ORIGINAL CONTRIBUTION

Combination of Intravenous Ascorbic Acid Administration and Hypothermia After Resuscitation Improves Myocardial Function and Survival in a Ventricular Fibrillation Cardiac Arrest Model in the Rat Min-Shan Tsai, MD, PhD, Chien-Hua Huang, MD, PhD, Chia-Ying Tsai, Huei-Wen Chen, PhD, Hsaio-Ju Cheng, Chiung-Yuan Hsu, MD, Wei-Tien Chang, MD, PhD, and Wen-Jone Chen, MD, PhD

Abstract Objectives: Intravenous (IV) administration of ascorbic acid during cardiopulmonary resuscitation (CPR) was reported to facilitate defibrillation and improves survival in ventricular fibrillation (VF) cardiac arrest. We investigated whether IV administration of ascorbic acid after return of spontaneous circulation (ROSC) can improve outcomes in VF cardiac arrest in a rat model and its interaction with therapeutic hypothermia. Methods: Ventricular fibrillation–induced cardiac arrest followed by CPR and defibrillation was performed in male Wistar rats. After ROSC, the animals were equally randomized to the normothermia (NormoT), hypothermia (HypoT), ascorbic acid (AA+NormoT), and ascorbic acid plus hypothermia (AA+HypoT) groups. The AA+NormoT and AA+HypoT groups received IV ascorbic acid (100 mg/kg). In the HypoT and AA+HypoT groups, therapeutic hypothermia was maintained at 32°C for 2 hours. Results: There were 12 rats in each group. Within 4 hours after ROSC, the HypoT, AA+NormoT, and AA+HypoT groups had significantly lower myocardial lipid peroxidation than the NormoT group. Within 4 hours following ROSC, the AA+NormoT group had a significantly better systolic function (dp/dt40) than the NormoT group (6887.9 mm Hg/sec, SD  1049.7 mm Hg/sec vs. 5953.6 mm Hg/sec, SD  1161.9 mm Hg/sec; p < 0.05). The AA+HypoT group also showed a significantly better diastolic function (–dp/dtmax) than the HypoT group (dp/dt40: 8524.8, SD  1166.7 mm Hg/sec vs. 7399.8 mm Hg/sec, SD  1114.5 mmHg/sec; dp/dtmax: –8183.4 mm Hg/sec, SD  1359.0 mm Hg/sec vs. –6573.7 mm Hg/sec, SD  1110.9 mm Hg/sec; p < 0.05) at the fourth hour following ROSC. Also at 4 hours, there was less myocytolysis in the HypoT, AA+NormoT, and AA+HypoT groups than the NormoT group. The HypoT, AA+NormoT, and AA+HypoT groups had significantly better survival rates and neurologic outcomes than the NormoT group. Compared with only five surviving animals in the NormoT group, there were nine, eight, and 10 in the HypoT, AA+NormoT, and AA+HypoT groups, respectively, with good neurologic outcomes at 72 hours. Conclusions: Intravenous ascorbic acid administration after ROSC in normothermia may mitigate myocardial damage and improve systolic function, survival rate, and neurologic outcomes in VF cardiac arrest of rat. Combination of ascorbic acid and hypothermia showed an additive effect in improving both systolic and diastolic functions after ROSC. ACADEMIC EMERGENCY MEDICINE 2014; 21:257–265 © 2014 by the Society for Academic Emergency Medicine

From the Department of Emergency Medicine, National Taiwan University Medical College and Hospital (MST, CHH, CYT, HJC, CYH, WTC, WJC), Taipei, Taiwan; and the Graduate Institute of Toxicology, College of Medicine, National Taiwan University (HWC), Taipei, Taiwan. Dr. Wen-Jone Chen is currently with the Department of Emergency Medicine, Lotung Poh-Ai Hospital, Yilan County, Taiwan. Received July 15, 2013; revision received September 3, 2013; accepted September 8, 2013. Presented at the American Heart Association Scientific Sessions, Los Angeles, CA, November 2012. The study was supported by a research grant from the Taiwan National Science Council, No. 100-2314-B-002-130-MY3. The authors have no potential conflicts of interest to disclose. Supervising Editor: Richard Sinert, DO. Address for correspondence and reprints: Wen-Jone Chen, MD, PhD; e-mail: [email protected].

