Hypoxic cardiopulmonary-cerebral resuscitation fails to improve neurological outcome following cardiac arrest in dogs

RESUSCITATION

Resuscitation 29(1995)225-236

ELSEVIER

Hypoxic cardiopulmonary-cerebral resuscitation fails to improve neurological outcome following cardiac arrest in dogs Charles F. Zwemer, Steven E. Whitesall, Louis G. D’Alecy* Department

of Physiology,

The University of Michigan Medical Ann Arbor, MI 48109-0622,

School, USA

7799 Medical

Science Building

II,

Received11November1994;accepted 2 January1995

Hyperoxic cardiopulmonaryresuscitation(CPR) is associated with an increasein neurologicdysfunction upon successfulresuscitationwith much of the damageattributable to an increasein reperfusionoxidant injury. We hypothesized that by contrast, hypoxic ventilation during resuscitationwould improve neurologic outcome by reducing availablesubstratenecessaryfor oxidant injury. Specifically,this study investigatedthe effectsof 2 levelsof hypoxic ventilation during resuscitation:Ftor = 0.085, Paoz= 26.6 * 3.4 mmHg, (HY8), and Fro2= 0.12, Paoz: = 33.0 * 4.2 mmHg,(HY 12),and normoxic resuscitation:Ftor = 0.21,Paoz= 60.6 * 17.0mmHg,(N) on survival and neurological outcome following 9 min of normothermic cardiac arrest. Concentrations of malonaldehyde(MDA) and 4hydroxynonenal(4-OH) in plasmaand concentrationsof glutathione(GSH) in erythrocyte lysatesweremeasuredto quantify possibleradical damage.Physiologicalvariablesincluding arterial blood gaseswerefollowed for 24 h after resuscitation.Neurologicoutcomewasassessed usinga standardizedscoringsystem.Hypoxically (HY8) resuscitated dogstendedto havea greaterneurologicdeficit than normoxicallyresuscitated dogsandhad a reducedoverall survival (16.9 f 8.9h) comparedto N dogs(24.0 * 0.0 h). Overall survival timecorrelatednegatively(-0.693) andsignificantly (P = 0.0018)with plasmaglucoseconcentration.Arterial plasmaglucoseconcentrationswerehigherin the HY8 group comparedto the N group immediately(HYS, 312 f 86 mg/dL; N, 196f 82 mg/dL; P= 0.17) and 30 min (HY8, 331f 109mg/dL; N, 187rt 74 mg/dL; P = 0.077)following resuscitation.No statistically discernibledifferencesin markersof oxidant injury wereapparentamongthe 3 groups,but pooleddata increasedsignificantly with time for MDA and 4-GH. Pooleddata for GSH showeda significantdrop at 1 h following resuscitationand returnedto normal by 6 h. Data from thesemarkerssuggested attendantoxidant injury in all groups.Thus, hypoxic ventilation at 2 depths of hypoxia during resuscitationfailed to improve neurologicoutcomebeyond that achievedby ventilation with air, suggesting that normoxiarather than hyperoxia or hypoxia is the idealtarget for arterial oxygenationduring resuscitation. Keyword:

Hypoxia; Normoxia; Global &hernia and reperfusion;Cardiopulmonaryresuscitation

* Corresponding author. 0300-9572/95/$09.05 0 I995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-9572(94)00848-N

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C.F. Zwemer et al. /Resuscitation 29 (1995) 225-236

1. Introduction Oxidant injury plays a significant role in the pathophysiology of ischemic/reperfusion brain injury [l-3]. Briefly, oxidant injury is thought to be the direct result of partially-reduced oxygen species (PROS) formation which, in part, is driven by reperfusion oxygen’s interaction with accumulated ischemic metabolites [l-3]. Arguably, the extent of oxidant damage in this setting may be directly related to the amount of oxygen available for PROS conversion. By delivering an ‘overabundance’ of oxygen to previously ischemic tissue in the immediate reperfusion period, an exacerbation of oxidant injury could occur. A recent study from our laboratory has supported this notion empirically by demonstrating that hyperoxic reperneurologic dysfunction fusion exacerbated following global cerebral ischemia, while normoxia and antioxidant pretreatment, even in the presence of hyperoxia, improved outcome [4]. By reducing reperfusion oxygen delivery to normoxic levels in the immediate reperfusion period, we showed that oxidant injury was curtailed. Further testing the theory of oxygen deliverydependent PROS damage, we proposed in this study to investigate the possible benefits of moderate to severe hypoxic resuscitation. Presumably, by further reducing substrate oxygen to lower levels, a reduction in PROS would result. We specifically hypothesized that hypoxic resuscitation would result in an improved neurologic outcome following 9 min of normothermic arrest. To test this we investigated the effects of providing 15 min of immediate post-resuscitation hypoxia at 2 levels (severe, 8.5% 0, and moderate, 12% 02) and normoxia on outcome and markers of oxidant injury. 2. Materials and methods 2.1. General experimental protocol The 17 dogs used in this study were divided into groups of 5, 5 and 7. Each group underwent precisely the same housing, induction, instrumentation, cardiac arrest, resuscitation and recovery procedures with the specific exceptions of frac-

