- Email: [email protected]
Effect of allopurinol and dimethylsulfoxide on long-term survival in rats after cardiorespiratory arrest and resuscitation
Effect of Allopurinol and Dimethylsulfoxide on Long-term Survival in Rats After Cardiorespiratory Arrest and Resuscitation CHARLES
STEPHEN F. BADYLAK, DVM, PhD, MD, F. BABBS, MD, PhD, CONSTANTINA KOUGIAS, KARI BLAH0
The effects of allopurinol and dimethylsulfoxide (DMSO) upon reperfusion injury were tested in separate studies that utilized a rat model of cardiorespiratoty arrest and resuscitation. The rats were subjected to 7 minutes of arrest followed by resuscitation, and then were alternately assigned to either a drug-treated group or a vehicle-treated group (n = 22 for all groups). Drug treatment was given after the return of spontaneous circulation, and survival was monitored for a ten-day period. Study 1 utilized DMSO (50% solution, 1 ml/kg) as the test drug and saline solution as the vehicle. The percentages of surviving rats in the DMSO-treated and vehicle-treated groups were never statistically significantly different. There were 59% (13/22) of the DMSOtreated rats and 63% (14122) of the vehicle-treated rats alive at one hour after resuscitation. Survival rates decreased to 18% (4122) of DMSO-treated rats and 22% (5/22) of vehicle-treated rats on days 3 through 10. Allopurinol (25 mg/kg) was the test drug in study 2, and 0.18-M sodium hydroxide was the vehicle. The survival rate of resuscitated rats was statistically significantly greater at two days in the drug-treated group (68%, 15/22) than in the vehicle-treated group (36%, 8/22) (x2 = 4.46, df = 1, P < 0.05). The difference increased to a maximum of 68% (15122) of the allopurinol-treated group versus 27% (6/22) of the vehicle-treated group at days 7 and 8 (x2 = 7.38, df = 1, P < 0.01) and remained statistically significant at day 10, with 64% (14/22) of allopurinol-treated versus 27% (6/22) of vehicletreated rats alive (x2 = 5.87, df = 1, P < 0.05). Neurological deficit scores were not different between the drug-treated and vehicle-treated groups at day 10 in either study. It is concluded that reperfusion injury is preventable with allopurinol, but not DMSO, when given after successful resuscitation from cardiopulmonary arrest in the rat model. It is also speculated that allopurinol is the more effective drug because it blocks an early step in the initiation of free-radical-mediated injury. (Am J Emerg Med 1986;4:313-318)
Individuals surviving cardiac arrest and cardiopulmonary resuscitation (CPR) are likely to sustain addiFrom the Biomedical Engineering Center, A. A. Potter Building, Purdue University, West Lafayette, Indiana 47907. Manuscript received uary 31, 1986.
November
23, 1985; revision
accepted
Jan-
This study was supported by grant HL29398 from the National Heart, Lung, and Blood Institute, Bethesda, Maryland. Address
reprint
requests
0735-6757186 $00.00
+ .25
MATERIALS Two
to Dr. Badylak.
Key Words: Cardiac arrest, cardiopulmonary resuscitation, peroxides, reperfusion injury, superoxide ion.
tional serious complications, including major organ dysfunction and death. Fewer than half of the patients resuscitated from pre-hospital cardiac arrest survive to leave the hospital. 1-3 Central nervous system damage and hemo-dynamic abnormalities have been found to cause 59% and 31%, respectively, of post-resuscitation, in-hospital deaths.3*4 From 10 to 40% of survivors are reported to suffer permanent functional neurological disability.4 The pathogenesis of morbidity and mortality after resuscitation may be related to known underlying pathology in some patients. However, an increasing body of evidence suggests that many problems that arise after resuscitation are caused by the return of circulation to transiently ischemic tissue (i.e., reperfusion) by a series of destructive cellular reactions that involve oxygen-derived free radicals such as superoxide (0,-s), and the hydroxyl radical (OH.).5-8 These radicals may be responsible for a significant portion of the ensuing tissue damage, which has been called “reperfusion injury.” Allopurinol, an inhibitor of xanthine oxidase, may provide protection against free-radical-mediated reperfusion injury by inhibiting the formation of 0,-w, which is a byproduct of xanthine oxidase activity.8,9 The conversion of 0,-e ions into highly deleterious OH* radicals is thought to be a major chemical mechanism leading to reperfusion injury.5-1’ Dimethylsulfoxide (DMSO) has been described as a potential freeradical scavenger, and has been shown to block the increase in vascular permeability of reperfused cat intestine9 and to have a protective effect following acute bowel ischemia in the rat.i2 These studies were undertaken to test the effect of allopurinol and DMSO on acute and chronic survival and on neurological function when administered after the return of spontaneous circulation in a model of cardiorespiratory arrest and resuscitation.
lipid
AND METHODS
separate
but identically
designed
studies
were
done. The test drug for study 1 was DMSO, and the test drug for study 2 was allopurinol. Each study had separate controls. 313
AMERICAN
TABLE 1.
JOURNAL
Neurological
OF EMERGENCY
Deficit
Function Level of consciousness
Scoring
MEDICINE
n Volume
4, Number
4 n July 1966
for Rats’
Criteria
0 2 4 6
Normal Clouded Delerium Coma
Function
Score Inclined
plane response
Criteria Normal Sluggish Absent w/feeble Absent
Score
attempts
0 2 4 6
0 2 4
Spontaneous locomotor activity (open field)
Normal Reduced Absent
0 4 6
Ataxia
Normal Sluggish Absent
0 2 4
Normal Patrial (2 limbs or less) Severe
0 3 6
Respiration
Normal Sluggish Absent
0 2 4
Rate
Normal Decreased/increased Absent
0 3 6
Cornea1 reflex
Normal Sluggish Absent
0 2 4
Pattern
Response
Normal Sluggish Absent
0 2 4
Normal Labored Gasping Absent
0 2 4 6
Normal Sluggish Absent
0 2 4
Normal Absent
0 4
Normal Sluggish Absent
0 2 4
Normal Diminished Absent
0 2 4
Sniffing and whisker activity
Normal Diminished Absent
0 2 4
Ability
to stand
Normal Diminished Absent
0 2 4
Ability
to drink
Normal Absent
0 2
Ability
to eat
Normal Absent
0 2
Normal Absent
0 2
Reflexes
and motor function Normal Slightly Dilated
Pupil Size
Pupillary reaction to light
Extensor
Response
Response of air
reflex
to touch
to pain
to burst
Muscle tone
Paralysis
Righting
reflex
Normal Slightly Absent
dilated
flaccid
0 2 4
Normal Partial Total
0 2 4
Normal Present but sluggish Absent w/feeble attempts Absent
0 2 4 6
Miscellaneous Defecation
Response
Grooming
to noise
behavior
’ Normal rat = 0; brain death = 100 (modified from the neurological deficit scoring system for dogs of Safar P, Stezoski EM. Amelioration of brain damage after 12 hours’ cardiac arrest in dogs. Arch Neurol 1976;33:91-99).
