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The neuroprotective effects of intraperitoneal injection of hydrogen in rabbits with cardiac arrest
Resuscitation 84 (2013) 690–695
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Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Experimental paper
The neuroprotective effects of intraperitoneal injection of hydrogen in rabbits with cardiac arrest夽,夽夽 Guoqing Huang a,b,d , Jun Zhou c,d , Wei Zhan a , Yan Xiong a , Chunlin Hu a , Xiangmin Li b , Xin Li a , Yingqing Li a , Xiaoxing Liao a,∗ a
Department of Emergency, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China Department of Emergency, Xiangya Hospital of Central South University, Changsha 410008, China c Department of Anesthesiology, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China b
a r t i c l e
i n f o
Article history: Received 9 July 2012 Received in revised form 11 October 2012 Accepted 18 October 2012
Keywords: Cardiac arrest Cardiopulmonary resuscitation Oxidative stress Hydrogen
a b s t r a c t Objective: The purpose of this study was to investigate the neuroprotective effects of intraperitoneal injection of hydrogen (H2 ) in rabbits with cardiac arrest (CA). Methods: A rabbit model of CA was established by the delivery of alternating current between the esophagus and chest wall to induce ventricular fibrillation. Before CA, the animals were randomly divided into four groups: a sham group (no CA), a CA group, a CA + low dose (10 ml/kg) H2 group (CA + H2 group 1), and a CA + high dose (20 ml/kg) H2 group (CA + H2 group 2). In the first experiment, animals were observed for 72 h after the restoration of spontaneous circulation (ROSC). The neurological scores were assessed at 24, 48 and 72 h after ROSC. The rabbits that survived until 72 h were sacrificed using an overdose of anesthetic, and the brain tissues were collected and Nissl-stained to observe nerve cell damage in the hippocampal CA1 area. In addition, TUNEL assay was performed to detect apoptosis. In the second experiment, animals were observed for 6 h after ROSC. Blood samples and brain hippocampal tissues were collected, and differences in oxidative stress indicators were compared among the four groups. Results: Intraperitoneal injection of H2 improved the 72-h survival rate and neurological scores, reduced neuronal injury and inhibited neuronal apoptosis. Intraperitoneal injection of H2 reduced oxidative stress indicators in the plasma and hippocampal tissues and enhanced antioxidant enzyme activity. No significant difference was observed between the two CA groups treated with different doses of H2 . Conclusions: Intraperitoneal injection of H2 is a novel hydrogen administration method and can reduce cerebral ischemia-reperfusion injury and improve the prognosis of cardiopulmonary cerebral resuscitation in a rabbit model of CA. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: H2 , hydrogen; CA, cardiac arrest; ROSC, restoration of spontaneous circulation; CPR, cardiopulmonary resuscitation; HIS, immunohistochemical score; TUNEL, TdT-mediated dUTP nick-end labeling; 8-OHDG, 8-hydroxydeoxyguanosine; MDA, malondialdehyde; TBA, thiobarbituric acid; WST1, water-soluble tetrazolium salt; CAT, catalase; SD, standard deviation; ANOVA, analysis of variance; LSD, least significant difference; • OH, hydroxyl radical; ROS, reactive oxygen species; ONOO-, peroxynitrite; O2 • , superoxide anion; Nrf2, Nuclear factor-E2 related factor 2; AMI, acute myocardial infarction. 夽 A Spanish translated version of the summary of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2012.10.018. 夽夽 Institutional protocol number: [2012]146. ∗ Corresponding author. Tel.: +86 20 87755766 8500/13922132259; fax: +86 020 87750632. E-mail addresses: [email protected], [email protected] (X. Liao). d These two authors contributed equally to this work as co-first authors. 0300-9572/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.resuscitation.2012.10.018
