Derek M. Yellon

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)

Sustained limitation of myocardial necrosis 24 hours after coronary artery occlusion: Verapamil infusion in dogs with small myocardial infarcts

Studies were undertaken to ascertain whether verapamil infusion affords a sustained limitation of myocardial injury in the dog after a 24-hour period of coronary artery occlusion. Regional myocardial ischemia was induced by an embolization procedure which did not involve thoracotomy. Immediately after embolization radioactive microspheres were administered intraventricularly to define any area of myocardial underperfusion (zone at risk of infarction). Verapamil (0.005 mg/kg/min) was then administered and maintained for 24 hours during which time the dogs were allowed to recover from anesthesia. The control group received a corresponding infusion of saline solution. After 24 hours the dogs were killed and transverse myocardial sections (3 mm) were prepared. The resulting area of necrosis was visualized by tetrazolium staining, and risk zones were visualized by autoradiography. In the control heart, 62 +/- 7% of the zone at risk deteriorated to necrotic tissue, whereas in the verapamil-treated group only 18 +/- 4% of the tissue in the zone at risk became necrotic. Verapamil appeared to exert a significant (p less than 0.001) tissue-sparing effect that was sustained for at least 24 hours after the onset of ischemia.

Effects of flurbiprofen in altering the size of myocardial infarcts in dogs: Reduction or delay?

Anti-inflammatory agents such as flurbiprofen have been claimed to reduce infarct size in a number of models of coronary artery occlusion. However, several of the studies are controversial and also do not allow the critical distinction between reducing and delaying injury. In the present study, a closed chest method of coronary occlusion was used to generate small areas of regional myocardial ischemia in dogs. The method involved cannulation of the coronary ostium by way of the carotid artery and coronary embolization with 2.5 mm diameter beads. Flurbiprofen (1 mg/kg) was given immediately after occlusion and thereafter every 6 hours. Groups of dogs were subjected to either 6 or 24 hours of elapsed ischemia, after which time the hearts were removed and sectioned. Frozen-tissue slices were stained with triphenyl tetrazolium in order to delineate infarct size. After staining the tissue slices were subjected to autoradiography in which microspheres given immediately after occlusion were visualized to delineate the perfusion bed served by the occluded coronary artery (zone at risk). Risk zone to infarct size ratios for drug treated and control animals revealed that flurbiprofen treatment had no effect upon infarct size as determined 24 hours after occlusion. Despite significant residual coronary flow in the ischemic area, virtually all of the risk zone deteriorated to necrotic tissue. By contrast, after 6 hours of elapsed ischemia, infarct size was considerably reduced in the flurbiprofen-treated group. With the proviso that the drug might have affected only the sensitivity to tetrazolium staining, these results indicate that in severe ischemia, flurbiprofen can greatly delay but not prevent tissue necrosis.

Temporal and spatial characteristics of evolving cell injury during regional myocardial ischemia in the dog: the "border zone" controversy.

An open chest dog heart with multiple coronary ligations was used to define the temporal and spatial characteristics of injury evolving during regional ischemia. With the use of a multiple (40 sample) biopsy device, adjacent transmural biopsy specimens were obtained from the transition zone between normal and ischemic tissue after 5, 30, 45, 60 and 120 minutes of ischemia. The first 1.8 mm of epicardial tissue was taken for the analysis of flow and metabolites. The results confirmed the existence of a sharp interface of flow and metabolism in the epicardial lateral plane at the boundary of the ischemic zone. There was no significant zone of intermediate injury (flow and metabolism being depressed uniformly throughout the ischemic area). Comparison of the distribution of flow determined by radiolabeled gadolinium-153 at onset of ischemia with that indicated by radiolabeled tin-113 microspheres given at the end of various periods of ischemia revealed no change in the position or steepness of the flow interface at any time during the first 2 hours of ischemia. This observation, together with the absence of any major redistribution or enhancement of residual flow to the ischemic zone, indicated that there was little or no significant collateralization between 5 and 120 minutes. Analysis of the adenosine triphosphate (ATP) content revealed a rapid depletion during the first 5 minutes of ischemia; the content then remained essentially unchanged until 30 minutes, after which time a second phase of accelerated ATP depletion was observed until 45 minutes. ATP content then remained relatively constant up to 2 hours.