Experimental Model We used the rat model of cardiorespiratory arrest and resuscitation that was developed in this laboratory by deGaravilla et ~1,‘~ and validated in subsequent work. 14,15Male Wistar rats weighing between 350 and 500 g were the experimental animals for both studies. Following anesthesia with intraperitoneal ketamine (60 mg/kg), institution of electrocardiographic monitoring, tracheostomy, and neuromuscular blockade with succinylcholine (1.5 mg/kg), cardiorespiratory arrest (electromechanical dissociation) was induced in each rat by a percutaneous intracardiac injection of 0.4 to 0.6 ml of a cold 1% KC1 cardioplegic 314
W, Nemoto
solution, followed by digital compression of the thorax. The cardiorespiratory arrest began within 10 seconds of KC1 injection and was maintained for 7 minutes. Resuscitation was accomplished by interposed abdominal compression-cardiopulmonary resuscitation (IAC-CPR)16 and jet ventilation at 70 breaths per minute with room air. Following 1 to 3 minutes of IAC-CPR, the return of a palpable apex heartbeat, and the return to sinus rhythm as documented by the electrocardiogram, the rats were alternately sorted into either a drug-treated group or a vehicle-treated group. The drug-treated rats were given the test drug and the vehicle-treated rats were given the vehicle in which the test drug was dissolved.
BADYLAK
ET AL n ALLOPURINOL
z
Treatment Regimen
4=
All rats were placed on a 37°C heating pad after resuscitation, and 5 ml each of 5% dextrose solution and lactated Ringer’s solution were given by subcutaneous injection to maintain hydration. The ventilation frequency was gradually reduced to 20 per minute during the subsequent 60- 120 minutes and then discontinued when vigorous spontaneous respirations were observed. The rats were extubated and returned to individual
Cardiorespiratory Arrest Resuscitation Then Anesthesia Treatment
/
, 1
Test Neurological Function
,
7 min 3 min
10 Days
t
t
t
lntubation and ECG Monitor
Begin IAC. CPR and Ventilation
Monitor Survival Rate
Time
DMSO -Treated Vehicle-Treated
FIGURE 1. This “time line” illustration shows the sequential steps in the experimental protocol for studies that tested the effect of DMSO and allopurinol on the survival of rats when administered after resuscitation from 7 minutes of cardiorespiratory arrest.
75
0 _
-
ITi _ &
_
cl
50
53
Care and Evaluation Atter Resuscitation
ARREST
100 -
Treatments were given by slow intravenous infusion over a 2-minute period. If animals could not be resuscitated within 3 minutes of initiation of IAC-CPR, they were not entered into the study. Both studies were continued until 22 rats had been entered into each of the drug-treated and vehicle-treated groups.
Study 1. The drug-treated rats were given DMSO (I ml/kg of a 50% solution diluted with physiological saline; Fisher Scientific Co., Springfield. New Jersey) by jugular intravenous infusion. The placebo-treated rats were given the vehicle for DMSO (saline solution) by identical methods. Dilution of DMSO with saline solution prior to infusion was necessary to liberate a majority of the heat of solution, which otherwise produced hemolysis when 100% DMSO was added to whole blood. ,Study 2. The drug-treated rats were given allopurinol (25 mg/kg; Sigma Chemical Co., St Louis, Missouri) by jugular intravenous infusion. The allopurinol was dissolved in equimolar amounts (136 mg/ml) of 1.O N sodium hydroxide and diluted in 0.90% saline solution to a concentration of 25 mg/ml. The placebotreated rats were given the vehicle for allopurinol (0.18 N sodium hydroxide) by identical methods.
AND DMSO AFTER CARDIOPULMONARY
-
25- L
fn
-
OL 60 m”
12 Hrs
TIM1 :
AFTER
RESUSCITATION
(days)
FIGURE 2. The sequential survival incidence of DMSO-treated versus placebo-treated rats for ten days following resuscitation from 7 minutes of cardiorespiratory arrest (n = 22 for each group).
cages following resuscitation and stabilization, and were observed daily. The number of survivors of resuscitation was recorded at one hour, 12 hours, 24 hours, and at daily intervals for ten days. Chi-square statistics were calculated for each study to test the null hypothesis that at each recording time the percentage of rats surviving in the drug-treated and vehicle-treated groups was the same.” Neurological deficits at ten days after resuscitation were evaluated in each rat by applying a modification of Safar’s neurological scoring method for dogs. l8 The specific tests and the corresponding point values are listed in Table 1. A score of 0 represents no detectable neurological deficits, and a score of 100 points represents maximum neurological deficit (brain death). The experimental protocol is illustrated in Figure 1. RESULTS Study I (DMSO). Approximately 72% of rats that experienced arrest were successfully resuscitated and entered into the study. All rats entered into the study regained a spontaneous sinus rhythm and palpable heart beat within 90-150 seconds of beginning IACCPR. The percentage of surviving resuscitated rats in the drug-treated and vehicle-treated groups was not statistically significantly different at any time throughout the study. There were 59% (13122) of the rats alive in the DMSO-treated group versus 63% (14/22) in the vehicle-treated group at one hour after resuscitation. The percentage of surviving animals decreased by day 3 to 18% (4/22) for the DMSO-treated group and 22% (5/22) for the vehicle-treated group, and these percentages remained stable thereafter (Fig. 2). Neurological deficit scores were not different between the DMSO-treated and vehicle-treated groups at ten days after resuscitation. The mean score for 315
AMERICAN
JOURNAL
OF EMERGENCY
MEDICINE
Allopurinol
n Volume
-Treated
Vehicle-Treated
4, Number
n I
ni
4 W July 1986
cardiorespiratory arrest and resuscitation. These experiments were based on the hypothesis that reperfusion injury is caused by a specific sequence of pathological chemical events. These chemical events begin with the generation of O?-*. This might be produced as a byproduct of the action of xanthine oxidase on its substrates (xanthine or hypoxanthine), which accumulate during ischemia.5.19-”
llli-ll Xanthine or Hypoxanthine
0
60 m,n
12 Hrs
TIME
I
2
AFTER
2
4
5
6
RESUSCITATION
7
0
0:.