© 2014 by the Society for Academic Emergency Medicine doi: 10.1111/acem.12335

ISSN 1069-6563 PII ISSN 1069-6563583

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nly one-third of successfully resuscitated victims of out-of-hospital cardiac arrest patients survive to hospital discharge.1,2 Myocardial injury, resulting from preexisting etiology, ischemia/reperfusion injury, cardiopulmonary resuscitation (CPR), and electric shock, is one of the major causes accounting for in-hospital mortality. Reactive oxygen species are involved in ischemia and reperfusion injury,3–7 reperfusion arrhythmia,3–5 and electric shock.8,9 Increased plasma-reactive oxygen species levels are directly proportional to the delivered energy of an electric shock and are significantly attenuated with antioxidant pretreatment.8,9 Ascorbic acid, a water-soluble antioxidant, directly acts with hydroxyl free radical (OH); participates in enzymatic defense against hydrogen peroxide (H2O2) and oxygen (O2); and attenuates oxidative damage, myocardial injury, and arrhythmia during reperfusion.3–7 Our previous study demonstrates that administering ascorbic acid ameliorates Ca2+ disequilibrium and contractile impairment in cardiomyocytes after electric shock by eliminating reactive oxygen species.10 Ascorbic acid administered at the start of CPR facilitates defibrillation, reduces myocardial damage, and benefits resuscitation in animal models of ventricular fibrillation (VF) cardiac arrest.11,12 Whether ascorbic acid administration after the return of spontaneous circulation (ROSC) still benefits myocardial function and outcomes remains unknown. Ascorbic acid has failed to fail to elicit beneficial effects in animals of hemorrhagic shock.13,14 Additionally, redox signaling has been shown to be useful for cell survival. The possibility of disrupting such signaling versus attenuating oxidative injury needs to be clarified. Therapeutic hypothermia improves the survival rate and neurologic outcomes after cardiac arrest and is recommended for unconscious patients following ROSC. However, several studies in the literature report the influence of hypothermia on pharmacodynamics, pharmacokinetics, and drug effects.15–17 Whether cooling modifies the effect of ascorbic acid on myocardial function and outcomes in cardiac arrest survivors remains unclear. Therefore, we hypothesized that ascorbic acid administration after ROSC will improve myocardial function and outcomes. The secondary outcome was to evaluate the effect of hypothermia on ascorbic acid in improving myocardial function and outcomes after cardiac arrest. In this study, we used a rat model mimicking clinical VF cardiac arrest to evaluate the influence of ascorbic acid administered after ROSC on myocardial function, survival rate, and neurologic outcomes, as well as its interaction with therapeutic hypothermia. METHODS Study Design This was a laboratory study of VF-induced cardiac arrest in the murine model. The study was approved by the institutional review board of the National Taiwan University College of Medicine and Public Health, in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the U.S. National Institutes of Health.

Animal Preparation Male Wistar rats at the age of 14 weeks and weighing around 400 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). The animals were prepared as described previously.11 The trachea was orally intubated with a PE 200 catheter, and mechanical ventilation was initiated with a tidal volume of 0.65 mL/100 g body weight at a frequency of 100 breaths/min with a fraction of inspired oxygen (FiO2) of 1.0. Arterial blood pressure and left ventricle (LV) pressure were measured with saline-filled PE-50 tubes inserted through the right femoral artery and through the right carotid artery and advanced into the LV. The tube in the right jugular vein was for fluid and drug administration and pressure monitoring. A thermodilution-tipped catheter (ADInstruments, Sydney, Australia) was inserted through the left femoral artery and advanced 10 cm into the abdominal aorta to monitor temperature changes. Blood pressure, LV pressure, central venous pressure, body temperature, and needleprobe electrocardiogram monitoring data were recorded with a computer-based data-acquisition system (ADInstruments). After surgical preparation, the animals were observed for 30 minutes to ensure hemodynamic stability. Before the experiment, body temperature was maintained at 37°C (standard deviation [SD]  0.5 °C) using an incandescent heating lamp. The sham group received the same preparation. Study Protocol Current-induced Cardiac Arrest Animal Model. Baseline data were recorded 15 minutes before inducing VF with a guidewire advanced from the right jugular vein into the right ventricle. An alternating current progressively increasing at 60 Hz to a maximum of 1 mA was delivered to the endocardium and continued for 1.5 minutes to prevent spontaneous defibrillation. Mechanical ventilation was discontinued after VF onset, and the animals were left untreated for 3.5 minutes. After 5 minutes of VF, chest compressions were delivered by a pneumatically driven mechanical chest compressor at 200 beats/ min, and mechanical ventilation was restarted. CPR was synchronized to provide a compression:ventilation ratio of 2:1 with equal compression–relaxation duration. The compression depth was initially adjusted to secure a coronary perfusion pressure under 20 mm Hg. This typically yielded an end-tidal partial pressure of carbon dioxide (ETCO2) of 13 mm Hg (SD  2 mm Hg). After 1 minute of CPR, one 3-J monophasic electric shock was administered, and then a sequence of 30 seconds of CPR followed by one 5-J electric shock. Resuscitation was declared to have failed when ROSC could not be achieved after a total of four shocks. All successfully resuscitated animals were closely monitored for 4 hours following ROSC, wounds were surgically closed, and surviving animals were subsequently extubated and returned to their cages. Survival status was recorded, and mortality was confirmed by loss of heartbeat and spontaneous respiratory movement for 2 minutes. Experimental Design. All successfully resuscitated animals were randomized into four groups: normothermia