tional inspired oxygen concentration (FIOZ) during resuscitation and for 15 min following. The groups were: (1) Normoxia: (N) resuscitation with room air Fro* = 0.21, (n = 5), (2) Hypoxia 8.5%: (HYB), resuscitation with F102 = 0.085, (n = 7), and (3) Hypoxia 12%: (HY12) resuscitation with Fro2 = 0.12 (n = 5). To assess expected changes and possible derangements in hemodynamic variables and blood gas transport, arterial blood was sampled at 10 time-points before and after cardiac arrest. Along with these indices, a well-standardized neurological deficit score [4- 1 l] was assigned at 1,2,6, 12, and 24 h post-arrest and resuscitation in all groups. 2.2. Instrumentation Adult male mongrel dogs weighing 15-22 kg were fasted for 24 h. They were pre-medicated with 1.5 mg/kg S.C. morphine sulfate and 20 min later were anesthetized with 5% halothane in oxygen via face mask (Foregger, Compact-75, PuritanBennett Corp., Westmont, IL). Upon reaching Stage III Plane 2 of surgical anesthesia, they were orotracheally intubated and mechanically ventilated (Air Shields Ventimeter Ventilator, Hatboro, PA) with 0.5- 1.O% halothane in oxygen for maintenance of surgical anesthesia and suppression of palpebral reflexes. No paralytic agents were used. End-tidal expired CO* concentration was continuously monitored and maintained between 4 and 5% (Beckman LB-2, Fullerton, GA) and arterial blood pH was measured (IL 1304 Blood Gas Analyzer, Lexington, MA) and maintained between 7.38 and 7.41 by adjustment of ventilation and/or administration of sodium bicarbonate. Deep esophageal temperature was measured and maintained at 39.0 f 1.O”C before and for at least 1 h after resuscitation with a homeothermic blanket system (Model 50-7095, Harvard Apparatus, South Natick, MA) and heating lamps. Urinary output was monitored through use of a urethral catheter and graduated collecting reservoir. During instrumentation, each dog received 500 ml of 0.45% sodium chloride (Abbott laboratories, North Chicago, IL) containing 2.52 g of sodium bicarbonate to assure adequate hydration and nor-

C.F. Zwemer et al. /Resuscitation 29 (1995) 225-236

mal arterial pH before circulatory arrest. Two venous catheters were inserted; one passed through the left external jugular vein to the right atrium for resuscitation drug administration and the other passed into the proximal caudal branch of the left femoral vein for post-operative/recovery fluid administration (0.45% sodium chloride, delivered at 2 m&kg x h) via continuous intravenous drip (IVAC 530, San Diego, CA). Arterial blood pressure was measured directly and continuously from a catheter placed in the proximal caudal branch of the left femoral artery (Statham P23XL transducer, Gould Inc., Oxnard, CA) and Lead II ele$rocardiogram (ECG) was continuously monitored by placement of subcutaneous disk electrodes (Grass Instrument Co. ESSH Silver Cup Electrodes, Quincy, MA). All catheters and electrical leads were passed subcutaneously to exit the skin at a dorsal midscapular incision for subsequent attachment to a dog jacket and hydraulic/electric swivel (Alice Ring Chatham Medical Arts, Los Angeles, CA). A left, fifth, inter-space thoracotomy and pericardiectomy were performed to provide direct access for both the initiation of cardiac arrest and for circumferential cardiac compression during resuscitation. Proper placement of the jugular catheter was established prior to cardiac arrest by direct palpation of the superior vena cava and right atrium. Pulsatile and mean arterial blood pressure (MAP), ECG, and end-expiratory COz tensions were continuously recorded on a 6channel oscillograph (Gould-Brush Model 200, Cleveland, OH).

2.3. Cardiac arrest

At the conclusion of instrumentation, halothane administration was halted while ventilation was continued with room air (Model 607, Hazard Apparatus, South Natick, MA). This was done to reduce and standardize the level of anesthesia at which ventricular fibrillation was induced. Upon return of palpebral reflexes and or limb withdrawal (Stage III, Plane 1 of surgical anesthesia), the heart was fibrillated by the delivery of a lo- 15 s, 60 Hz, 2-ms square-wave stimulus (Model S-9, Grass Medical Instruments, Quincy, MA) to the left ventricular epicardium. Ventilation was

221

discontinued and circulatory arrest was confirmed by ECG, MAP and direct observation of the heart. 2.4. Resuscitationand recovery

Immediately after 9 min of normothermic ventricular fibrillation, ventilation with room air or hypoxic gas, dependent on experimental group, commenced with direct cardiac compression. Specifically, the compression was done with the right hand by wrapping fanned fingers around the heart such that the fingers lay across the right ventricle and the palm of the hand and thumb lay across the left ventricle. The heart was then compressed with sufficient force and frequency to maintain MAP above 75 mmHg. During the compressions, vasopressor support was initiated by central venous administration of 40 &kg epinephrine (Berlex Laboratories Inc., Wayne, NJ) and an initial peripheral venous infusion of 20 hg/(kg x min) dopamine (Abbott Laboratories). The central venous bolus of epinephrine was followed in rapid succession by 1 mg/kg lidocaine (Elkins-Sinn Inc., Cherry Hill, NJ), 4 meqkg sodium bicarbonate, and 25 mg/lcg calcium chloride (American Reagent Laboratories Inc., Shirley, NY). Cardiac countershocking was then attemp ted by delivery of a 20-50-J charge with 31-cm2 paddles placed on right and left ventricular surfaces (Lifepak 6 defibrillator/monitor, PhysioControl, Redmond, WA). Additional drugs and or charges were delivered as indicated by MAP and ECG monitoring. Post-resuscitation dopamine infusion was continued to maintain MAP above 75 mmHg as long as necessary but no longer than 6 h (typi~a~y < 30 min). A rubber catheter was passed through the chest wall and was sealed in place with a purse suture and connected to intermittent suction (Gomco Thoracic Pump, Gomco Corp., Buffalo, NY) for the removal of excess air and serosanguineous fluid after the chest closure was completed. Mechanical ventilation was continued until spontaneous ventilation resumed (typically 25 min post-resuscitation) and no later than 6 h postarrest. Ventilation, from the start of resuscitation out to 15 min, was through the ventilator from a nondiffusing gas bag (120 liter Quintron, Milwaukee, WI) containing either room air for