0,
9
(days)
FIGURE 3. The sequential survival incidence of allopurinoltreated versus placebo-treated rats for ten days following resuscitation from 7 minutes of cardiorespiratory arrest (n = 72 for each group).
survivors in both the DMSO-treated and vehicletreated groups was one out of a possible 100 points, with a range of 0 to 3. Although no formal neurological testing was done prior to ten days, many rats that later died showed ataxia, lethargy, poor grooming behavior, and convulsive disorders 24-48 hours before death. Study 2 (allopnrinol). Approximately 70% of rats that experienced arrest were successfully resuscitated and entered into the study. As in study I 1 all rats entered into the study regained a spontaneous sinus rhythm and palpable heart beat within 90- 150 seconds of beginning IAC-CPR. The percentage of surviving, resuscitated rats was statistically significantly greater at two days in the allopurinol-treated group (68%, 15/22) than in the vehicle-treated group (36%, 8/22; x’ = 4.46, df = 1, P < 0.05) (Fig. 3). The difference in survival increased slightly during the subsequent days to a maximum of 68% (lY22) for the allopurinol-treated group versus 27% (6/22) for the vehicle-treated group at days 7 and 8 (x2 = 7.38, df = 1, P < 0.01). This difference in survival remained statistically significant at day 10 of the study, in that 64% (14122) were alive in the allopurinol-treated group versus 27% (6/22) in the vehicletreated group (x2 = 5.87, df = 1, P < 0.05’). Neurological deficit scores were not different between the allopurinol-treated and vehicle-treated groups at ten days after resuscitation. The mean score for survivors in both the allopurinol-treated and vehicle-treated groups was 1 out of a possible 100 points, with a range of 0 to 2. DISCUSSION The present studies indicate that allopurinol, but not DMSO, enhances survival when given after the return of spontaneous circulation in a rat model of 316
xanthine oxidase
It might also be produced by the action of NAD(P)Hcytochrome-c reductase on reducing equivalents, which also accumulate during ischemia.iO*” 20, + NADPH -+ NADP+
+ H+ + 20,-m
The sudden flush of O2 during reperfusion drives these reactions to generate a burst of 02-* and other free radicals that exceeds the capacity of physiological defense systems, such as superoxide dismutase. catalase, and glutathione peroxidase, to remove them. The activity of these defense enzymes has been shown to be reduced during hypoxia.24-26 The second chemical step involves conversion of the relatively stable 02-* to highly reactive and deleterious OH* radicals by the superoxide-driven. iron catalyzed Haber-Weiss reaction:iO+*’ 02-. + Fe++++02 + Fe++ (k = 3 x lo9 l/mole-set [est]) 20,-e + 2H+ + H20z + 0, (k = 3 x IO5 l/mole-set) H+ + Fe++ + H,O,+ HO* + Fe+++ (k = 76 l/mole-set)
+ H,O
for which k denotes estimated or measured rate constants for the reactions in rdr.o.28,29 The final chemical reactions in the proposed pathogenesis of oxidative reperfusion injury involve the attack of cellular lipids and proteins by the highly reactive OH* radicals with resulting damage to mitochondrial, lysosomal. nuclear, and plasma membranes. Each of the ensuing peroxidative reactions can not only cause one-for-one alterations of cellular macromolecules, but also create secondary radicals capable of propagating damaging chain reactions.8-” Several available and relatively non-toxic drugs provide possible pharmacologic approaches to the prevention of reperfusion injury by this mechanism. Prevention of 02-* formation might be accomplished by administration of a xanthine oxidase inhibitor such as allopurinol. The present study provides supporting evidence that this intervention is effective. Moreover. the increased probability of long-term survival in animals treated with allopurinol in study 2. compared
BADYLAK
ET AL n ALLOPURINOL
with the control animals, confirms that some form of preventable reperfusion injury does indeed exist. The lack of a protective effect in the vehicle-treated control group by the solvent for allopurinol, NaOH, minimizes the possibility of any contribution the injected base (either NaOH or the allopurinol sodium salt) may have had on the survival of these animals. A second approach to prevention of reperfusion injury involves blockade of the iron-catalyzed HaberWeiss reaction by chelation of free iron with a drug such as deferoxamine. This approach may be especially important in view of the four-fold elevation of non-protein bound, free iron in the brain after ischemia and reperfusion recently reported by Krause and co-workers.30 Iron chelation has been tested in our laboratory in the same rat model of circulatory arrest and CPR as was used for the present study. There was a statistically significant improvement in long-term survival, from 36 to 64% without neurological deficit, which is similar to the effect of allopurinol in study 2.r4 In addition, Myers and her colleagues have recently shown that CPK release from isolated, perfused rabbit hearts subjected to hypoxia and re-oxygenation is reduced 50 to 70% by treatment of the hearts with deferoxamine.31 A third strategy for the pharmacological prevention of reperfusion injury involves trapping OH. radicals with any of a number of putative “hydroxyl radical trapping” compounds such as mannitol, cysteine, and DMSO. We selected DMSO because its combined lipid and water solubility properties and rapid distribution to peripheral tissues3* offered hope that it would penetrate the blood-brain barrier quickly and efficiently. Dimethylsulfoxide has been shown to provide protection against intestinal reperfusion injury in cats that were pre-treated with the drug,9 and in rats following 30 minutes of bowel ischemia.12 Of the various pharmacological interventions just described, study 1 showed DMSO to be the least effective for protection against reperfusion injury. Upon considering the extreme reactivity of OH* radicals, it is not surprising that the relatively low concentrations of a free radical trapping agent like DMSO, which can be safely given as a therapeutic intervention, would be ineffective in protecting against free radical mediated injury. Rate constants for abstraction reactions involving HO* radicals, HO. + RH + R. + HOH range from lo* to 1Oro I/mole-sec.29 These values approach the rate constant for the collision of these radicals with other molecules (10” I/mole-sec).33 This extreme reactivity means that effective trapping of HO* radicals by DMSO would require that the probability of a radical encountering a DMSO molecule rather than a biological target molecule be relatively high. In
AND DMSO AFTER CARDIOPULMONARY
ARREST
other words, even if one assumes that every collision of a DMSO molecule and a OH- radical traps the radical permanently, extremely high concentrations of DMSO would be required to effectively prevent OH* radical interaction with cell macromolecules. Actually. although DMSO does react readily with HO. radicals, (k = 7 x lo9 l/mole-sec),34 the product of this reaction is the still highly reactive methyl radical, CH3*.35*36which can proceed to abstract hydrogen atoms from unsaturated fatty acids and other biological molecules, producing methane gas and initiating harmful radical chain reactions.37 Alternatively, the methyl radicals can react with oxygen in physiological concentrations to produce methylperoxy radicals (CH,OO* ), which can either continue radical chain oxidation of unsaturated fatty acids33 or react with each other to produce formaldehyde and methanol by a Russel reaction mechanism (2 CH,O@ + CH,O + CH,OH + 02).38 Thus, although DMSO may act somewhat selectively to “trap” hydroxyl radicals, the products of the trapping reaction are by no means biologically harmless, and in retrospect we should not have expected DMSO to provide effective protection against HO. attack in ho. We conclude, therefore, that effective strategies for drug therapy to prevent reperfusion injury must block the sequence of radical formation before deleterious OH. radicals are created. Both deferoxamine and allopurinol show this desired effect in a whole animal model of circulatory arrest and reperfusion, when administered at the beginning of the reperfusion phase. The beneficial effects of these drugs as treatments for reperfusion injury may be further enhanced if combined with sustained intensive care during the chronic recovery period.