Protection afforded by allopurinol in the first 24 hours of coronary occlusion is diminished after 48 hours

Experiments were performed to test whether the reduction in infarct size afforded by allopurinol following 24 h of permanent coronary artery occlusion is sustained over the subsequent 24 h. A dogs coronary artery was occluded with an embolus followed by injection of radiomicrospheres into the left ventricle to mark the ischemic region and to measure regional blood flow. Dogs were sacrificed either 24 h or 48 hours after embolization. The infarcts were delineated by failure to stain with triphenyl tetrazolium chloride and the ischemic zones were visualized by autoradiography of the heart slices. Dogs in the treatment groups received 600 mg of allopurinol PO 18 h before surgery, and a 10 mg/kg IV bolus 15 minutes before embolization followed by constant IV infusion of 55 mg/kg/24 h until sacrifice. A close correlation in the control animals between the percent of the ischemic zone which infarcted and collateral blood flow was used to predict a nonintervention infarct size in each treatment animal. Allopurinol treatment caused 17.9 +/- 3.3% less of the risk zone to be tetrazolium negative after 24 hours of ischemia than that seen in untreated animals. Less allopurinol induced salvage was observed in the 48 hour drug group with only a 11.1 +/- 3.3% limitation in infarct size. Furthermore, the effect was inconsistent at 48 h with only 2 dogs showing salvage. We conclude that allopurinol delays but does not prevent infarction in the permanent occlusion model.

Nifedipine limits infarct size for 24 hours in closed chest coronary embolized dogs

SummaryWe studied the ability of nifedipine, a calcium antagonist, to limit infarct size in the closed chest, coronary embolized dog. Immediately after embolization141Ce labelled microspheres were administered into the left ventricle. Myocardium not receiving microspheres was considered to be the region at risk. The nifedipine group (10 dogs) received a bolus (16 μg/kg i.v. over 8 minutes as a loading dose) followed by continuous infusion (1,000 μg/24 hours) 10 min after embolization. The control group (9 dogs) received an equal volume of saline. Twenty-four hours after embolization the dogs were sacrificed, the heart sectioned into 4-mm slices and the slices were stained with tetrazolium to reveal the infarct. The region at risk was determined by autoradiography of the microspheres in the heart slices. Infarct and risk zone volume were determined by planimetric methods. The nifedipine group had a significantly smaller infarct volume to risk zone voluem ratio than the control group (38.7±4.7% vs. 79.5±4.3%, p<0.001). We conclude that nifedipine produces a sustained limitation of infarct size following permanent occlusion of a dogs coronary artery.

A High Velocity Impact Device for Obtaining Multiple, Contiguous, Myocardial Biopsies

Abstract A method has been devised which allows multiple contiguous transmural biopsies to be obtained simultaneously from the left ventricular wall of the dog heart. The device is based upon the principle of high velocity impact. Cutting matrices can be constructed to retrieve any number of biopsies of any size over any defined area. Sampling and freezing can be achieved in less than 3 s. Preliminary studies have confirmed the adequacy of freezing (preservation of labile metabolites). Each biopsy sample can be further subfractionated and micoanalytical procedures are described which allow flow and multiple metabolites to be measured in each biopsy. The device may be used to study the metabolic and flow geometry of an area of regional ischemia during evolving myocardial infarction.

Adenosine-induced increase in myocardial ATP: are there beneficial effects for the ischaemic myocardium?