228

CF. Zwemer et al. /Resuscitation 29 (1995) 225-236

normoxically-resuscitated animals or 8.5% or 12.0% oxygen (balance nitrogen) for hypoxicallyventilated animals. Precise mixtures of the hypoxic gases were directed into the bag by way of gasmixing flowmeters (GF-3, Cameron Instrument Co., Port Aransas, TX). The bag was refilled with either mixture as necessary. At the end of 15 min, all animals breathed room air. The airway was extubated upon return of the gag reflex. All dogs were given Spectinomycin (Ceva Laboratories Inc., Overland Park, KS), 10 mg/kg i.m., intra-operatively, and morphine sulfate was provided for analgesia if post-operative attention to wound sites or aggressive behaviors suggested the presence of pain (no animals in this study, however, required such analgesia). Post-operatively, each dog was placed in a jacket and swivel which allowed for free movement about the recovery cage. This arrangement permitted 3 electrical and 3 hydraulic connections to be made into the dog for continuous sampling and hemodynamic measurement. Dogs surviving 24 h post-arrest were euthanized with 120 mg/kg i.v. sodium pentobarbital following the final neurological deficit scoring and sampling procedures. In every dog, a post-mortem examination of the heart, lungs and wound sites was conducted to identify possible iatrogenic complications. Dogs were excluded from this study if their deaths could be attributed to non-neurologic consequences, such as aberrant catheter placement, pneumothorax, and excessive hemorrhage. 2.5. Neurological deficit assessment

A well-standardized neurologic scoring system was assigned to animals in each group at 1, 2, 6, 12 and 24 h post-arrest to assessneurologic deficit [4-l 11. Of a possible 100 points, 18 were assigned to level of consciousness, 18 to respiratory function, 16 to cranial nerve function, 20 to spinal nerve function, and 28 to motor function. Maximum neurological deficit assessed was 100 points and minimal or non-detectable deficit scored 0. 2.6. Blood sampling

During the experiment, 10 arterial blood samples were removed from the dogs and analyzed for blood gases, pH, hematocrit, and plasma glucose

concentrations. The first sample was withdrawn directly after installation of the femoral artery catheter and served as the control. The second and third samples were taken immediately prior to ventricular fibrillation (pre-arrest) and immediately following resuscitation (post-fibrillation), respectively. The 7 other samples were removed at 0.5, 1, 2, 4, 6, 12, and 24 h post-arrest. At each of these sampling points, mean arterial blood pressure (MAP), heart-rate (HR), total urine output volume (up to no later than 6 h post-arrest), and body temperature (deep esophageal temperature while on operating table; rectal when in recovery cage) were recorded. 2.7. Malonaldehyde, Chydroxynonenal, reducedglutathione measurements

and

Whole blood samples (3 ml/sample) were withdrawn at control, 1, 6, 12, and 24 h following resuscitation using unheparinized plastic syringes and placed into glass test tubes containing 50 ~1 of 0.17 M KsEDTA (Aldrich) used as an anticoagulant and 49 ~1of 0.28 1 M butylated hydroxytoluene (Sigma) used to halt further oxidation of samples in -7O’C storage. Whole blood samples were gently mixed with the above solutions and centrifuged at 2500 g for 10 min at 4’C in a refrigerated centrifuge (Beckman GPR centrifuge, Palo Alto, GA). Following centrifugation, the supematant was pipetted off into glass test-tubes and was rapidly frozen in a methanol/dry ice slurry (-68’C) prior to storage at -70°C until analyzed for malonaldehyde and 4-OH concentrations. A 0.5-g aliquot of the erythrocyte pellet from the sample tube was then diluted with 5% metaphosphoric acid, well mixed and centrifuged at 1700 g for 10 min. The resulting supematant was then pipetted off into plastic test tubes, flash frozen in a methanol/dry ice slurry and stored at -70°C until analyzed for reduced glutathione concentrations. Malonaldehyde and Ghydroxynonenal

assay.

Using spectrophotometric kits (LPO-586, Bioxytech S.A., Bonneul sur Mame, France, supplied by Cayman Chemical, Ann Arbor, MI) plasma samples (duplicates/time-point) were measured for MDA and 4-OH. Briefly, samples were thawed and 200 ~1 were mixed in a glass test tube with 650 ~1 of proprietary chromogenic reagent in

229

C.F. Zwemer et al. /Resuscitation 29 (1995) 22S-236

acetonitrile (11.4 mM) and 150 ~1 of 10.4 M methanesulfonic acid, for 4-OH and MDA measurement (combined total aldehyde assay) or 37% hydrochloric acid for MDA (exclusive MDA assay). Test-tubes were capped and incubated in a stirred water bath for 40 min at 45 f 1’C. They were then placed in an ice-water slurry for 10 min, decanted into microcentrifuge tubes and spun at 10 000 g for 10 min. The supernatant was decanted and its absorbance was read at 586 nm. The concentration of 4-OH was calculated as the difference between total measured aldehyde (MDA plus 4-OH) from the combined assay and measured MDA from the exclusive MDA assay. Reduced glutathione assay. Using spectrophotometric assay kits (GSH-400, Bioxytech S.A., Bonneuil sur Marne, France, supplied by Cayman Chemical, Ann Arbor, MI) erythrocyte lysate samples (duplicates/time-point) were analyzed for GSH. Briefly, samples were thawed and an 100-111aliquot was thoroughly mixed with 800 ~1of buffer (200 mM potassium phosphate, pH 7.8, containing 0.2 mM DPTA and 0.025% Lubrol), 50 ~1 of proprietary chromogenic reagent, and 50 ~1 of 30% aqueous sodium hydroxide. They were then incubated in the dark for 10 min at 25°C. Following incubation, the samples were decanted

and absorbance was measured at 400 nm, extinction coefficient generated from a curve relating known concentrations of absorbances was then used to determine trations of GSH in samples.