REFERENCES 1. DeBard ML. Cardiopulmonary resuscitation: Analysis of six years’ experience and review of the literature. Ann Emerg Med 1981;10:408-416. 2. Eisenberg MS, Bergner L, Hallstrom A. Cardiac resuscitation in the community: Importance of rapid provision and implications for program planning. JAMA 1979;241:1905-1907. 3. Myerburg RJ, Conde CA, Sung RJ, et al. Clinical, electrophysiologic, and hemodynamic profile of patients resuscitated from pre-hospital cardiac arrest. Am J Med 1980;68:568-576. 4. Liberthson R, Nagel E, Hirschmann J. Pre-hospital ventricular fibrillation: Prognosis and follow-up course. N Engl J Med 1974;291:317-321. 5. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159-163. 6. Meerson FZ, Kagan VE, Kozlov YP, et al. The role of lipid peroxidation in the pathogenesis of ischemic damage and the antioxidant protection of the heart. Basic Res Cardiol 1982;77:465-485. 7. Granger DN, Rutili G, McCord JM. Superoxide radicals in 317
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8.
9.
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14.
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16.
17.
18.
19.
20.
21.
22.
318
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OF EMERGENCY
MEDICINE
m Volume
feline intestinal ischemia. Gastroenterology 1981;81:2229. Parks DA, Bulkley GB, Granger DN, et al. lschemic injury in the cat small intestine: Role of superoxide radicals. Gastroenterology 1982;82:9-15. Parks DA, Bulkley GB, Granger DN. Role of oxygen-derived free radicals in digestive tract diseases. Surgery 1983;94:415-422. Aust SD, Svingen BA. The role of iron in enzymatic lipid peroxidation. In Free Radicals in Biology. New York: Academic Press, 1982. Morehouse LA, Thomas CE, Aust SD. Superoxide generation by NADPH-cytochrome P-450 reductase, the effect of iron chelators, and the role of superoxide in microsomal lipid peroxidation. Arch Biochem Biophys 1984;232:366377. Demetriou AA, Kagoma PK, Kaiser S, et al. Effect of dimethyl sulfoxide and glycerol on acute bowel ischemia in the rat. Am J Surg 1985;149:91-94. deGaravilla L, Babbs CF, Tacker WA. An experimental circulatory arrest model in the rat to evaluate calcium antagonists in cerebral resuscitation. Am J Emerg Med 1984;2:321-326. Kompala SD, Babbs CF. Effect of deferoxamine on late deaths following CPR in rats. Ann Emerg Med 1986 (in press). Badylak SF, Babbs CF. Effects of carbon dioxide, lidoflazine, and deferoxamine administered after cardiorespiratory arrest and CPR in rats. Ann Emerg Med 1986 (in press). Voorhees WD, Ralston SH, Babbs CF. Regional blood flow during cardiopulmonary resuscitation with abdominal counterpulsation in dogs. Am J Emerg Med 1984;2:123128. Dunn OJ (ed). Basic Statistics: A Primer for the Biomedical Sciences. New York: John Wiley and Sons, 1977:122135. Safar P, Stezoski W, Nemoto EM: Amelioration of brain damage after 12 minutes, cardiac arrest in dogs. Arch Neurol 1976;33:91-99. Roy RS, McCord JM. Superoxide and ischemia: Conversion of xanthine dehydrogenase to xanthine oxidase. In Greenwald R, Cohen G (eds). Oxyradicals and Their Scavenger Systems, Vol. 2: Cellular and Molecular Aspects. New York: Elsevier Science, 1983:145-153. DeWall RA, Vasko KA, Stanley EL, et al. Responses of the ischemic myocardium to allopurinol. Am Heart J 1971;82:362-370. Jones DE, Crowell JW, Smith EE. Significance of increased blood uric acid following extensive hemorrhage. Am J Physiol 1968;214:1374-1377. Sackler ML. Xanthine oxidase from liver and duodenum of the rat: Histochemical localization and electrophoretic heterogeneity. J Histochem Cytochem 1966;14:326-333.
4, Number
4 n July 1986
23. Betz AL. Identification of hypoxanthine transport and xanthine oxidase activity in brain capillaries. J Neurothem 1985;44:574-579. 24. Guarnieri C, Flamigni F, Caldarera CM. Role of oxygen in the cellular damage induced by re-oxygenation of hypoxic heart. J Mol Cell Cardiol 1980;12:797-808. 25. Guarnieri C, Flamigni F, Rossoni-Caldarera C. Glutathione peroxidase activity and release of glutathione from oxygen-deficient perfused rat heart. Biochem Biophys Res Commun 1979;89:678-684. 26. Hearse DJ, Humphrey SM. Chain FB. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: A study of myocardial enzyme release. J Mol Cell Cardiol 1973;5:395-407. 27. Crichton RR. Interactions between iron metabolism and oxygen activation. Excerpta Medica 1979:57-76. 28. Allen AO, Bielski BHJ. Formation and disappearance of superoxide radicals in aqueous solutions. In Oberley LW (ed). Superoxide Dismutase, Vol. 1. Boca Raton, Florida: CRC Press, Inc., 1982:125-141. 29. Walling C. Fenton’s reagent revisited. Act Chem Res 1975;8:125-131. 30. Krause GS, Joyce KM, Nayini NR, et al. Cardiac arrest and resuscitation: Brain iron delocalization during reperfusion. Ann Emerg Med 1985;11:1037-1043. 31. Myers CL, Weiss SJ, Kirsh MM, et al. Involvement of hydrogen peroxide and hydroxyl radical in the oxygen paradox: Reduction of creatinine kinase dismutase. J Mol Cell Cardiol 1985;17:675-684. 32. Jacob SW. Pharmacology of DMSO. In Dimethyl Sulfoxide, Vol. 1. New York: Marcel Dekker, 1971:99-l 12. 33. Semenov NN. In Some Problems of Chemical Kinetics and Reactivity, Vol. 1. New York: Permagon Press, 1958:3. 34. Dorfman LM, Adams GE. Reactivity of the hydroxyl radical in aqueous solutions. US National Bureau of Standards, National Standard Reference Data Series 46 (NSRDSNBS46). Washington, DC: U.S. Government Printing Office, 1973. 35. Dixon WT, Norman ROC, Bulev AL. Electron spin resonance studies of oxidation: Part II. Aliphatic acids and substituted acids. J Am Chem Sot 1964:3625-3634. 36. Lagercrantz C, Forshult S. Trapping of short-lived free radicals as nitroxide radicals detectable by ESR spectroscopy: The radicals formed in the reaction between OHradicals and some sulphoxides and sulphones. Acta Chem Stand 1969;23:81 l-817. 37. Klein SM, Cohen G, Cederbaum Al. Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical generating systems. J Biochem 1981;20:6006-6012. 38. Russell GA. Deuterium-isotope effects in the autoxidation aralkyl hydrocarbons. Mechanism of the interaction peroxy radicals. J Am Chem Sot 1957;79:13711377.