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(NormoT), hypothermia (HypoT), ascorbic acid plus normothermia (AA+ NormoT), and ascorbic acid plus hypothermia (AA+HypoT). Immediately after ROSC, the animals in the AA+NormoT and AA+HypoT groups received IV ascorbic acid at 100 mg/kg (1 mL), while the NormoT and HypoT groups received 0.9% saline (1 mL) at room temperature. The animals in the hypothermia groups were cooled externally with ice water spray and electric fans after ROSC. The target temperature was 32°C, which was maintained for 2 hours by adjusting the fans or using the heating lamp. After cooling, the rewarming process was maintained at the rate of 0.5°C/ hour during intubation. The temperature recovered naturally after extubation with temperature monitored hourly. The target temperature of rewarming was 37°C. The temperature of animals receiving hypothermia usually achieved 37°C at the 10th through 12th hours following ROSC. The body temperatures of the animals in the NormoT and AA+NormoT groups were kept at 37°C. There was a sham group receiving preparation without inducing cardiac arrest, experimental drug, or hypothermia. Myocardial Injury (Malondialdehyde [MDA] Assay/ Histologic Exam). The MDA-586 method (OxisResearch, Portland, OR) was used to determine oxidative damage in the myocardium. Briefly, MDA forms a conjugate with N-methyl-2-phenylindole, resulting in a blue chromogenic signal measured at 586 nm. The MDA concentration was corrected for protein concentration between samples, and blank sample values were subtracted to correct for any variance due to sample turbidity.18 A pilot study was conducted before the current study to determine the time point of the most severe myocardial damage in animals receiving normothermia. The planned sacrifices were made at the first, second, fourth, 24th, and 72nd hours following ROSC, and the most severe myocardial damage was found at the fourth hour. Therefore, in the current study, we selected the fourth hour as the time point for histologic examination. The apex, septum, and lateral wall of the LV were examined. For hematoxylin and eosin (HE) staining, the LV was embedded in paraffin, cut into sections, and observed under an optical microscope. Myocytolysis was counted in five independent randomly selected microscopic fields at 200 9 magnification in each specimen. Six specimens were counted in each animal. The morphologic and histologic results were examined by two independent anatomists blinded to the grouping. An independent assessment by a third investigator and the majority was chosen if there were any discrepancies.

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measurements, 0.2 mL of isotonic saline indicator at room temperature was injected intravenously (IV) into the right atrium. The change in temperature was recorded by the thermodilution-tipped catheter (ADInstruments) in the abdominal aorta mentioned above, and cardiac output was calculated with a Cardio-Max II computer (Columbus Instruments, Columbus, OH). The cardiac output was measured at baseline and hourly after ROSC until the fourth hour following cardiac arrest. Evaluation of Survival and Neurological Outcomes. The neurologic outcomes of the studied animals were evaluated with neurologic function scoring at the sixth, 24th, 48th, and 72nd hours following ROSC (Table 1).19 Assessments were performed independently by two investigators. Any discrepancies were resolved by an independent assessment by a third investigator, and the score chosen by the majority was accepted. Data Analysis Assuming that the ascorbic acid administration might increase the survival rate from 30% to 90%, the required sample size to achieve an 80% power at a = 0.05 for correctly detecting such difference was 11. We chose 12 animals for each group and used block randomization in each batch of animals. Binomial variables were analyzed with the chi-square test and Fisher’s exact test. Continuous values are presented as mean  SD. The Kruskal-Wallis test was used for the comparison for continuous values. The significance of differences regarding the neurologic function scoring was also evaluated with the Kruskal-Wallis test. Survival curves were determined by the Kaplan-Meier method and compared by the log-rank test. p-values < 0.05 were considered statistically significant when we compared

