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

Free Radical Biology & Medicine, Vol. 4, pp. 25-30, 1988

0891-5849/88 $3.00+ .00 © 1988 PergamonJournalsLtd.

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Conference Proceedings PROTECTION AFFORDED BY ALLOPURINOL IN THE FIRST 24 HOURS OF CORONARY OCCLUSION IS DIMINISHED AFTER 48 HOURS* TETSUJI MIURA, DEREK M. YELLON~:, JOHN KINGMA:~, and JAMES M. DOWNEYt Department of Physiology, College of Medicine, University of South Alabama, Mobile, AL 36688, U.S.A.; and :~The Heart Research Unit, The Rayne Institute, St. Thomas' Hospital, London, SE1, U.K. (Received 20May 1987)

Abstract--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 dog's 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 -4- 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. Keywords--Collateral blood flow, Myocardial infarction, Xanthine oxidase, Ischemia

INTRODUCTION

The oxidase form o f this e n z y m e which is present in the ischemic dog heart 3 can directly transfer the excess electrons from the o x i d a t i o n o f h y p o x a n t h i n e to molecular o x y g e n in the tissue p r o d u c i n g the free radical superoxide and h y d r o g e n p e r o x i d e , both o f which are cytotoxic, O x y g e n could a p p e a r in the ischemic tissue either as a result o f collateral perfusion or during reestablishment o f antegrade perfusion (reperfusion). It is b e l i e v e d that in some organs xanthine oxidase is the m a j o r source o f free radical injury. Support for the xanthine o x i d a s e hypothesis in the dog heart is p r o v i d e d by the finding that inhibition o f this e n z y m e by allopurinol reduces infarct size in both the reperfused 3,7 and the p e r m a n e n t l y o c c l u d e d s canine heart. In the latter study infarcts in the treatment group were found to be a p p r o x i m a t e l y half the size o f those in the control dogs. It was c o n c l u d e d that collateral flow, which is r e l a t i v e l y high in this species 9 brought sufficient o x y g e n to m o d e r a t e l y ischemic tissue to produce toxic free radicals throughout the ischemic pc-

M y o c a r d i a l i s c h e m i a in the d o g is b e l i e v e d to be a free radical m e d i a t e d form o f tissue injury. ~,2 A l t h o u g h the source and quantity o f free radicals p r o d u c e d in ischemic m y o c a r d i u m still remain controversial, 3-6 the e n z y m e xanthine o x i d a s e appears to be a m a j o r source o f free radicals in the ischemic dog heart. 3 During ischemia intracellular ATP is d e g r a d e d to adenosine and then to h y p o x a n t h i n e . The latter is substrate for the e n z y m e xanthine o x i d a s e which o x i d i z e s it to urate.

*This paper was presented at a symposium entitled "The Role of Oxygen Free Radicals in Myocardial Ischemia." The symposium took place on March 30, 1987, in Washington, DC, U.S.A., as part of the 71st Annual Meeting of the Federation of American Societies for Experimental Biology (FASEB). The symposium was organized by Dr. Robert A. Kloner, under the auspices of the American Physiological Society (APS). tAddress correspondence to: James M. Downey, Ph.D., MSB 3024, Department of Physiology, College of Medicine, University of South Alabama, Mobile, AL 36688.

25

26

T. MIURA, D. M. YELLOrq,J. KINGMA,and J. M. DOWNEY

riod. Infarcts in that study were measured only 24 h after embolization. The possibility that an agent could significantly limit infarct size even in the absence of a reperfusion intervention would have major clinical implications and thus warranted further examination. The present study was initiated to see if the apparent limitation of infarct size seen 24 h after coronary occlusion persists for longer time periods. Another shortcoming of our previous study was the failure to incorporate collateral flow into the analysis of infarct size which increases the accuracy and reliability of the method. ~°'~ Those measurements are included in the present protocol. MATERIALS AND METHODS

Surgical procedures Fifty-nine dogs of either sex, weighing between 1233 kg, were anesthetized with sodium pentobarbital, 30 mg/kg IV, and penicillin, one million units IM, was injected for infection prophylaxis. Dogs were intubated but were allowed to spontaneously breathe room air. Coronary artery occlusion, risk zone and infarct delineation were performed as previously described. 12 Briefly, a percutaneous catheter was placed in the jugular vein for administration of drug to treatment groups or saline to controls. After injection of 10,000 units of heparin through this route, the carotid artery was exposed via a midline cervical incision and a rigid steel cannula 13was inserted into the carotid artery. The cannula tip was manipulated into the left coronary ostium and a teflon bead, either 2.5 or 3 mm in diameter, was flushed through the cannula and into the coronary artery with 10 ml of saline. The cannula was then withdrawn and repositioned into the left ventricle where 20 million 15 txm microspheres labeled with 141-cerium were injected to mark the zone at risk. The cannula was removed, the cervical wound closed and the dogs were returned to the recovery room. All surgery was performed under sterile conditions.