of of
STEPHEN F. BADYLAK, DVM, PhD, MD, F. BABBS, MD, PhD, CONSTANTINA KOUGIAS, KARI BLAH0
The effects of allopurinol and dimethylsulfoxide (DMSO) upon reperfusion injury were tested in separate studies that utilized a rat model of cardiorespiratoty arrest and resuscitation. The rats were subjected to 7 minutes of arrest followed by resuscitation, and then were alternately assigned to either a drug-treated group or a vehicle-treated group (n = 22 for all groups). Drug treatment was given after the return of spontaneous circulation, and survival was monitored for a ten-day period. Study 1 utilized DMSO (50% solution, 1 ml/kg) as the test drug and saline solution as the vehicle. The percentages of surviving rats in the DMSO-treated and vehicle-treated groups were never statistically significantly different. There were 59% (13/22) of the DMSOtreated rats and 63% (14122) of the vehicle-treated rats alive at one hour after resuscitation. Survival rates decreased to 18% (4122) of DMSO-treated rats and 22% (5/22) of vehicle-treated rats on days 3 through 10. Allopurinol (25 mg/kg) was the test drug in study 2, and 0.18-M sodium hydroxide was the vehicle. The survival rate of resuscitated rats was statistically significantly greater at two days in the drug-treated group (68%, 15/22) than in the vehicle-treated group (36%, 8/22) (x2 = 4.46, df = 1, P < 0.05). The difference increased to a maximum of 68% (15122) of the allopurinol-treated group versus 27% (6/22) of the vehicle-treated group at days 7 and 8 (x2 = 7.38, df = 1, P < 0.01) and remained statistically significant at day 10, with 64% (14/22) of allopurinol-treated versus 27% (6/22) of vehicletreated rats alive (x2 = 5.87, df = 1, P < 0.05). Neurological deficit scores were not different between the drug-treated and vehicle-treated groups at day 10 in either study. It is concluded that reperfusion injury is preventable with allopurinol, but not DMSO, when given after successful resuscitation from cardiopulmonary arrest in the rat model. It is also speculated that allopurinol is the more effective drug because it blocks an early step in the initiation of free-radical-mediated injury. (Am J Emerg Med 1986;4:313-318)
Individuals surviving cardiac arrest and cardiopulmonary resuscitation (CPR) are likely to sustain addiFrom the Biomedical Engineering Center, A. A. Potter Building, Purdue University, West Lafayette, Indiana 47907. Manuscript received uary 31, 1986.
November
23, 1985; revision
accepted
Jan-
This study was supported by grant HL29398 from the National Heart, Lung, and Blood Institute, Bethesda, Maryland. Address
reprint
requests
0735-6757186 $00.00
+ .25
MATERIALS Two
to Dr. Badylak.
Key Words: Cardiac arrest, cardiopulmonary resuscitation, peroxides, reperfusion injury, superoxide ion.
tional serious complications, including major organ dysfunction and death. Fewer than half of the patients resuscitated from pre-hospital cardiac arrest survive to leave the hospital. 1-3 Central nervous system damage and hemo-dynamic abnormalities have been found to cause 59% and 31%, respectively, of post-resuscitation, in-hospital deaths.3*4 From 10 to 40% of survivors are reported to suffer permanent functional neurological disability.4 The pathogenesis of morbidity and mortality after resuscitation may be related to known underlying pathology in some patients. However, an increasing body of evidence suggests that many problems that arise after resuscitation are caused by the return of circulation to transiently ischemic tissue (i.e., reperfusion) by a series of destructive cellular reactions that involve oxygen-derived free radicals such as superoxide (0,-s), and the hydroxyl radical (OH.).5-8 These radicals may be responsible for a significant portion of the ensuing tissue damage, which has been called “reperfusion injury.” Allopurinol, an inhibitor of xanthine oxidase, may provide protection against free-radical-mediated reperfusion injury by inhibiting the formation of 0,-w, which is a byproduct of xanthine oxidase activity.8,9 The conversion of 0,-e ions into highly deleterious OH* radicals is thought to be a major chemical mechanism leading to reperfusion injury.5-1’ Dimethylsulfoxide (DMSO) has been described as a potential freeradical scavenger, and has been shown to block the increase in vascular permeability of reperfused cat intestine9 and to have a protective effect following acute bowel ischemia in the rat.i2 These studies were undertaken to test the effect of allopurinol and DMSO on acute and chronic survival and on neurological function when administered after the return of spontaneous circulation in a model of cardiorespiratory arrest and resuscitation.
lipid
AND METHODS
separate
but identically
designed
studies
were
done. The test drug for study 1 was DMSO, and the test drug for study 2 was allopurinol. Each study had separate controls. 313
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TABLE 1.
JOURNAL
Neurological
OF EMERGENCY
Deficit
Function Level of consciousness
Scoring
MEDICINE
n Volume
4, Number
4 n July 1966
for Rats’
Criteria
0 2 4 6
Normal Clouded Delerium Coma
Function
Score Inclined
plane response
Criteria Normal Sluggish Absent w/feeble Absent
Score
attempts
0 2 4 6
0 2 4
Spontaneous locomotor activity (open field)
Normal Reduced Absent
0 4 6
Ataxia
Normal Sluggish Absent
0 2 4
Normal Patrial (2 limbs or less) Severe
0 3 6
Respiration
Normal Sluggish Absent
0 2 4
Rate
Normal Decreased/increased Absent
0 3 6
Cornea1 reflex
Normal Sluggish Absent
0 2 4
Pattern
Response
Normal Sluggish Absent
0 2 4
Normal Labored Gasping Absent
0 2 4 6
Normal Sluggish Absent
0 2 4
Normal Absent
0 4
Normal Sluggish Absent
0 2 4
Normal Diminished Absent
0 2 4
Sniffing and whisker activity
Normal Diminished Absent
0 2 4
Ability
to stand
Normal Diminished Absent
0 2 4
Ability
to drink
Normal Absent
0 2
Ability
to eat
Normal Absent
0 2
Normal Absent
0 2
Reflexes
and motor function Normal Slightly Dilated
Pupil Size
Pupillary reaction to light
Extensor
Response
Response of air
reflex
to touch
to pain
to burst
Muscle tone
Paralysis
Righting
reflex
Normal Slightly Absent
dilated
flaccid
0 2 4
Normal Partial Total
0 2 4
Normal Present but sluggish Absent w/feeble attempts Absent
0 2 4 6
Miscellaneous Defecation
Response
Grooming
to noise
behavior
’ Normal rat = 0; brain death = 100 (modified from the neurological deficit scoring system for dogs of Safar P, Stezoski EM. Amelioration of brain damage after 12 hours’ cardiac arrest in dogs. Arch Neurol 1976;33:91-99).