Table 1 Neurologic Scaling Score Neurologic Sign Consciousness level Corneal reflex Respiration

Score

Description

0

No reaction to pinching of the tail Poor response to tail pinch Normal response to tail pinch No blinking Sluggish blinking Normal blinking Irregular breathing pattern Decreased breathing frequency with normal pattern Normal breathing frequency and pattern No turning attempts Sluggish turning Turns over spontaneously and quickly No movement Moderate ataxia Normal coordination No spontaneous movement Sluggish movement Normal movement

1 2 0 1 2 0 1 2

Hemodynamic Monitoring. The measurement of myocardial function in this study included LV-positive dp/dt40 (dp/dt40), maximal LV-negative dp/dt (–dp/dtmax), and cardiac output. The dp/dt40, which is the rate of LV pressure rise at LV pressure of 40 mm Hg, reflects systolic function. The –dp/dtmax, which is the maximal rate of LV pressure fall, reflects diastolic function. The needle-probe electrocardiogram, dp/dt40, and –dp/dtmax were continuously recorded and analyzed by the data acquisition system (ADInstruments). For cardiac output

Righting reflex

0 1 2

Coordination

0 1 2 0 1 2

Movement/ activity

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differences between the AA+NormoT and NormoT groups. The application of an alpha value of 0.05 in the subgroup analyses may inflate the possibility of type I error. All statistical analyses were performed with SPSS 12.0 software (IBM SPSS Inc., Armonk, NY). RESULTS A total of 105 animals were initially used in the hemodynamic and survival studies, with a successful resuscitation rate of 45.7%. Forty-eight successfully resuscitated animals were equally randomized into the four groups. There were no significant differences with respect to body weight, body temperature, or hemodynamic or metabolic data at ROSC between groups. The numbers of electric shocks, coronary perfusion pressure, ETCO2 during CPR, and CPR duration did not differ between groups (Table 2). Temperature and Hemodynamic Change The HypoT and AA+HypoT groups achieved the target body temperature within 30 minutes after cooling started (Figure 1). Immediately after ROSC, the heart rates of all resuscitated animals decreased significantly compared with the baseline. The heart rates of the NormoT and AA+NormoT groups recovered gradually. The heart rates of the animals receiving hypothermia, including HypoT and AA+HypoT groups, were significantly slower than those in the NormoT and AA+ NormoT groups (Figure 2A). The dp/dt40 and –dp/dtmax, which were representative of the systolic and diastolic functions of the heart, decreased after resuscitation and recovered gradually in all groups. Within 4 hours after ROSC, the AA+NormoT group had a significantly better dp/dt40 than the NormoT group. However, there was no significant difference in –dp/dtmax between the AA+ NormoT and NormoT groups. In contrast, the AA+HypoT group demonstrated better dp/dt40 and –dp/dtmax when compared with the HypoT group. The hemodynamic results suggested AA administrated after ROSC may improve only systolic function in normothermia,

Figure 1. Body temperature by group. AA = ascorbic acid; CPR = cardiopulmonary resuscitation; HypoT = hypothermia; NormoT = normothermia; ROSC = return of spontaneous circulation.

but improve both systolic and diastolic functions in hypothermia (Figures 2B and 2C). The AA+NormoT group did not show a significantly higher cardiac output than the NormoT group in the early post–cardiac arrest period. The AA+HypoT group exhibited a better cardiac output than the HypoT group only at the second hour after ROSC (Figure 2D). There were no differences between the HypoT and AA+NormoT groups with respect to dp/dt40, –dp/dtmax, and cardiac output. Survival and Neurologic Outcomes There were five animals surviving at 72 hours in the NormoT group, whereas there were 10, nine, and 10 animals in the HypoT, AA+NormoT, and AA+HypoT groups surviving for 72 hours, respectively. The HypoT, AA+NormoT, and AA+HypoT groups had better survival rates than the NormoT group (p < 0.05). However, there were no significantly differences among the HypoT, AA+NormoT, and AA+HypoT groups (Figure 3A). The HypoT, AA+NormoT, and AA+HypoT groups had significantly higher neurologic function