Experimental groups Dogs were divided into 4 groups: a 24 hour control group (n = 9); a 24-h allopurinol group (n = 16); a 48 hour control group (n = 17); and a 48-h allopurinol group (n = 17). The drug treated groups received allopurinol 600 mg PO 24 hour before surgery and allopurinol, 10 mg/kg IV bolus 15 min prior to coronary occlusion. This was followed by a continuous infusion of allopurinol at 55 mg/kg/24 h until sacrifice. A1lopurinol for intravenous infusion was prepared as 2 mg/ml in normal saline according to the method of Shatney et al.~4 Control groups received an equal v o l -

ume of saline. Ten thousand units of heparin were added to the daily IV infusate in all groups to prevent thrombus formation around the embolus.

Infarct size and risk zone size determination Following either the 24 or 48 hour occlusion period, the animals were reanesthetized, the hearts were removed and weighed and the location of the embolus was noted. Each heart was then frozen and sliced transversely into 4 mm thick sections. The slices were incubated in triphenyl tetrazolium chloride (1% w/v) in phosphate buffer (pH 7.4) at 37°C for 20 min to delineate the necrotic from the normal tissue, t516 The tissue slices were then pressed against a sheet of Xray film (Kodak no screen NS-ZT) and stored at - 20°C for a 4 8 - 7 2 h exposure period. The films were then removed and developed. After autoradiography, the outline of the sections and the tetrazolium negative region were traced. Autoradiograms clearly revealed a perfusion defect which was designated the risk zone. The volumes of the zone at risk and tetrazolium negative tissue were calculated by planimetry and multiplying by the thickness of each slide. We then calculated the percent of the risk zone which was tetrazolium negative which wilt be designated as %I.

Regional blood flow Three transmural samples ( 0 . 5 - 1 . 0 g each) from the risk region and three from the non-ischemic region were taken from each heart. The ischemic and nonischemic samples were cut out from the same slice using the autoradiogram as a guide. To avoid contamination with well perfused normal tissue, samples from the risk area were taken at least 5 mm inside the border of the zone at risk. Each transmural sample was divided into endocardial, midmyocardial, and epicardial thirds of approximately equal size. The samples from each layer were weighed and counted in a gamma counter. Collateral blood flow was expressed as a percentage of the flow of the corresponding normally perfused layer.

Statistics All results were expressed in terms of mean ± one standard error unless otherwise indicated. An unpaired Student's t test was used to compare the hemodynamic data between the drug and the respective control groups. Significance was considered whenever p < 0.05. The regression line between %1 and collateral blood flow in each group was determined by a linear least squares fit. We tested for significant differences

Myocardial infarct size limitation by allopurinol Table 1. Survival Figures for the 4 Groups

Group 48 48 24 24

h h h h

Survived

Early Death

Total

11 13 9 14

6 4 0 1

17 17 9 15"

Control Drug Control Drug

27

The remaining 47 dogs contributed to this study. Blood pressure, heart rate, and double product (systolic blood pressure × heart rate) at the time of coronary occlusion were not significantly different between control and allopurinol treated groups as shown in Table 2.

*One dog was eliminated due to a technical error in the autoradiogram.