Experimental Model We used the rat model of cardiorespiratory arrest and resuscitation that was developed in this laboratory by deGaravilla et ~1,‘~ and validated in subsequent work. 14,15Male Wistar rats weighing between 350 and 500 g were the experimental animals for both studies. Following anesthesia with intraperitoneal ketamine (60 mg/kg), institution of electrocardiographic monitoring, tracheostomy, and neuromuscular blockade with succinylcholine (1.5 mg/kg), cardiorespiratory arrest (electromechanical dissociation) was induced in each rat by a percutaneous intracardiac injection of 0.4 to 0.6 ml of a cold 1% KC1 cardioplegic 314
W, Nemoto
solution, followed by digital compression of the thorax. The cardiorespiratory arrest began within 10 seconds of KC1 injection and was maintained for 7 minutes. Resuscitation was accomplished by interposed abdominal compression-cardiopulmonary resuscitation (IAC-CPR)16 and jet ventilation at 70 breaths per minute with room air. Following 1 to 3 minutes of IAC-CPR, the return of a palpable apex heartbeat, and the return to sinus rhythm as documented by the electrocardiogram, the rats were alternately sorted into either a drug-treated group or a vehicle-treated group. The drug-treated rats were given the test drug and the vehicle-treated rats were given the vehicle in which the test drug was dissolved.
BADYLAK
ET AL n ALLOPURINOL
z
Treatment Regimen
4=
All rats were placed on a 37°C heating pad after resuscitation, and 5 ml each of 5% dextrose solution and lactated Ringer’s solution were given by subcutaneous injection to maintain hydration. The ventilation frequency was gradually reduced to 20 per minute during the subsequent 60- 120 minutes and then discontinued when vigorous spontaneous respirations were observed. The rats were extubated and returned to individual
Cardiorespiratory Arrest Resuscitation Then Anesthesia Treatment
/
, 1
Test Neurological Function
,
7 min 3 min
10 Days
t
t
t
lntubation and ECG Monitor
Begin IAC. CPR and Ventilation
Monitor Survival Rate
Time
DMSO -Treated Vehicle-Treated
FIGURE 1. This “time line” illustration shows the sequential steps in the experimental protocol for studies that tested the effect of DMSO and allopurinol on the survival of rats when administered after resuscitation from 7 minutes of cardiorespiratory arrest.
75
0 _
-
ITi _ &
_
cl
50
53
Care and Evaluation Atter Resuscitation
ARREST
100 -
Treatments were given by slow intravenous infusion over a 2-minute period. If animals could not be resuscitated within 3 minutes of initiation of IAC-CPR, they were not entered into the study. Both studies were continued until 22 rats had been entered into each of the drug-treated and vehicle-treated groups.
Study 1. The drug-treated rats were given DMSO (I ml/kg of a 50% solution diluted with physiological saline; Fisher Scientific Co., Springfield. New Jersey) by jugular intravenous infusion. The placebo-treated rats were given the vehicle for DMSO (saline solution) by identical methods. Dilution of DMSO with saline solution prior to infusion was necessary to liberate a majority of the heat of solution, which otherwise produced hemolysis when 100% DMSO was added to whole blood. ,Study 2. The drug-treated rats were given allopurinol (25 mg/kg; Sigma Chemical Co., St Louis, Missouri) by jugular intravenous infusion. The allopurinol was dissolved in equimolar amounts (136 mg/ml) of 1.O N sodium hydroxide and diluted in 0.90% saline solution to a concentration of 25 mg/ml. The placebotreated rats were given the vehicle for allopurinol (0.18 N sodium hydroxide) by identical methods.
AND DMSO AFTER CARDIOPULMONARY
-
25- L
fn
-
OL 60 m”
12 Hrs
TIM1 :
AFTER
RESUSCITATION
(days)
FIGURE 2. The sequential survival incidence of DMSO-treated versus placebo-treated rats for ten days following resuscitation from 7 minutes of cardiorespiratory arrest (n = 22 for each group).
cages following resuscitation and stabilization, and were observed daily. The number of survivors of resuscitation was recorded at one hour, 12 hours, 24 hours, and at daily intervals for ten days. Chi-square statistics were calculated for each study to test the null hypothesis that at each recording time the percentage of rats surviving in the drug-treated and vehicle-treated groups was the same.” Neurological deficits at ten days after resuscitation were evaluated in each rat by applying a modification of Safar’s neurological scoring method for dogs. l8 The specific tests and the corresponding point values are listed in Table 1. A score of 0 represents no detectable neurological deficits, and a score of 100 points represents maximum neurological deficit (brain death). The experimental protocol is illustrated in Figure 1. RESULTS Study I (DMSO). Approximately 72% of rats that experienced arrest were successfully resuscitated and entered into the study. All rats entered into the study regained a spontaneous sinus rhythm and palpable heart beat within 90-150 seconds of beginning IACCPR. The percentage of surviving resuscitated rats in the drug-treated and vehicle-treated groups was not statistically significantly different at any time throughout the study. There were 59% (13122) of the rats alive in the DMSO-treated group versus 63% (14/22) in the vehicle-treated group at one hour after resuscitation. The percentage of surviving animals decreased by day 3 to 18% (4/22) for the DMSO-treated group and 22% (5/22) for the vehicle-treated group, and these percentages remained stable thereafter (Fig. 2). Neurological deficit scores were not different between the DMSO-treated and vehicle-treated groups at ten days after resuscitation. The mean score for 315
AMERICAN
JOURNAL
OF EMERGENCY
MEDICINE
Allopurinol
n Volume
-Treated
Vehicle-Treated
4, Number
n I
ni
4 W July 1986
cardiorespiratory arrest and resuscitation. These experiments were based on the hypothesis that reperfusion injury is caused by a specific sequence of pathological chemical events. These chemical events begin with the generation of O?-*. This might be produced as a byproduct of the action of xanthine oxidase on its substrates (xanthine or hypoxanthine), which accumulate during ischemia.5.19-”
llli-ll Xanthine or Hypoxanthine
0
60 m,n
12 Hrs
TIME
I
2
AFTER
2
4
5
6
RESUSCITATION
7
0
0:.