Table 2 Baseline Characteristics and Resuscitation Events of the Survival Study

Baseline Characteristics Weight (g) Heart rate at ROSC (min 1) sBP at ROSC (mm Hg) dp/dt40 at ROSC (91000 mm Hg/sec) –dp/dt max at ROSC (91000 mm Hg/sec) pH at ROSC HCO3 (mEq/L) at ROSC Resuscitation events Electric shock no. CPP (mm Hg) after 1 min of CPR ETCO2 after 1 min of CPR CPR duration (s)

NormoT (n = 12)

HypoT (n = 12)

AA+NormoT (n = 12)

AA+HypoT (n = 12)

417.8  22.4 312.5  59.3 67.4  14.4 4.36  1.66 –4.18  1.51 7.08  0.09 11.83  4.45

421.7  17.2 309.3  53.1 65.6  18.7 4.37  1.52 –4.03  1.36 7.10  0.06 12.02  3.83

413.1  23.8 318.5  49.5 70.8  15.8 4.20  1.49 –4.10  1.58 7.10  0.11 13.14  2.87

434.4  17.5 323.7  53.9 68.6  12.6 4.4  1.77 –4.23  1.60 7.10  0.09 12.29  3.83

2.5  1.1 25.10  9.62 14.82  2.31 94.8  15.2

2.2  0.9 26.34  10.29 14.37  2.28 110.0  22.9

2.4  1.1 26.75  13.52 15.35  2.30 86.4  20.6

2.3  0.8 26.23  13.60 14.63  2.17 105.5  17.7

Data are reported as mean  SD. AA = ascorbic acid; CPP = coronary perfusion pressure; CPR = cardiopulmonary resuscitation; ETCO2 = end-tidal CO2; HypoT = hypothermia; NormoT = normothermia; ROSC = return of spontaneous circulation; sBP = systolic blood pressure.

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Figure 2. Hemodynamics by group. (A) The HypoT and AA+HypoT groups had lower heart rates than the AA and NormoT groups. (B) The AA+NormoT group had a significantly better dp/dt40 than the NormoT group within 4 hours of ROSC. Also, the AA+HypoT group had a significantly better dp/dt40 than the HypoT group. (C) The AA+ HypoT groups had a significantly improved –dp/dtmax than the HypoT group within 4 hours of resuscitation. (D) The AA+HypoT group exhibited a better cardiac output than the HypoT group only at the second hour after ROSC. AA = ascorbic acid; CPR = cardiopulmonary resuscitation; HypoT = hypothermia; NormoT = normothermia; ROSC = return of spontaneous circulation. *p < 0.05 when the AA+NormoT group was compared with the NormoT group; §p < 0.05 when the AA+HypoT group was compared with the HypoT group.

scores than the NormoT group at the sixth, 24th, 48th, and 72nd hours after ROSC. When defining neurologic function scores ≥10 points as good neurologic outcome, nine animals in the HypoT, eight in the AA+NormoT, 10 in the AA+HypoT, and five in the NormoT group had good neurologic outcomes at 72 hours. There were no significant differences between the HypoT, AA+NormoT, and AA+HypoT groups with respect to neurologic function scores (Figure 3B).

sections in all groups exhibited myocytolysis and waving and transverse contraction bands. There was significantly less myocytolysis in the AA+NormoT, HypoT, and AA+HypoT groups than in the NormoT group (Figure 5). However, there was no significant difference in myocytolysis between the AA+NormoT, HypoT, and AA+HypoT groups.