The assessment of infarct size The effect of allopurinol on myocardial infarct size was evaluated by its effect on the infarct size-collateral blood flow relationship. As can be seen in Figures 1 and 2 there is a close correlation between the percent of the risk zone which was tetrazolium negative (%1) and the subepicardial collateral flow (CF) in both the 24 and 48 hour control groups. %1 = 86.4 - 0.94 x CF in the 24 h group (r = - . 8 6 , p < 0.01) and %I = 92.1 - 1.05 x CFqn the 48 hour group (r = - . 9 1 , p < 0.01). The relationships between %1 and CF were not significantly different for the two control groups both in slopes and intercepts. The drug groups also showed a significant correlation between %1 and CF (r = - . 7 2 , p < 0.01 in 24 hour group, and r = - . 6 0 , p < 0.05 in 48 h group). The regression lines of the 24 and 48 h drug groups (%1 = 65.12 0.87 x C F a n d %I = 81.00 - 1.13 x C F r e s p e c tively) had significantly smaller intercepts than corresponding control lines (p < 0.01 in 24 h groups and p < 0.05 in 48 h groups). Because the slopes were not significantly different from the control regressions it would appear that allopurinol caused a parallel shift downward in the %I-CF relationship in this group. In the 24 h evaluation group, all but one of the allopurinol experiments fell well below the control regression line for that series, indicating that allopurinol consistently limited the size of the tetrazolium negative region in the first 24 h of ischemia (see Fig. 1). On the other hand, Figure 2 reveals that most of the 48 h experiments fell much closer to the control regression line for that series and only one drug point was markedly displaced from the control line. Co-

in infarct size and thus, tissue salvage, by two methods. First, the difference between the regression lines was analyzed by an analysis of covariance.17 Secondly, an index of salvage was calculated for each animal as follows: The observed %1 for each dog was subtracted from the %1 which would be predicted on the basis of that dog's collateral flow. The predicted %1 was determined from the regression line for the corresponding control group. Drug induced salvage is indicated when this index is greater than zero and extension is indicated when it is less than zero. We used an index based on a subtraction since it was our observation.that drug treatment caused parallel shifts in the collateral flowinfarct size relationship. We tested for significance between the salvage indexes from the groups with an unpaired Student's t test. A salvage index was calculated for both control groups in order to perform the comparison. Although the mean salvage index for the controls will be near zero by definition this was necessary in order to appreciate the variability in the control groups.

RESULTS

Survival of the animals following coronary artery embolization in each group is shown in Table 1. The survival rate was similar between the control and the allopurinol groups in both the 24 h and 48 h studies. In addition to the dogs which died prematurely, one dog in the 24 hour allopurinol group was excluded from the study because of an unsatisfactory autoradiogram.

Table 2. Hemodynamic Data and Infarct Size Data for the 4 Groups Group

SYS BP

H RATE

DBL PROD

END CF%

MID CF%

EPI CF%

INFARCT

RISK

I%

24 h Control SE 24 h Drug SE 48 h Control SE 48 h Drug SE

182 8 175 4 158 6 159 5

171 6 172 6 178 9 170 6

30852 1730 30428 1761 28396 1998 27187 1533

8.0 2.1 7.3 0.8 6.6 1.1 3.7 1.1

16.7 3.6 11.5 1.4 9.5 1.6 6.1 1.4

27.5 6.1 30.8 4.0 20.8 4.5 15.8 2.4

11.3 1.3 5.7 0.7 27.4 5.5 21.8 3.6

17.7 1.2 15.3 0.7 36.9 5.6 33.1 4.1

60.5 5.8 38.3 4.8 70.2 5.2 63.1 4.4

SYS BP = systolic blood pressure (mmHg); H Rate = heart rate (bpm); DBL PROD = double product; END CF% = endocardial collateral blood flow (% normal flow); MID CF% = midmyocardial collateral blood flow; EPI CF% = subepicardial collateral blood flow; INFARCT = the infarct size (cm3); RISK = the risk zone size (cm3); 1% = the percentage of risk zone infarcted.

28

T. MIURA, D. M. YELLOn, J. KINGMA and J. M. DOWr~EY DISCUSSION

1oo

i ~

o7

©

4o

t

~ 2o

0

20

40 60 80 COLLATERAL FLOW (EPI)

100

Fig. 1. A plot of the percentage of the risk zone which infarcted vs. subepicardial collateral flow for the 24 hour occlusion dogs. The line is a least squares fit to the control data (open circles). Note that the allopurinol points (solid circles) are all displaced well below the line indicating that for any level of collateral flow, the treatment hearts consistently had smaller infarcts.

