypothesis that secondary ischemic damage is caused by the formation of oxygen-derived free radicals during reperfusion.

Measurement of malonyldialdehyde production during sodium arachidonate-induced polymorphonuclear leukocyte aggregation.

Sodium arachidonate stimulated canine polymorphonuclear leukocytes (PMNs) to aggregate and produce malonyldialdehyde (MDA). The MDA production was due to cellular processes during the aggregation, as boiled PMN suspension neither aggregated nor produced MDA. Aggregation and MDA production were not due to platelet contamination because epinephrine and ADP were unable to stimulate either of these responses in the PMN suspensions. Finally, use of the aggregatory modifiers indomethacin, 1-methylimidazole, and vitamin E dissociated aggregation from MDA production. These results suggest that the cellular process(es) resulting in MDA production is/are not responsible for canine PMN aggregation.

Tumor necrosis factor-alpha pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury.

We have demonstrated that tumor necrosis factor-alpha (TNF-alpha) pretreatment protected the rat heart from ischemia-reperfusion injury. This effect was monitored by assaying for lactate dehydrogenase (LDH), an enzyme whose release correlates with loss of cell membrane integrity. Intact hearts removed from rats pretreated with TNF-released significantly lower amounts of LDH compared to control hearts after 20 min. of total global ischemia followed by reperfusion. Hearts from TNF-alpha-pretreated animals contained higher levels of manganous superoxide dismutase (MnSOD) mRNA than hearts from untreated rats. Because oxygen free radicals have been implicated as a major cause of reperfusion damage and the function of MnSOD is to detoxify superoxide anions in the mitochondria, a possible protective mechanism for TNF-alpha may be to induce expression of MnSOD in the heart and thus confer resistance to oxygen free radicals generated during reperfusion.

Improved lactate extraction in dogs with coronary artery ligation following administration of a thromboxane synthetase inhibitor.

Increased thromboxane A2 formation has been implicated in the pathophysiology of acute myocardial ischemia. The present study was designed to investigate the action of imidazole, a thromboxane synthetase inhibitor, during acute myocardial ischemia induced in anesthetized dogs. Regional ischemia was produced by ligation of the left anterior descending coronary artery (LAD), which caused a decrease in myocardial lactate extraction across the ischemic area, decreases in myocardial high energy phosphate contents and an increase in myocardial lactate in the center of the ischemic region. Intravenous administration of imidazole prior to LAD ligation attenuated the decrease in myocardial lactate extraction, but did not alter the depression in high energy phosphates or the rise in myocardial lactate content in the center of the ischemic myocardium. The results of this study support the suggested protective action of imidazole during an ischemic insult; perhaps by maintaining the metabolic status of the moderately ischemic border zone.

Circulating dopamine levels and a protective action of naloxone in canine splanchnic arterial occlusion shock.

The possible beneficial actions of naloxone (NAL) were evaluated in canine splanchnic arterial occlusion (SAO). MEan arterial pressure (MAP), cardiac output (CO) and left ventricular contractility (LV dP/dt max) were measured in pentobarbital anesthetized dogs. Animals received intravenously NAL (8 mg/kg) or an equivalent volume of 0.9% saline prior to occlusion or sham occlusion, followed by infusion of 8 mg/kg/hr. The occlusion was released after 2 hr and the dogs monitored for an additional 3 hr or until MAP fell to 40 mmHg. Blood was drawn during the experiments for analysis of dopamine (DA), norepinephrine (NE) and epinephrine (E). SAO and SAO + NAL dogs had decreased MAP and CO after release. However, SAO + NAL animals had a higher LV dP/dt max. NAL in sham and SAO dogs produced significantly higher plasma DA and E levels than in their respective control groups. 67% of SAO + NAL survived 3 hr post release, compared to 43% of SAO dogs. Although the mechanism of NAL protection remains undefined, it may relate to altered vascular response to endogenous opiates or catecholamines.

Lack of a direct metabolic effect of fructose, 1,6-diphosphate in ischemic myocardium.