MDA and Histologic Results After resuscitation, all groups had significantly elevated MDA concentrations when compared to the sham group. Within 4 hours after ROSC, the MDA concentrations in the HypoT, AA+NormoT, and AA+HypoT groups were significantly lower than that in the NormoT group; there were no significant differences between the HypoT, AA+NormoT, and AA+HypoT groups. At 24 and 72 hours after resuscitation, MDA concentrations did not differ among any groups (Figure 4). Four hours after ROSC, the HE-stained LV

This study suggests that intravenous ascorbic acid administration after ROSC in normothermia ameliorates lipid peroxidation and tissue injury in the myocardium after VF cardiac arrest and improves systolic function, survival rate, and neurologic outcomes in a rat model. Combining therapeutic hypothermia and ascorbic acid seemed to have an additive effect toward improving systolic and diastolic functions in the early postarrest period. Ischemia and reperfusion injury during cardiac arrest and CPR cause oxidative stress and contribute to

DISCUSSION

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Figure 3. Survival analysis and neurologic scaling scores by group. (A) The HypoT, AA+NormoT, and AA+HypoT groups showed better survival than the NormoT group, but there was no difference between the HypoT, AA+NormoT, and AA+HypoT groups. (B) The HypoT, AA+NormoT, and AA+HypoT groups had significantly higher neurologic function scores than the NormoT group at the sixth, 24th, 48th, and 72nd hours following ROSC. No difference was noted among the AA+NormoT, HypoT, and AA+HypoT groups. AA = ascorbic acid; HypoT = hypothermia; NormoT = normothermia; ROSC = return of spontaneous circulation. *p < 0.05 versus the normothermia group.

post–cardiac arrest syndrome, which includes both neurologic and myocardial dysfunction.20 Furthermore, electric shocks increase reactive oxygen species generation and are one of the major causes of myocardial injury after cardiac arrest.8,11,21 Our previous study demonstrated that administering ascorbic acid eliminated reactive oxygen species caused by electric shock in cardiomyocytes and preserved contractile function.10 In another animal study, we observed increased lipid peroxidation and tissue injury in the myocardium after VF cardiac arrest and electric shock, and IV ascorbic acid administration at the start of CPR ameliorated these injuries, facilitated defibrillation and resuscitation, and improved the survival rate.11 At physiologic concentrations, free radicals participate in several signal transduction pathways and play crucial roles in biological processes.22–24 However, the elevation of intracellu-

lar free radicals is one of the most critical triggers for necrotic and apoptotic cell death.25–27 The increased lipid peroxidation observed in this study implies an increase of free radicals after ischemia/reperfusion injury and electric shock, which may eventually cause myocardial damage. This study shows that ascorbic acid administered in normothermia after ROSC decreases lipid peroxidation and tissue injury in the myocardium and improves cardiac systolic function in the early post–cardiac arrest period, as well as the survival rate and neurologic outcomes. Ascorbic acid plays a critical role in redox homeostasis and reduces oxidative stress during ischemia and reperfusion.3–7 It also attenuates reperfusion arrhythmia3–5,7 and recurrent atrial fibrillation.28 In this study, ascorbic acid administration diminished MDA generation and myocardial damage after VF cardiac

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Figure 4. MDA assay. Within 4 hours of ROSC, the MDA concentration was significantly lower in the myocardium of the AA+NormoT, HypoT, and AA+HypoT groups than in the NormoT group, but there was no difference between the AA+NormoT, HypoT, and AA+HypoT groups. AA = ascorbic acid; HypoT = hypothermia; MDA = malondialdehyde; NormoT = normothermia; ROSC = return of spontaneous circulation. *p < 0.05 versus the normothermia group.

arrest and electric shock, suggesting that it reduces post–cardiac arrest myocardial injury by eliminating oxidative stress. However, the difference in myocardial injury failed to demonstrate differences in diastolic function and cardiac output. In contrast, the AA+HypoT group showed significantly improved systolic and diastolic functions compared to the HypoT group. These findings suggest that hypothermia may benefit the effect of ascorbic acid in improving both systolic and diastolic functions. There were no significant differences between the HypoT, AA+NormoT, and AA+HypoT groups in reducing MDA concentrations and myocytolysis, suggesting that both hypothermia and ascorbic acid can eliminate reactive oxygen species generation, and hypothermia and ascorbic acid may reduce myocardial damage via other