A molar standard GSH to concen-

2.8. Statistics

Comparisons of all physiologic variables were assessed with one way analysis of variance (ANOVA-Scheffe) and neurological deficit scores were compared non-parametrically first using Kruskal-Wallis analysis and then using MannWhitney U-analysis to determine individual differences. Significance was set at P I 0.05. All average data are expressed as mean f one standard deviation (S.D.). All statistical calculations were performed on a computer (Apple Quadra 700) using a commercial software package (Statview III SE + Graphic). 2.9. Experimental approval

The entire experimental procedure conformed to the guidelines set by the American Physiological Society and was approved by the University Committee on the Use and Care of Animals (Approval No. 3271A).

Table 1 Pre-arrest and resuscitation variables Variable

HY 8.5% (n = 7)

HY 12% (n = 5)

N (n = 5)

Body weight (kg) Operative time (mm) Resuscitation time (min) Number of countershocks Time on ventilator post-resuscitation (min) Time to extubation (min) Epinephrine (&kg) Lidocaine (mg/kg) Sodium bicarbonate (meq/kg) Calcium chloride (mg/kg) Duration of dopamine infusion (min), 10 pg/(kg x min) Survival (h)

18.9 36.0 3.6 2.9 40.2

..---..

f f f f f

2.1 3.2 2.1 2.0 6.6

18.2 30.6 2.5 1.2 38.2

f f f f f

3.2 4.3 1.2 0.4 5.7

19.6 f 37.2 f 5.9 * 3.8 zt 40.6 f

3.9 4.4 4.4 2.8 4.9

199 f 56* 2.0 l 5.1 f 34 f 27.1 f

76 18

240 f 48 f 1.0 f 5.0 f 25.0 f 20.5 f

25 11 0.0 1.4 0.0 5.8

244 66 1.8 5.8 33 33.1

40 30 0.8 2.1 16.8 12.7

1.0

1.6 16.1 10.6

16.9 f 8.9

23.4 f 1.4

f f zt rt f f

24 f 0.0

Average pre-arrest and resuscitation data (mean f SD.). Groups were compared by pairwise comparison using ANOVA (Scheffe). Resuscitation time = time until MAP > 75 mmHg without mechanical assistance; n = sample size.

114 * 31 108 zt 15 106 f 13

7.41 * 0.04 7.40 l 0.03 7.38 f 0.06

34.8 f 3.3 38.8 f 2.5 41.6 zt 4.3

32.8 f 7.3 34.5 l 6.8 32.9 f 3.5

39.0 l 0.4 39.2 zk 0.3 38.6 f 0.6

136.0 zt 94 45.0 f 45 75.0 l 71

MAP (mmHg) Hypoxic FIoz 8.5% Hypoxic FIoz 12.0% Normoxic F,oz 2 1.O%

Arterial pH Hypoxic FIoz 8.5% Hypoxic F,oz 12.0% Normoxic F,* 21.0%

Arterial Pco2 (mmHg) Hypoxic F,ol 8.5% Hypoxic F,q 12.0% Normoxic F102 21.0%

Hematocrit (%) Hypoxic F,* 8.5% Hypoxic FIo2 12.0% Normoxic F,q 21 .O%

Temperature (“C) Hypoxic F,Gz 85% Hypoxic F,* 12.0% Normoxic F,oz 2 1.O%

Total Urine Output (ml) Hypoxic F,q 85% Hypoxic FIo2 12.0% Normoxic F,* 21 .O% 143.0 f 93 50.0 f 50 90.0 EIZ84