variate analysis revealed that the intercept of the regression line for the 24 h allopurinol group was smaller than that of 48 hour allopurinol group, but this difference was not statistically significant (0.05 < p < . 1). When the one hyper-responding animal in the 48 hour group was excluded, the new regression was virtually unchanged (%1 = 64.0 - 0.85 x CF) but the difference in intercepts between the 24 and 48 h drug groups becomes highly significant (p < 0.01). It is interesting to note that the hyper-responding animal mentioned above failed to exhibit a transmural gradient of flow in the ischemic zone in that the endocardial flow equaled the epicardial flow. In all of the other animals in this series subendocardial flow was approximately I / 5 subepicardial flow. It is our judgment that the unusual collateral flow distribution rather than the drug accounted for the unexpectedly small infarct in that heart and on that basis we have eliminated it from the group. To further appreciate the shifts in the curves we examined a salvage index. In the 24 h allopurinol group, the salvage index averaged 17.9 --- 3.3% and was significantly different from the salvage index calculated for the control group, 0.2 --- 4.6 (p < 0.02). The salvage index fell to 11.1 +-- 3.3% in the 48 hour allopurinol group, which was not significantly different than that in the 24 hour allopurinol group but was significantly smaller than that for the 48 hour control group 0.0 ± 4.6 (p < 0.03). When the one hyperresponding point in the 48 hour drug group was dropped, however, the salvage index fell further to 8.0 ± 2.0 which was significantly smaller than that seen in the 24 hour allopurinol treatment group (p < 0.03). That was also significantly larger than the salvage index for the 48 h controls (p < 0.05).

This study indicates that most of the limitation of infarct size that was consistently afforded by allopurinol 24 h after permanent coronary artery occlusion was lost over the subsequent 24 h. Allopurinol caused an additional 18% of the risk zone to be salvaged when the evaluation was made 24 h after occlusion. The salvage index calculations would indicate that by 48 h only 8% of the risk zone had been spared from infarction because of the drug. There is no proof that the infarct size was stable at 48 h either. It may well be that given enough time all protection would have been lost in all of the hearts. Does allopurinol actually delay the onset of cell death or does it simply alter the staining characteristics of otherwise dead tissue? One explanation of these results is that allopurinol may have spared the myocyte from the ravages of free radical attack early in the ischemic process only to have the tissue succumb to a second phase of cell death later. That second phase of cell death might either be the result of a chronic energy deficit due to the failure of blood flow to return to the preischemic level or it could result from an active phase of injury, perhaps neutrophil attack. 4 Simply restoring blood flow doesn't seem to sustain the protective effect of allopurinol either. Allopurinol significant limited infarct size in dog hearts which were reperfused after a period of ischemia only when infarcts were analyzed in the first few hours following reperfusion. 3,7 When Reimer and Jennings n examined infarct size 96 h after reperfusion, however, no evidence of salvage from allopurinol was found. Although there admittedly are other important differences between the above three ischemia/reperfusion models, an attractive

100. Z

O 80.

Z Z~

60. O N U3

40

E:

•

L,,J

20 0 0

I 20

I 610 40 80 COLLATERAL FLOW (EPI)

100

Fig. 2. A plot of the percentage of the risk zone which infarcted vs. subepicardial collateral flow for the 48 hour occlusion dogs. The line is a least squares fit to the control data (open circles). In contrast to the 24 hour data, only 1 of the allopurinol treatment points (solid circles) is appreciably displaced below the regression. The rest of the points cluster close to the control line indicating that by 48 hours post occlusion little salvage persisted in most of these hearts.

Myocardial infarct size limitation by allopurinol explanation would be that the protection afforded by allopurinol is not sustained in the reperfusion setting either. That would seemingly refute the contention that a late phase of cell death is simply the result of a chronic energy deficit alone. It is known that the loss of tetrazolium staining lags the appearance of ultrastructural indicators of irreversible injury by several hours. 18 Still, it is difficult to envision how allopurinol might only be altering the tetrazolium staining characteristics of the tissue without affecting cellular viability, especially for a full 24 h. Furthermore, allopurinol is seen to preserve the heart's mechanical function following ischemia/reperfusion in both the dog 19 and the rat 2° suggesting again that the early effects of the drug are truly protective. We propose that some active process, perhaps inflammation, occurs some 24 h after the onset of the ischemic process which proceeds to kill cells which were initially salvaged by allopurinol. The present findings in the 24 h group confirm our previous report on the effects of allopurinol on infarct size following a 24 h permanent coronary artery occlusion, s By including collateral flow along with risk zone size in the determination, an extremely accurate prediction of infarct size in the absence of treatment can be made in each animal. In studies where only the infarct to risk zone ratios are obtained, analysis is limited to a simple comparison of the means of the drug and control groups. Information relating to the success or failure of the intervention in any single animal is therefore unavailable with that approach. The salvage index allows salvage in each dog to be assessed. AIlopurinol caused an additional 17% of the risk zone to be salvaged in the 24-h group. While we would have expected an average of 55% of the risk zone to infarct in the 24-h group, only 38% actually did infarct. This represents a 31% limitation of infarct size which is less dramatic than the 69% limitation which we reported in our previous study with allopurinol. 7 The most likely explanation for the difference is that a few dogs with higher collateralization were fortuitously included in that previous study's allopurinol treated group. The difference between that study and the present one illustrates the importance of measuring collateral flow in the dog model. It was necessary to eliminate one case from the 48 h drug group before a significant difference could be obtained between the 24 and 48 h drug groups. The elimination was done on the basis of an unusual distribution of collateral flow. Flow to the subepicardium in that case was 16.8% of that in the well perfused (normal) region. Flow to the subepicardium in that case was 16.7% of normal. Every other case in the study experienced both a steep transmural blood flow gra-