Fructose 1,6-diphosphate (FdP) reportedly protects ischemic myocardium. To determine whether this is a direct action on the heart, we used a canine model in which two coronary arteries were perfused at identical but reduced rates. Into one artery we infused FdP (total doses of 400 mg or 1.8 g) while the other received 0.9% NaCl. After 1 h, biopsies were taken from a normal region and the two ischemic regions and were analyzed for ATP, phosphocreatine (PC), and lactate content. In the 0.9% NaCl-treated ischemic tissue, ATP and PC fell to half the nonischemic levels. The FdP-treated tissue exhibited high-energy phosphate levels similar to the 0.9% NaCl-treated tissue with no significant differences between the two ischemic areas. Lactate levels in both ischemic areas were elevated threefold above nonischemic levels. Contractility studies showed that infusion of FdP directly into the coronary artery depressed contractility in both nonischemic and ischemic conditions. Our data show that, if FdP does have a protective action in ischemia, it is not through a direct action on the heart.

Sympathetic neuroeffector transmission to pulmonary vascular smooth muscle in porcine superior mesenteric arterial occlusion (SMAO) shock.

Splanchnic arterial occlusion shock results in pulmonary endothelial damage and depression of porcine intralobar pulmonary artery and vein contractility. This study evaluates the functional integrity of the adrenergic nerves innervating intralobar pulmonary arteries and veins and the changes in neurotransmission following 1) superior mesenteric artery occlusion (SMAO) shock in swine; 2) sequential inhibition of prostacyclin, thromboxane, and prostaglandin synthesis; and 3) mechanical stripping of the endothelium. Rings of porcine intralobar pulmonary arteries and veins were obtained from sham and SMAO shocked swine. They were suspended in muscle baths and stimulated transmurally at 1-32 Hz, 2 msec duration, 2 msec delay at 7.5-10V. Some experiments were performed on rings of intralobular pulmonary arteries and veins in which the endothelium was stripped with a razor blade. Appropriate inverted-reverted controls were used to account for any deleterious effects of the preparatory techniques involved in stripping. Intralobar pulmonary arteries and veins from sham swine contracted in response to 1 Hz, with maximum responses at 32 Hz. The responses to nerve stimulation were enhanced by cocaine and inhibited by phentolamine, an alpha-receptor antagonist. Inhibition of endothelial prostacyclin synthesis, as well as endothelial stripping, diminished by 30-40% the responses to nerve stimulation. The responses to nerve stimulation were depressed in both intralobar pulmonary arteries and veins in SMAO shocked swine. The data demonstrate physiologic regulation of neural control in porcine pulmonary blood vessels. Furthermore, the data suggest that prostaglandin, the vascular endothelium, and shock, may modify this process.

Direct metabolic effects of isoproterenol and propranolol in ischemic myocardium of the dog.

The effect of either isoproterenol or propranolol on the metabolism of ischemic myocardium was examined. To ensure that all changes were due to changes in metabolism and not drug-induced changes in residual flow to the ischemic regions, we devised a preparation in which two coronary branches on the same heart were simultaneously perfused at a low flow rate. Microsphere measurements verified that the two ischemic regions were receiving identical blood flow rates. One branch received an infusion of 0.9% NaCl and the other received the drug. After 1 h both regions were biopsied and the high-energy phosphate levels in each region were determined. ATP and phosphocreatine each fell to about 50% of their starting values in the 0.9% NaCl-treated regions, and isoproterenol did not further depress the high-energy phosphate concentrations. Propranolol, on the other hand, significantly preserved the high-energy phosphate concentrations. We conclude that although isoproterenol seemed incapable of accelerating energy utilization in ischemic myocardium, propranolol is apparently capable of reducing it.

Aortic prostacyclin release is lowered by superior mesenteric artery occlusion (SMAO) shock in pigs.