38.3 f 0.3 38.6 zt 0.5 38.0 f 0.3

36.1 f 5.1 37.7 f 7.1 42.8 f 4.0

57.4 f 8.4 64.8 f 5.8 67.2 f 12

7.41 l 0.06 7.43 l 0.07 7.40 f 0.12

132 ct 26 114 zt 18 111 i 26

210 l 50 185 f 48 189 t 64

Post-CA

171.0 l 91 70.0 * 57 90.0 zt 84

38.5 + 0.4 38.6 zt 0.4 38.5 ct 0.4

37.4 Et 9.7 37.0 t 5.9 38.0 zt 3.8

36.0 f 7.6 34.6 i 3.4 42.8 ziz 11.6

7.40 * 0.08 7.51 l 0.02 7.48 l 0.05

93 l 21 88 * 12 88 zt 6

173 f 56 173 l 40 143 l 19

30 min

96 f 12 89* 11 110 zt 36

177 + 48 153 f 19 160* 17

221.0 ct 125 150.0 l 94 120.0 l 110

38.6 zt 0.5 38.3 zt 0.6 38.5 f 0.2

37.2 zt 6.9 37.1 l 6.5 39.2 f 2.0

37.6 f 7.0 37.4 l 7.0 40.5 ZIG5.2

7.42 zk 0.06 7.49 f 0.06 7.48 l 0.05

lh

671.0 * 147 590.0 f 228 700.0 l 283

37.9 f 0.9 37.7 * 0.5 37.7 + 1.0

43.5 zt 6.3 44.5 l 5.5 39.7 f 2.6

41.3 l 6.8 45.8 f 4.3 42.7 h 6.9

7.43 Et 0.09 7.39 f 0.03 7.43 l 0.06

110 l 15 106* 11 119 Et 11

121 l 60 125 zt 40 126 f 16

4h

750.0 l 71 825.0 zt 35 NA

38.5 f 0.7 38.2 f 0.6 37.8 + 0.9

46.3 f 8.6 46.6 l 5.1 45.1 f 3.8

41.4 l 6.6 44.1 * 3.5 41.9 f 4.3

7.38 i 0.06 7.38 f 0.04 7.41 f 0.04

115 f 17 111 f 16 122 l 10

151 f 36 132 f 16 153 f 22

6h

Groups were compared pairwise using ANOVA (Scheffe). Significance was set at the 95% level. NA = not available.

88 zt 28 76 f 18 86* 11

Pre-CA

Heart rate (beats/m+ Hypoxic F,oz 8.5% Hypoxic FIoz 12.0% Normoxic F,oz 2 1.O%

Table 2 Physiologic variables

NA NA NA

39.8 EIE1.3 39.8 f 1.4 38.6 f 0.9

l 6.6 49.3 f 4.2 47.6 zt 3.5 44.1

27.1 zt 8.9 33.3 ct 5.6 33.0 * 5.9

7.44 l 0.07 7.42 zt 0.08 7.45 l 0.07

125 f 12 118 zt7 120 * 12

153 f 72 151 zt 38 178 f 37

12 h

NA NA NA

39.1 zt 0.8 39.7 f 0.4 39.1 l 0.5

40.8 f 8.0 48.3 * 7.0 45.8 f 3.1

28.5 zt 5.3 28.7 f 2.7 27.3 EIZ4.4

7.40 f 0.04 7.37 f 0.04 7.41 f 0.06

108 * 10 104 * 19 114 l 17

l 13 152 f 30 163 EIE23 146

24 h

C.F. Zwemer

et al. /Resuscitation

3. Results

3.1. Pre-arrest and resuscitation variables All 17 dogs in this study were successfully resuscitated from cardiac arrest as defined by return of spontaneous circulation and maintenance of MAP greater than 75 mmHg. No dogs from this study were excluded. Analysis of variance (Scheffe) failed to detect any differences among these groups in pre-arrest or resuscitation variables (Table 1). Survival time did tend to separate among the groups and almost reached statistical significance (P = 0.16, using ANOVA Scheffe, P = 0.11, using an unpaired student’s ttest, and had a Fisher’s Exact P = 0.2) between HY8 (16.9 f 8.9 h) and N (24.0 f 0.0 h) groups. In addition, analysis of variance (Scheffe) was unable to detect statistically-significant trends among the 3 groups in heart rate (HR), MAP, arterial Pcoz (Paco2), and temperature. Hematocrit increased over time in all groups. However, no significant differences were found among them. While we anticipated a decrease in arterial pH in the 2 hypoxic groups following resuscitation, this was also not observed. The lack of an additional pH burden in the hypoxic groups was further sup-

1251

-O-f

/

..---

Normoxic n-5 Hyporic (8.5%) nyporic

29 (1995)

225-236

‘3 I

ported by data from Table 1 which revealed that no additional sodium bicarbonate was necessary in either hypoxic group during the acute p&ases of resuscitation. All of these physiologic variables appear in tabular form in Table 2. 3.2. Arterial Pol Animals in groups HY8 and HY12 were maintained at statistically-significant hypoxic states relative to normoxic controls immediately following resuscitation from cardiac arrest as seen in Fig. 1. Using a paired Student’s t-test, Paot was significantly lower at the immediate postresuscitation time-point in both the HY8 (P = 0.0001) and HY12 (P = 0.0017) groups as compared to normoxic controls. 3.3. Arterial blood glucose Significant differences in arterial blood glucose among groups were detectable using ANOVA (Scheffe). Specifically, arterial plasma glucose concentrations were higher in the HY8 group compared to the N group immediately (HY8: 312 f 86 mg/dL, N: 196 f 82 mg/dL, P = 0.17) and 30 min (HY8: 331 f 109 mg/dL, N: 187 f 74 mg/dL, P = 0.077) following resuscitation. Using a

n=7

(12%) n4 -3 *

1

Normoxic Reausc. “A tiyporic (8.5%) ResJsc. fK7 Hypoxic (12%) Resusc. 03

l

‘1,

25 / 01

0

0.25

0.75

1

Tlmeqc)

Fig. I. Mean f S.D. of P,@ for all 3 groups versus time. ANOVA (Scheffe) confirmed that both HY8 and HY 12 group animals bad arterial hypoxemia maintained at a lower Paq for I5 tin during resuscitation from card&c arrest than nonnoxic controls. The sample size of each group is noted next to its group symbol.

L..--0.5

__ 0

0.5

- _.- ~ 1

. . .--~_.1.5

2

Time (hr)

2.5

~ . .T__~ ; 3

3.5

4

Fig. 2. Mean f S.D. of arterial blood glucose for all 3 groups versus time. Using ANOVA (ScheRe), blood @case concentration was determined to be significaauy higher in HY8 as compared to N group 0.5 h following resuscitation. Tbe sample size of each group is noted next to its group symbol.