0,
9
(days)
FIGURE 3. The sequential survival incidence of allopurinoltreated versus placebo-treated rats for ten days following resuscitation from 7 minutes of cardiorespiratory arrest (n = 72 for each group).
survivors in both the DMSO-treated and vehicletreated groups was one out of a possible 100 points, with a range of 0 to 3. Although no formal neurological testing was done prior to ten days, many rats that later died showed ataxia, lethargy, poor grooming behavior, and convulsive disorders 24-48 hours before death. Study 2 (allopnrinol). Approximately 70% of rats that experienced arrest were successfully resuscitated and entered into the study. As in study I 1 all rats entered into the study regained a spontaneous sinus rhythm and palpable heart beat within 90- 150 seconds of beginning IAC-CPR. The percentage of surviving, resuscitated rats was statistically significantly greater at two days in the allopurinol-treated group (68%, 15/22) than in the vehicle-treated group (36%, 8/22; x’ = 4.46, df = 1, P < 0.05) (Fig. 3). The difference in survival increased slightly during the subsequent days to a maximum of 68% (lY22) for the allopurinol-treated group versus 27% (6/22) for the vehicle-treated group at days 7 and 8 (x2 = 7.38, df = 1, P < 0.01). This difference in survival remained statistically significant at day 10 of the study, in that 64% (14122) were alive in the allopurinol-treated group versus 27% (6/22) in the vehicletreated group (x2 = 5.87, df = 1, P < 0.05’). Neurological deficit scores were not different between the allopurinol-treated and vehicle-treated groups at ten days after resuscitation. The mean score for survivors in both the allopurinol-treated and vehicle-treated groups was 1 out of a possible 100 points, with a range of 0 to 2. DISCUSSION The present studies indicate that allopurinol, but not DMSO, enhances survival when given after the return of spontaneous circulation in a rat model of 316
xanthine oxidase
It might also be produced by the action of NAD(P)Hcytochrome-c reductase on reducing equivalents, which also accumulate during ischemia.iO*” 20, + NADPH -+ NADP+
+ H+ + 20,-m
The sudden flush of O2 during reperfusion drives these reactions to generate a burst of 02-* and other free radicals that exceeds the capacity of physiological defense systems, such as superoxide dismutase. catalase, and glutathione peroxidase, to remove them. The activity of these defense enzymes has been shown to be reduced during hypoxia.24-26 The second chemical step involves conversion of the relatively stable 02-* to highly reactive and deleterious OH* radicals by the superoxide-driven. iron catalyzed Haber-Weiss reaction:iO+*’ 02-. + Fe++++02 + Fe++ (k = 3 x lo9 l/mole-set [est]) 20,-e + 2H+ + H20z + 0, (k = 3 x IO5 l/mole-set) H+ + Fe++ + H,O,+ HO* + Fe+++ (k = 76 l/mole-set)
+ H,O
for which k denotes estimated or measured rate constants for the reactions in rdr.o.28,29 The final chemical reactions in the proposed pathogenesis of oxidative reperfusion injury involve the attack of cellular lipids and proteins by the highly reactive OH* radicals with resulting damage to mitochondrial, lysosomal. nuclear, and plasma membranes. Each of the ensuing peroxidative reactions can not only cause one-for-one alterations of cellular macromolecules, but also create secondary radicals capable of propagating damaging chain reactions.8-” Several available and relatively non-toxic drugs provide possible pharmacologic approaches to the prevention of reperfusion injury by this mechanism. Prevention of 02-* formation might be accomplished by administration of a xanthine oxidase inhibitor such as allopurinol. The present study provides supporting evidence that this intervention is effective. Moreover. the increased probability of long-term survival in animals treated with allopurinol in study 2. compared
BADYLAK
ET AL n ALLOPURINOL
with the control animals, confirms that some form of preventable reperfusion injury does indeed exist. The lack of a protective effect in the vehicle-treated control group by the solvent for allopurinol, NaOH, minimizes the possibility of any contribution the injected base (either NaOH or the allopurinol sodium salt) may have had on the survival of these animals. A second approach to prevention of reperfusion injury involves blockade of the iron-catalyzed HaberWeiss reaction by chelation of free iron with a drug such as deferoxamine. This approach may be especially important in view of the four-fold elevation of non-protein bound, free iron in the brain after ischemia and reperfusion recently reported by Krause and co-workers.30 Iron chelation has been tested in our laboratory in the same rat model of circulatory arrest and CPR as was used for the present study. There was a statistically significant improvement in long-term survival, from 36 to 64% without neurological deficit, which is similar to the effect of allopurinol in study 2.r4 In addition, Myers and her colleagues have recently shown that CPK release from isolated, perfused rabbit hearts subjected to hypoxia and re-oxygenation is reduced 50 to 70% by treatment of the hearts with deferoxamine.31 A third strategy for the pharmacological prevention of reperfusion injury involves trapping OH. radicals with any of a number of putative “hydroxyl radical trapping” compounds such as mannitol, cysteine, and DMSO. We selected DMSO because its combined lipid and water solubility properties and rapid distribution to peripheral tissues3* offered hope that it would penetrate the blood-brain barrier quickly and efficiently. Dimethylsulfoxide has been shown to provide protection against intestinal reperfusion injury in cats that were pre-treated with the drug,9 and in rats following 30 minutes of bowel ischemia.12 Of the various pharmacological interventions just described, study 1 showed DMSO to be the least effective for protection against reperfusion injury. Upon considering the extreme reactivity of OH* radicals, it is not surprising that the relatively low concentrations of a free radical trapping agent like DMSO, which can be safely given as a therapeutic intervention, would be ineffective in protecting against free radical mediated injury. Rate constants for abstraction reactions involving HO* radicals, HO. + RH + R. + HOH range from lo* to 1Oro I/mole-sec.29 These values approach the rate constant for the collision of these radicals with other molecules (10” I/mole-sec).33 This extreme reactivity means that effective trapping of HO* radicals by DMSO would require that the probability of a radical encountering a DMSO molecule rather than a biological target molecule be relatively high. In
AND DMSO AFTER CARDIOPULMONARY
ARREST
other words, even if one assumes that every collision of a DMSO molecule and a OH- radical traps the radical permanently, extremely high concentrations of DMSO would be required to effectively prevent OH* radical interaction with cell macromolecules. Actually. although DMSO does react readily with HO. radicals, (k = 7 x lo9 l/mole-sec),34 the product of this reaction is the still highly reactive methyl radical, CH3*.35*36which can proceed to abstract hydrogen atoms from unsaturated fatty acids and other biological molecules, producing methane gas and initiating harmful radical chain reactions.37 Alternatively, the methyl radicals can react with oxygen in physiological concentrations to produce methylperoxy radicals (CH,OO* ), which can either continue radical chain oxidation of unsaturated fatty acids33 or react with each other to produce formaldehyde and methanol by a Russel reaction mechanism (2 CH,O@ + CH,O + CH,OH + 02).38 Thus, although DMSO may act somewhat selectively to “trap” hydroxyl radicals, the products of the trapping reaction are by no means biologically harmless, and in retrospect we should not have expected DMSO to provide effective protection against HO. attack in ho. We conclude, therefore, that effective strategies for drug therapy to prevent reperfusion injury must block the sequence of radical formation before deleterious OH. radicals are created. Both deferoxamine and allopurinol show this desired effect in a whole animal model of circulatory arrest and reperfusion, when administered at the beginning of the reperfusion phase. The beneficial effects of these drugs as treatments for reperfusion injury may be further enhanced if combined with sustained intensive care during the chronic recovery period.