We have previously demonstrated altered platelet reactivity subsequent to splanchnic artery occlusion (SAO) shock in pigs: decreased reactivity, such as might be seen subsequent to submaximal stimulation, was observed in the postrelease period. Prostacyclin (PGI2) formed and released by vessel endothelium may function as a circulating hormone and regulate platelet reactivity by opposing the stimulatory influence of other factors. We have therefore investigated PGI2 release by aortas taken at "death" (systolic blood pressure = 30 mmHg) from pigs subjected to shock induced by a 2-hr occlusion of the superior mesenteric artery (SMAO). This shock is analogous to, but with a longer time course than, SAO shock. Aortas from sham-operated animals (matched for "death" time to the SMAO animals) served as controls. Thoracic aortas were removed, placed in cold 50 mM pH 7.5 tris buffer, cleaned of adhering tissue or clots, cut into rings and suspended in room temperature 50 mM pH 7.5 tris buffer into which PGI2 was released. Aliquots of the latter were quantitated for PGI2 by bioassay using human platelets for which a dose-response relationship for pure, synthetic PGI2 had been established: aliquot PGI2 quantities were determined graphically from the log dose-response (percentage inhibition of aggregation) curves, and converted arithmetically to amount per weight tissue. After 10 min incubation of tissue in buffer, the mean +/- SEM PGI2 for the shock animals (N = 8) was 0.053 +/- 0.02 ng/mg, and for the sham animals (N = 7), 0.174 +/- .06; the P value for t-test of means was 0.083. For 30-min incubation, the mean PGI2 for the shock group was 0.052 +/- 0.02 and for the sham, 0.283 +/- 0.11; the P value was 0.029. Inhibitory activity of the buffer aliquots declined in parallel with authentic PGI2. Thus, the occurrence of SMAO shock resulted in a significant decrease in aortic release of PGI2. Altered platelet reactivity as a consequence of shock, such as demonstrated by us and by others, could thus be in part a result of lowered endothelial release of PGI2, either alone or in combination with the occurrence of activation by other factors.

Free radical-producing enzyme, xanthine oxidase, is undetectable in human hearts.

Samples from four human hearts were analyzed for both their xanthine dehydrogenase and xanthine oxidase content. We used the conventional spectrophotometric assay and a more sensitive fluorometric assay to determine the content of enzyme in these samples. In no case could any activity be detected. We conclude that human hearts must contain less than 2.0 nU/g of activity. This makes it unlikely that xanthine oxidase is a significant source of O2 free radicals in the ischemic human heart or that xanthine oxidase inhibitors will be of therapeutic value in that setting.

Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart.

The xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in ischemic and reperfused myocardium. A spectrophotometric assay was employed to measure the xanthine oxidase activity of rat and rabbit hearts exposed to varying durations of global ischemia. In the rat 24.6 +/- 4.8 mIU/g wet wt of xanthine dehydrogenase + xanthine oxidase activity were detected in both ischemic and normally perfused myocardium. In the non-ischemic state only 6% of this activity was associated with the free radical-producing oxidase form. After 5 min of ischemia however about 25% of the enzyme was in the oxidase form, a value which remained unchanged over the following 25 min. Neither xanthine dehydrogenase nor xanthine oxidase could be detected in the rabbit heart. Failure of allopurinol, an inhibitor of xanthine oxidase, to limit infarct size in a rabbit model of ischemia/reperfusion provides further evidence that this species has insignificant amounts of xanthine oxidase in its heart. Anesthetized rabbits were subjected to coronary artery ligation for 45 min and 3 h of reperfusion. The volume of the zone of underperfusion was assessed with fluorescent microspheres and infarct size was assessed by tetrazolium staining. In control animals 67.5 +/- 3.8% of the zone of underperfusion became necrotic. In rabbits given superoxide dismutase (15000 IU/kg) + catalase (50,000 IU/kg) for 90 min starting 15 min before occlusion, infarct size was only 35.4 +/- 3.3% of the zone of underperfusion. However, in rabbits pretreated with allopurinol (75 mg p.o. 24 h before study + 30 mg/kg 5 min before occlusion) infarct size was 65.8 +/- 8.7%.(ABSTRACT TRUNCATED AT 250 WORDS)