232

CF. Zwemer et 01./Resuscitation 29 (1995) 225-236

Fisher’s r to z analysis,30-min post-resuscitation plasma glucose concentrations were found to correlate negatively (-0.693) and significantly (P < 0.0018) with survival time. Fig. 2 describesthe arterial blood glucose values for the 3 groups in the first 4 h following cardiac arrest. 3.4. Neurologic deficit

All animals survived at least 6 h post-arrest. Three animals in the HY8 group and 1 in the HY12 group died between 6 and 24 h and were assessedan NDS of 100 at 24 h, as their deaths were directly attributable to a primary neurological insult. Deaths attributable to neurologic insult were determined by direct observation. Specifically,signsof fixed and dilated pupils, profound extensor rigidity and convulsive activity followed by a decreasein MAP to levels below 20 mmHg were considered to be indicative of neurologic death. No animals in the N group died prior to euthanasia at 24 h. A Kruskal-Wallis (non-parametric) analysis detected no significant differences in neurological deficit score (NDS) among the 3 groups. While the averageNDS tended to be lower (better outcome) in the N and HY12 groups than the HY8 group, the pattern did not reach statisticalsignificanceat either the 12and 24 100

1

I

-mt

75

2.57 !-2%d,3 252

3

t

tlypoxk

(8.5%) n.7

t -D-

Hypork (12%) n=S Namwxk 18~5

*

tina6 I-

1.5.

z +2

1.

4 a

0.5.

E 4 p

0. -03

. 0

4

a

Ttn$hr)

16

(8SX) (12%)

ns7 Il.5

24

Fig. 4. Mean f S.D. for plasma concentrations of 4-OH for all 3 groups in PM versus time. Using paired btests uncorrected for multiple comparisons, the 12- and 24-h pooled samples were signitkantly greater than control at P=O.O46 and 0.035, respectively. The sample size of each group is noted next to its group symbol.

h points. Theseresults appear graphically as ND6 versus time in Fig. 3. 3.5. Plasma concentrations of 4-OH

Plasma concentrations of 4-OH were not different amongindividual groups throughout the 24 h experiment as seen in Fig. 4. As such, means lo-

Hypdc tlypoxk

20

p-0.002 * ,"ncorrect.d,

I

g-7.5. t

t

Mypoxk

t

llypoxk (12%) II& Nommxkndi

-0..

(8.5%) 41.7

II

V) 50 4 2s

C

”

4

8

12

16

20

24

Time (hr) Fig. 3. Mean f SD. of post-arrest neurologic deficit score (NDS) (0, no deficit; 100, maximum deficit) for all 3 groups versus time. Mean NDS scores had a tendency to be lower for animals in N and HY 12 groups. The sample sixe of each group is noted next to its group symbol.

i

i

i

.

Time

16

do

24

&)

Fig. 5. Mean i S.D. for plasma concentrations of malonaldehyde for all 3 groups in PM versus time. Using paired t-tests uncorrected for multiple comparisons, the 1, 6, 12, and 24 h pooled samples were signitkantfy greater than control at P = O.ooOl. 0.0001,0.OttO4, and 0.002, respectively. The sample size of each group is noted next to its group symbol.

C.F. Zwemer

et al. /Resuscitation

29 (1995j

225-236

233

4. Dlsamh

1

0

4

8 Time

12 (hr)

f +

Hypoxic Hyporic

-I3.-

Nwmork

16

(8.5%) u-3=7 (12%) n=5

” _

n=S

20

Fig. 6. Mean f S.D. for eiythrocyte coaantrations of reduced dutrthioaef~rll3lpoupsinIrMvenustimc.Usiagpairedrt&swroorPdaD#Sfard~~ns,thcl-hpookdsampks were aigniwtly lower than that of control. The sample size of each group is noted next to its group symbol.

were pooled per time-point and, using paired ttests (uncorrected for multiple comparisons), were compBfcd against the co&o1 mean. This post-hoc fxmpMm reveakd that the plasma levels of 4 OH incmased over time and, by 12 and 24 h, reached statistical separation. 3.6. Ph concentrations of malonal&hyde Using pooled means and paired t-tests (uncorrected for multiple comparisons) the data revealed a consistent and sign&ant increase in plasma MDA concentration at every time-point following resuscitation, as is seen in Fig. 5. Plasma concentrations of MDA were not discernibly different among groups throughout the 24-h experiment. 3.7. Erythrocyte glutathione concentration Pooled mean GSH levels for all groups dropped significantly (P = 0.0123) from a control mean of 604 f 292 PM to 72% of baseline (436 f 162 PM) at 1 h following resuscitation from cardiac arrest depicted in Fig. 6. By 6 h, levels had returned to baseline. In addition, no discernible differences among individual groups were found in erythrocyte GSH concentration.

24

The data presented here lead us to reject the hypothesis that hypoxic reperfusion reduces the severity of the ischemidreperfusian insult. While post-ischemic hypoxia (P,o#O-45 mmHg> similar to what we delivered in this study, has been shown to improve the met&&c and functional recovery of the ischemic spinal cord in rabbits [12,13], in ischemic dog skeletal muscle lI4} and in explanted rat livers [15], it was clearly not the case in our study. A possible exphmation for this ap parent contradiction of current literature may be due to differences above studies investigated the reperfusion in single organ&&sue systems while our model relies upon a global normothermic ischemia and reperfusion. Thus, our data do not necessarily conflict with their fiadiog but apply the same hypothesis in a distinctly di@erent type of reperfusion injury (global versus local). The data we obtained testing a similar hypothesis in our systemic cardiac arrest and resuscitation model lead us to reject this hypothesis. It is however that other systemic responses such as postresuscitation hyperglycemia may have contributed to the adverse outcome in our model. Hyperglycemia in the setting of ischemia and reperfusion injury appears to have ovetwbelmingly deleterious effects on outcome. The data supporting this are plentiful. In a retrospective study, Longstreath and Inui identifil a statistical correlation between systemic hyperg&cemia upon hospital admission in humans to poor neurological outcume [16]. WbiIe in cardiac arrest and resuscitation models of global cerebral is&em& prearrest [17] and post-arrest 15J hyperglycemia has been associated with profound 24-h mortality rates. Interestingly, both the HY8 and HY12 groups in this study sustained hyperglycemia following resuscitation. Furthermore, systemic hyperglycemia has been shown to exace&te outcome following regional/organ i&e&a and reperfusion. Models of focal brain in&y [18], spinal cord 119,201 and renal 121) injury all identify a causal link between hyperglycemia and poor ischemi&qerfirsion outcome. It is possible then that the post-resuscitation hyperglycemia observed