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mechanisms in addition to reducing reactive oxygen species. Within the early post–cardiac arrest period, there were no differences in cardiac output between the AA+NormoT and NormoT groups. The AA+HypoT group only showed a significantly better cardiac output than the HypoT group at the second hour following cardiac arrest. Cardiac output is dependent on heart rate, and in the current study, heart rate did not differ between the AA+NormoT and NormoT groups or between the AA+HypoT and HypoT groups. Although bradycardia developed in animals receiving hypothermia, there was no significant difference in cardiac output between the AA+NormoT and AA+HypoT groups and between the NormoT and HypoT groups. Stroke volume may have increased during hypothermia. LIMITATIONS Malondialdehyde is a by-product produced when free radicals oxidize lipids, and it is recognized as a standard measurement for determining the degree of cell oxidation. However, MDA does not directly reflect the intracellular reactive oxygen species concentrations. Second, the improved neurologic outcomes in the ascorbic acid group may be attributable to the improved hemodynamics in the early postresuscitation period. Therefore, the direct effect of ascorbic acid on brain and neurologic outcomes needs further clarification. Third, we chose 2 hours as the cooling duration in this study based on our previous study, whereas the American Heart Association suggested hypothermia for 12 to 24 hours after ROSC to improve neurologic outcomes. The shorter cooling duration in our study may limit the interpretation of the cooling effect. Fourth, we used an alpha value of 0.05 when we evaluate subgroup analysis (the AA+HypoT group vs. the HypoT group); although most alpha values were less than 0.01, the possibility of type I error may inflate. Fifth, we used healthy animals, whereas in clinical practice, VF cardiac arrest usually

Figure 5. Histologic examination by group. At the fourth hour after ROSC, there was significantly less myocytolysis in the AA+NormoT group than in the NormoT group. There was no difference between the HypoT, AA+NormoT, and AA+HypoT groups. AA = ascorbic acid; HE stain = hematoxylin and eosin stain; HypoT = hypothermia; NormoT = normothermia; ROSC = return of spontaneous circulation. *p < 0.05 versus the normothermia group.

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Tsai et al. • ASCORBIC ACID IMPROVES OUTCOMES IN CARDIAC ARREST IN RATS

accompanies ischemic heart diseases and/or myocardial infarction. Because these clinical conditions are more complex than the animal models, the present results should be interpreted cautiously when being applied to clinical practice. In addition, the electric shock doses chosen in this study were based on rat resuscitation models in other laboratories and our previous pilot studies. The strength of these shocks is larger than that used on humans in clinical practice on a per-weight basis. Therefore, the injury caused by electric shocks may be accentuated in this study, potentially affecting the injury mechanisms. Last, ascorbic acid was administered as a single dose. Whether continuous infusion or repeated bolus injection of ascorbic acid maintains the benefits of improved hemodynamics needs further investigation. CONCLUSIONS Intravenous administration of ascorbic acid after return of spontaneous circulation in normothermia may reduce lipid peroxidation and myocardial tissue injury and improve systolic function, survival rate, and neurologic outcomes in a rat model of ventricular fibrillation cardiac arrest. Combining therapeutic hypothermia and ascorbic acid seemed to confer an additive effect in improving both systolic and diastolic functions during the early post–cardiac arrest period. References 1. Peatfield RC, Sillett RW, Taylor D, McNicol MW. Survival after cardiac arrest in the hospital. Lancet 1997;1:1223–5. 2. Lloyd-Jones D, Adams R, Carnethon M, et al. American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics–2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 2009;119:480–6. 3. Woodward B, Zakaria MN. Effect of some free radical scavengers on reperfusion induced arrhythmias in the isolated rat heart. J Mol Cell Cardiol 1985;17:485–93. 4. Tan DX, Manchester LC, Reiter RJ, Qi W, Kim SJ, El-Sokkary GH. Ischemia/reperfusion-induced arrhythmias in the isolated rat heart: prevention by melatonin. J Pineal Res 1998;25:184–91. 5. Karahaliou A, Katsouras C, Koulouras V, et al. Ventricular arrhythmias and antioxidative medication: experimental study. Hellenic J Cardiol 2008;49:320– 8. 6. Senthil S. Veerappan RM, Ramakrishna Rao M, Pugalendi KV. Oxidative stress and antioxidants in patients with cardiogenic shock complicating acute myocardial infarction. Clin Chim Acta 2004;348:131– 7. 7. Gao F, Yao CL, Gao E, et al. Enhancement of glutathione cardioprotection by ascorbic acid in myocardial reperfusion injury. J Pharmacol Exp Ther 2002;301:543–50.

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Combination of intravenous ascorbic acid administration and hypothermia after resuscitation improves myocardial function and survival in a ventricular fibrillation cardiac arrest model in the rat.

Intravenous (IV) administration of ascorbic acid during cardiopulmonary resuscitation (CPR) was reported to facilitate defibrillation and improves sur...
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