SummaryThe adenosine triphosphate (ATP) content of isolated Langendorff-perfused rat hearts may be increased by more than 40% above the normal value by a 2-h perfusion with adenosine (15 μmol/l). This metabolic manipulation was used to investigate the hypothetical relationship between total tissue ATP content and ischaemia-induced contractile failure, ischaemic contracture and post-ischaemic functional recovery.Adenosine perfused hearts were submitted to 20 min of normothermic ischaemia and reperfused for 45 min with or without adenosine. Control experiments were performed with adenosine-free preischaemic perfusion. In identically designed experiments the tissue-protective effect of diltiazem (0.5 μmol/l) was determined and compared with the experiments with adenosine.At the end of 120 min of preischaemic perfusion, the ATP content of the adenosine treated hearts was 34.3±1.8 μmol/g dry weight (control=23.6±1.9 μmol/g, p<0.01). After a period of 20 min of normothermic ischaemia, the ATP content of the adenosine hearts decreased to 13.3± .4 μmol/g, whereas ATP fell to 8.3±1.6 μmol/g in the control hearts. The creatine phosphate (CP) levels of adenosine hearts were significantly lower than those of the control group before ischaemia, but did not show major differences following ischaemia.During ischaemia, the contractile activity measured via an intraventricular balloon catheter, as well as ischaemic contracture did not differ between the adenosine and control hearts. The inclusion of diltiazem into the perfusate significantly delayed the onset of contracture.After 45 min of reperfusion, ATP contents of adenosine and control hearts reached similar values (8.4±2.3 and 8.3±2.9 μmol/g, respectively). Inclusion of adenosine (15 μmol/l in the reperfusion perfusate of the adenosine experiments prevented a further decrease, but did not increase tissue ATP content. CP values of all groups showed a partial recovery upon reperfusion, they did not differ significantly.Contractile recovery was equal in all experimental groups except for the diltiazem treated hearts, which showed during the first 10 min of reperfusion an improved mechanical performance.It is concluded that total tissue ATP is not necessarily a good indicator of functional capabilities under conditions of normothermic ischaemia and reperfusion in the isolated rat heart.

Abrupt withdrawal of chronic beta-blockade: Adaptive changes in cyclic AMP and contractility

Abstract The effect of the abrupt withdrawal of chronic beta-blockade on the sensitivity of the myocardium to catecholamine challenge has been investigated. Oxprenolol and metoprolol (200 mg/kg body wt/day) were administered orally to rats for 3 weeks. This dosage produced effective beta-blockade together with peak plasma drug levels within the normal clinical range. Hearts were perfused as isolated Langendorff preparations on various days following the abrupt termination of drug treatment. Response to perfusion with 10 −8 M isoprenaline was measured in terms of changes in dP d t max , heart rate and tissue cAMP content. On the last day of drug administration (day 0), at a time when plasma drug levels were high, hearts from neither the oxprenolol treated group nor the metoprolol treated group exhibited a significant blockade of isoprenaline induced increases in contractility or cAMP when compared with hearts from untreated animals. However, at 2 and 3 days after the termination of drug administration, there was a significant decline in the myocardial response to isoprenaline challenge. Thus, hearts from the drug-treated groups, perfused on day 3, showed a similar degree of betablockade as that shown by hearts obtained from rats having received only a single dose of either beta-blocker. After day 3, both chronically treated groups developed an increasing sensitivity to isoprenaline challenge characterized by a marked hypersensitive response (in d P d t max , and cAMP levels) which peaked 7 to 9 days after the withdrawal of the drugs. There followed a decline in sensitivity which in turn was followed by a second period of hypersensitivity. Under the conditions of this study, chronic beta-blockade induces an oscillatory, hypersensitive response to catecholamine challenge which is manifest in terms of d P d t max and cAMP levels. This adaptation, with its concomitant periods of increased and decreased sensitivity, may suggest a possible mechanism for the clinically observed propranolol “rebound” phenomenon.

Myocardial salvage by pharmacological interventions

During the last 15 years numerous therapeutic agents have been reported to reduce or limit myocardial infarct size in both experimental and clinical studies (Maroko et al., 1971; Maroko & Braunwald, 1973; Kloner & Braunwald, 1980; Rude et al., 1981). However, the interpretation of some of these studies is now open to question and the methods for measuring infarct size subject to criticism (Most et al., 1976; Vogel et al., 1977; Heng et al., 1978; Peter et al., 1978; Opie, 1980; Hearse & Yellon, 1981; Reimer & Jennings, 1982; Chambers et al., 1983). As a consequence, the ability of pharmacological agents to reduce the size of a myocardial infarct has become a highly controversial issue. Much of this controversy relates to the validity of various experimental models and to the relevance of the results to the clinical situation. With this in mind we have developed new methods which conform to certain criteria which we believe should be adopted when studying the effects of drug therapy on infarct size.