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in this study accounted for the trend of decreased survival and increased neurological deficit in the HY8 and even HY 12 groups. 4.1. Potential markers of oxidant injury

The overall pattern of neurologic damage in the 3 groups was paralleled by the pattern of change in the markers of oxidant injury which we chose to measure. The progressive increases in MDA and 4OH over time, coupled with transient reduction in GSH suggests the presence of oxidant injury in this cardiac arrest and resuscitation protocol. 4.2. I-hydroxynonenal and malonaldehyde

Both MDA and 4-OH compounds are breakdown products of lipid peroxides and as such, their appearance in plasma may serve to indicate the level of oxidant injury. Pooled 4-OH concentrations increased from baseline over 24 h following resuscitation and reached statistical significance at both 12- and 24-h time-points as seen in Fig. 4. The baseline values from this study (0.256 * 0.335 PM) were in close agreement with the range of published human values (0.3-0.7 PM) [22] and their increase over time following resuscitation supports the possible occurrence of lipid peroxidation and oxidant injury. Furthermore pooled plasma MDA increased significantly above baseline at all measured points following resuscitation (Fig. 5). The values we obtained for MDA were consistent with previously reported values. Wong et al. measured baseline healthy human plasma values of MDA at 0.60 f S.D. 0.13 PM, and control levels for rats at 1.4 * S.D. 0.30 PM (231. Two spectrophotometric studies from 1985-1986 also report similar human control values of MDA at 0.61 f S.D. 0.11 [24] and 0.95 f S.D. 0.09 PM [25], respectively. Our control concentrations of MDA (0.745 i 0.305 PM) agree very well with the results from these 3 studies. Moreover, the significant increase in MDA concentration suggested an ongoing release from the tissues, possibly being the result of progressive lipid peroxidation. These data are consistent with data generated by Elkstrom et al. [26] who showed a IO-fold increase in urine concentration of MDA from control values 24 h following chloroform treatment in rats, and Wong et al. [23]

who showed a 2.5-fold increase in MDA plasma concentration 24 h following NiCl poisoning. Thus, while our data did not show differences among groups, they did show significant increases in MDA concentration which, consistent with previously published patterns of oxidant injury, indicate that, in all groups, some degree of oxidant injury may have occurred following resuscitation. It is important to note that no outcome differences were detected either. 4.3. Erythrocyte glutathione concentration

Erythrocyte GSH concentration dropped significantly from control levels in all groups at 1 h following resuscitation and returned to normal at the 6-h point (Fig. 6). This pattern fits well with the early reperfusion oxidant injury theory which suggests that a majority of oxidant mediated injury occurs in the first 15 min of reperfusion [l-3]. Reduced glutathione can conceivably act as a ‘sacrificial’ antioxidant and in the presence of oxygen radicals will become readily oxidized. Erythrocytes containing normal concentrations of GSH, when introduced into ischemic and reperfused heart preparations, have been shown to decrease hydrogen peroxide levels and reperfusion injury [27]. Likewise, erythrocyte glutathione levels have been used as a clinical assessment of oxidant state in patients undergoing treatments which cause oxidative stress [28]. Generally then, it is possible that the significant and transient decrease in erythrocyte GSH concentration seen in our study may have been attributable to oxidation from tissues undergoing PROS production. Conceivably, as the RBCs passed through tissue undergoing radical production, erythrocyte GSH may have been oxidized. The return of erythrocyte GSH concentrations to control levels by the 6-h point may possibly be attributed to normal pathways of the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) by red cell glutathione reductase and NADH from red cell glycolysis. In summary of the oxidant injury marker data, it may be concluded that oxidant injury may have been occurring in all groups following resuscitation. Further supporting this notion is data from a previous study, using this model, which demon-

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strated a significant fall in plasma levels of vitamin E following resuscitation in animals resuscitated with air [7]. In the same study, animals treated at post-resuscitation with the antioxidant U74006F maintained plasma vitamin E levels and sustained significantly-improved outcomes over untreated counterparts. Furthermore, a recent study from our laboratory demonstrated that the severe neurologic dysfunction associated with 100°/o oxygen resuscitation can be ameliorated with antioxidant pre-treatment [4]. Together, the findings of these studies strongly suggest a component of oxidant injury in this cardiac arrest resuscitation model. 5. conelusions The central hypothesis of this study was that resuscitation with hypoxic ventilation (FIOZ values of 0.08 and 0.12) would improve outcome by reducing available substrate necessary for PROS production beyond that offered by normoxic resuscitation. The results of this study simply do not support that hypothesis. Resuscitation with hypoxic gases did not provide any protection from neurologic dysfunction beyond that offered by normoxic resuscitation. Post-resuscitation glucose concentration correlated inversely with survival and tended to be highest in those animals resuscitated with severe hypoxia. Thus, it may be concluded that hypoxic ventilation during resuscitation not only has no advantage, but is deleterious to outcome at severe levels. Likewise, hyperoxic ventilation during resuscitation is also deleterious to outcome, as demonstrated in an earlier paper [4]. We therefore suggest that normoxia be maintained during resuscitation in order to minimize neurologic dysfunction and improve outcome. Acknowledgments This work was supported, in part, by a grant from the American Heart Association of Michigan (G10934) and a gift from The Upjohn Company of Kalamazoo, Michigan. We thank the Bio-Tek Instruments Company of Winooski, Vermont for the generous donation of the QED-4 Defibrillator