REFERENCES 1. DeBard ML. Cardiopulmonary resuscitation: Analysis of six years’ experience and review of the literature. Ann Emerg Med 1981;10:408-416. 2. Eisenberg MS, Bergner L, Hallstrom A. Cardiac resuscitation in the community: Importance of rapid provision and implications for program planning. JAMA 1979;241:1905-1907. 3. Myerburg RJ, Conde CA, Sung RJ, et al. Clinical, electrophysiologic, and hemodynamic profile of patients resuscitated from pre-hospital cardiac arrest. Am J Med 1980;68:568-576. 4. Liberthson R, Nagel E, Hirschmann J. Pre-hospital ventricular fibrillation: Prognosis and follow-up course. N Engl J Med 1974;291:317-321. 5. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159-163. 6. Meerson FZ, Kagan VE, Kozlov YP, et al. The role of lipid peroxidation in the pathogenesis of ischemic damage and the antioxidant protection of the heart. Basic Res Cardiol 1982;77:465-485. 7. Granger DN, Rutili G, McCord JM. Superoxide radicals in 317
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m Volume
feline intestinal ischemia. Gastroenterology 1981;81:2229. Parks DA, Bulkley GB, Granger DN, et al. lschemic injury in the cat small intestine: Role of superoxide radicals. Gastroenterology 1982;82:9-15. Parks DA, Bulkley GB, Granger DN. Role of oxygen-derived free radicals in digestive tract diseases. Surgery 1983;94:415-422. Aust SD, Svingen BA. The role of iron in enzymatic lipid peroxidation. In Free Radicals in Biology. New York: Academic Press, 1982. Morehouse LA, Thomas CE, Aust SD. Superoxide generation by NADPH-cytochrome P-450 reductase, the effect of iron chelators, and the role of superoxide in microsomal lipid peroxidation. Arch Biochem Biophys 1984;232:366377. Demetriou AA, Kagoma PK, Kaiser S, et al. Effect of dimethyl sulfoxide and glycerol on acute bowel ischemia in the rat. Am J Surg 1985;149:91-94. deGaravilla L, Babbs CF, Tacker WA. An experimental circulatory arrest model in the rat to evaluate calcium antagonists in cerebral resuscitation. Am J Emerg Med 1984;2:321-326. Kompala SD, Babbs CF. Effect of deferoxamine on late deaths following CPR in rats. Ann Emerg Med 1986 (in press). Badylak SF, Babbs CF. Effects of carbon dioxide, lidoflazine, and deferoxamine administered after cardiorespiratory arrest and CPR in rats. Ann Emerg Med 1986 (in press). Voorhees WD, Ralston SH, Babbs CF. Regional blood flow during cardiopulmonary resuscitation with abdominal counterpulsation in dogs. Am J Emerg Med 1984;2:123128. Dunn OJ (ed). Basic Statistics: A Primer for the Biomedical Sciences. New York: John Wiley and Sons, 1977:122135. Safar P, Stezoski W, Nemoto EM: Amelioration of brain damage after 12 minutes, cardiac arrest in dogs. Arch Neurol 1976;33:91-99. Roy RS, McCord JM. Superoxide and ischemia: Conversion of xanthine dehydrogenase to xanthine oxidase. In Greenwald R, Cohen G (eds). Oxyradicals and Their Scavenger Systems, Vol. 2: Cellular and Molecular Aspects. New York: Elsevier Science, 1983:145-153. DeWall RA, Vasko KA, Stanley EL, et al. Responses of the ischemic myocardium to allopurinol. Am Heart J 1971;82:362-370. Jones DE, Crowell JW, Smith EE. Significance of increased blood uric acid following extensive hemorrhage. Am J Physiol 1968;214:1374-1377. Sackler ML. Xanthine oxidase from liver and duodenum of the rat: Histochemical localization and electrophoretic heterogeneity. J Histochem Cytochem 1966;14:326-333.
4, Number
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23. Betz AL. Identification of hypoxanthine transport and xanthine oxidase activity in brain capillaries. J Neurothem 1985;44:574-579. 24. Guarnieri C, Flamigni F, Caldarera CM. Role of oxygen in the cellular damage induced by re-oxygenation of hypoxic heart. J Mol Cell Cardiol 1980;12:797-808. 25. Guarnieri C, Flamigni F, Rossoni-Caldarera C. Glutathione peroxidase activity and release of glutathione from oxygen-deficient perfused rat heart. Biochem Biophys Res Commun 1979;89:678-684. 26. Hearse DJ, Humphrey SM. Chain FB. Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: A study of myocardial enzyme release. J Mol Cell Cardiol 1973;5:395-407. 27. Crichton RR. Interactions between iron metabolism and oxygen activation. Excerpta Medica 1979:57-76. 28. Allen AO, Bielski BHJ. Formation and disappearance of superoxide radicals in aqueous solutions. In Oberley LW (ed). Superoxide Dismutase, Vol. 1. Boca Raton, Florida: CRC Press, Inc., 1982:125-141. 29. Walling C. Fenton’s reagent revisited. Act Chem Res 1975;8:125-131. 30. Krause GS, Joyce KM, Nayini NR, et al. Cardiac arrest and resuscitation: Brain iron delocalization during reperfusion. Ann Emerg Med 1985;11:1037-1043. 31. Myers CL, Weiss SJ, Kirsh MM, et al. Involvement of hydrogen peroxide and hydroxyl radical in the oxygen paradox: Reduction of creatinine kinase dismutase. J Mol Cell Cardiol 1985;17:675-684. 32. Jacob SW. Pharmacology of DMSO. In Dimethyl Sulfoxide, Vol. 1. New York: Marcel Dekker, 1971:99-l 12. 33. Semenov NN. In Some Problems of Chemical Kinetics and Reactivity, Vol. 1. New York: Permagon Press, 1958:3. 34. Dorfman LM, Adams GE. Reactivity of the hydroxyl radical in aqueous solutions. US National Bureau of Standards, National Standard Reference Data Series 46 (NSRDSNBS46). Washington, DC: U.S. Government Printing Office, 1973. 35. Dixon WT, Norman ROC, Bulev AL. Electron spin resonance studies of oxidation: Part II. Aliphatic acids and substituted acids. J Am Chem Sot 1964:3625-3634. 36. Lagercrantz C, Forshult S. Trapping of short-lived free radicals as nitroxide radicals detectable by ESR spectroscopy: The radicals formed in the reaction between OHradicals and some sulphoxides and sulphones. Acta Chem Stand 1969;23:81 l-817. 37. Klein SM, Cohen G, Cederbaum Al. Production of formaldehyde during metabolism of dimethyl sulfoxide by hydroxyl radical generating systems. J Biochem 1981;20:6006-6012. 38. Russell GA. Deuterium-isotope effects in the autoxidation aralkyl hydrocarbons. Mechanism of the interaction peroxy radicals. J Am Chem Sot 1957;79:13711377.
of of