29

dient across the ischemic zone and a much lower subendocardial flow. The next highest subendocardial flow in that set was 4.2% of normal. The corresponding subepicardial flow in that case was 22.7% of normal. Collateral flow to the ischemic zone is the primary determinant of the extent of necrosis in the canine model.l° Infarction begins at the subendocardium and progresses upward until it reaches a level where collateral flow is sufficient to sustain the viability. 21 A tight correlation exists between subepicardial collateral flow and infarct size only because the blood flow gradient is reproducible from animal to animal. Should that gradient be absent due to a peculiar vascular anatomy, then, the correlation would obviously be lost. Table 2 reveals that the risk zones were significantly larger in the 48 hour drug group as opposed to the 24h drug group. Although it has been proposed that risk zone size is a primary determinant of the %1 in a somewhat different canine model,22 we have never seen such a relationship in this model.l° Similarly, there was a large overlap in the risk zone sizes in the two groups. The range for the 48-h group was 7 - 4 2 % of ventricular weight. For the 24 hour group it was 3 . 5 - 1 7 % of ventricular weight. Finally, a multiple linear regression against risk volume and collateral flow against %1 indicated no correlation with risk volume in any of the 4 groups. In non-drug treated dogs experiencing permanent occlusion of a coronary artery the ultimate size of the infarct is reached by 24 h.~° In light of the present findings, however, it would appear that 24 h is too early to evaluate any drug in the permanent occlusion setting with tetrazolium. It is impossible to discern between delay and sustained limitation of infarct size in a 24 h evaluation. These findings raise the question as to whether any drug can appreciably limit infarct size following permanent occlusion. We have recently found that the apparent protection afforded by nifedipine in a 24 h trial 23 is also attenuated by 48 hours. 24 Diltiazem demonstrated a significant limitation of infarct size in a 48 h trial 25 but that limitation was very modest relative to the protection afforded by either nifedipine 23 or verapami126 in 24 hour trials. To our knowledge, the only undisputed demonstration of appreciable salvage in a 48 hour permanent occlusion trial is that of Jugdutt et al. who report limitations in infarct size of about 50% with prostacyclin. 22 Recent evidence suggests that the dog and rat may differ from man as to the role xanthine oxidase plays in ischemia-reperfusion injury in the heart. Man 27 and rabbit 2s have at least a thousandfold less xanthine oxidase in their hearts than dog. Accordingly, allopurinol fails to protect the reperfused rabbit heart 2° even when the infarcts are measured very early after reperfusion. 2s

30

T. MIURA, D. M. YELLON, J. KINGMA and J. M. DOWNEY

It is interesting that the scavenger superoxide dismutase does limit infarct size in the rabbit model 2s so we still believe that free radicals contribute to ischemiareperfusion injury in this model. In light of those recent findings it is likely that xanthine oxidase inhibitors will be inappropriate therapy for myocardial ischemia in man and that allopurinol-induced protection is probably unique to the dog model. Nevertheless, we believe that there is value in characterizing the allopurinol effect in the dog heart since it is still a good model of free radical mediated injury. Furthermore, many organs in the human do contain xanthine oxidase 29 and allopurinol may yet become an important drug in the treatment of non-cardiac ischemia. Acknowledgment--We are grateful to Mr. Steven Marshall for his expert technical assistance.

REFERENCES

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