Analyzer and thanks to the Physio-control Corp. of Redmond, WA for the generous donation of the Lifepak-6 defibrillator. Dr. Zwemer was supported by an NIH, NRSA (F32 HL08792-01) postdoctoral fellowship. The authors also wish to thank Jason A. Pollack, A. Majid Khan, Natasha K. Eaddy, Erin M. O’Connor, Mary C. Lloyd, and Peter Chelune for their excellent and expert technical assistance throughout this project. References [1] Hall ED, Braughler JM. Free radicals in CNS injury. Chapter 6, In: Molecular and cellular approachesto the treatment of neurological disease edited by S.G. Waxman. New York: Raven Press, Ltd., 1993; 81-10.5. [2] Traystman RJ, Kirsch JR, Koehler RC. Oxygenradical mechanisms of brain injury following ischemia and reperfusion. J App Physiol 1991; 71(4): 1185-I lY5. [3] Hall ED, McCall NJ, MeansED. Therapeuticpotential of the lazaroids (21-Aminosteroids) in acute central nervous system trauma, ischemia, and subarachnnid hemorrhage. Adv in Pharmacol 28: 221-268. [4] Zwemer CF. Whitesall SE, D’Alecy LG. Cardiopulmonary-cerebral resuscitation with 100% oxygen exacerbates neurological dysfunction following nine minutes of normothermic cardiac arrest in dogs. Resuscitation 27(2): 159- 170. (51 Lundy EF. Kuhn JE, Kwon JM, Z&mock GB, D’Alecy LG. Infusion of 5% dextrose increases mortality and morbidity following six minutes of cardiac arrest in dogs. J Crit Care 1987; 2( 1): 4-14. [6] Natale JE, D’Alecy LG. Protection from ccrebrat &hernia by brain cooling without reduced lactate accumulation in dogs. Stroke 1989; 20: 770-777 [7] Natale JE, Schott RJ, Hall ED, Braughler JM, D’Alecy LG. Effect of the aminosteroid U74006F after cardiopulmonary arrest in dogs. Stroke 1988; 19(111: 1371-137X. [8] Natale JE, D’Alecy LG. Continued circulatory support: effect of epinephrine or dopamine in 24 h survival and neurologic function in dogs. Resuscitation 1989: 1.7. 273-286. [9] Schott RJ, Natale JE. Ressler SW. Burney. D‘Alecy LG Neutrophil depeletion fails to improve neurologic outcome after cardiac arrest in dogs. .Ann Emerr? Med 1989; 18: 517-522. [lo] Natale JE, Stante SM, D’Alecy LG. Elevated brain lactate accumulation and increased neurologic defecit are associated with modest hyperglycemia in global bram ischemia. Resuscitation 1990; 19: 271-289. [I I] Facktor MA. Mayor GH, Nachreiner RF. D’Alecy LG. Thyroid hormone replacement during resuscitation from cardiac arrest in dogs. Resuscitation 1993: 76, 141-162. [12] Marsala M. Danielsova V, Chavko M, Hornakova A, Marsala J. Improvement of energy stat< and basic

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Hyperglycemia exacerbates and insulin fails to protect in acute renal ischemia in the rat. J Surg Res 46.572-578. yU] Esterbauer H, ZoBner H, Schaur RJ. Akiehydes formed by lipid peroxidation: mechanisms of formation, occurance and determination. In: Vigo-Pelfrey G, editor. Membrane Lipid oxidation, vol. I. Boca Raton, FL: CRC pres& 1990; 239-283. [23] Wong SH, Knight JA, Hopfer SM, Zaharia 0, Leach CN, Sunderman FW. Lipoperoxides in plasma as mcasud by liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin Chem 1987; 33/2: 214-220. [24] Framxsco V, Cesare A. Lui8i I, Stefano F, Andrea G, Francesco B. Malondialdehyde-like material and betathromboglobulin plasma levels in patients suffering from transient ischemic attacks. Stroke 1985; 16: 14-16. [ZS] Ledwozyw A, Michalak J, Stepien A, Kadaiolka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986; 155: 275-284. [26] Ekstr6m T, St&l A, S&vardsson K, H6gberg J. Lipid peroxidation in vivo monitored as ethane exhalation and malondialdehyde excretion in urine after oral administration of chloroform. Acta Ph armad et Toxic01 1986; 58: 289-296. [271 Brown JM, Grosso MA, Terada LS, Beehler CJ, Toth KM, Whitman GJ, Harken AH, Repine JE. Erythrocytes decrcasc myocardial hydrogen peroxide levels and reperfusion injury. Am J Physiol1989; 256 (Heart Circ Physiol 25): H584-H588. [28] Sheriff IX. Erythrocyte glutathione concentration as a measure of oxidant stressin patients undergoing dapsone therapy. Ann Chn B&hem 1989; 26: 199-200.