Cardiac arrest (CA) is a medical emergency that is a serious threat to human life. Recently, with the advances in cardiopulmonary resuscitation (CPR) techniques, a continuous rise in the rate of restoration of spontaneous circulation (ROSC) has been achieved in patients with CA.1 However, the final survival rate remains very low, since only 7.9–8.5% of out-of-hospital patients with CA survive to hospital discharge.2,3 Furthermore, about 50% of patients that survive to hospital discharge are complicated by moderate or severe neurological dysfunction. Therefore, enhancement of the success rate of CPR and reduction of impairment of cerebral function is still a hot topic for research in emergency medicine. Systemic ischemia-reperfusion injury is the most important pathophysiological process after CA, while oxidative stress and inflammation are the leading causes of systemic ischemia-reperfusion injury.4 In 2007, inhalation of hydrogen (H2 ) was shown to reduce cerebral infarct area and improve prognosis in a rat model of stroke,
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and H2 was considered to act as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals.5 Since then, H2 has been demonstrated to protect tissues and organs from ischemiareperfusion injury in several other animal models.6–10 In addition to inhalation, some other administration techniques of H2 have been developed, such as intravenous injection of H2 -rich saline,10,11 intraperitoneal injection of H2 -rich saline8,12 and drinking H2 containing water.7,13 In the present study, a rabbit model of CA was treated with intraperitoneal injection of H2 to investigate the effect of H2 on the prognosis of cardiopulmonary cerebral resuscitation. 2. Materials and methods 2.1. Animals All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Sun Yat-Sen University (Institutional protocol number: [2012]146). New Zealand white rabbits were purchased from the Laboratory Animal Center of Sun Yat-Sen University and raised in individual cages. 2.2. Establishment of a CA animal model A rabbit model of CA was established by the delivery of alternating current between the esophagus and chest wall to induce ventricular fibrillation. All animals were fasted but given free access to water on the night prior to the experiment. A 24-guage venous needle was used for right auricular vein puncture, and pentobarbital sodium (Sigma, USA) at a dose of 30 mg/kg was injected intravenously for anesthesia. The left ear artery was punctured using a 22-gauge venous needle to monitor arterial blood pressure. A BL-420s multichannel physiological signal recording system (Chengdu Taimeng Science and Technology Co., Ltd., Chengdu, China) was used to record the electrocardiogram and arterial blood pressure. A tracheal tube with internal diameter of 3.0 mm was inserted via the mouth using a blind endotracheal intubation method and fixed at a distance of 11 cm between the distal end of the tube and the incisor. A 30-guage acupuncture needle (0.30 mm × 2 mm) was inserted subcutaneously into the precordial region where the apical pulse was strongest, and esophageal pacing electrodes were implanted in the esophagus. The distance from the location of the metal ring of the electrodes to the incisor was 16 cm. We induced ventricular fibrillation with 35-mA constant current at a frequency of 50 Hz. CA was identified using the following criteria: (1) the systolic arterial pressure after electrical stimulation gradually fell to below 25 mmHg; and (2) pulsations in the arterial pressure waveform disappeared. After reaching the criteria for CA, electrical stimulation was performed for 1 min followed by 4 min of observation without treatment. After 5 min of CA, CPR was performed as previously described.14 The indicators of ROSC included recovery of a supraventricular rhythm and a mean arterial pressure ≥60 mmHg that was sustained for >10 min. Animals without ROSC following standard cardiopulmonary resuscitation (CPR) for 15 min were defined as resuscitation failures. 2.3. Preparation of H2 A M177021 hydrogen generator (Beijing Midwest Yuanda Technology Co., Ltd., Beijing, China) was used to produce pure H2 (99.999% purity) by electrolysis of water. The H2 produced was stored in aseptic soft plastic infusion bags and was used the same day when they were prepared.
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2.4. Experimental design Sixty-six New Zealand white rabbits were randomly assigned to one of four treatment groups: a sham group (no CA), a CA group, a CA + H2 group 1, and a CA + H2 group 2. The core temperature of the rabbits was continuously measured with a rectal temperature probe, which was maintained at 39 ± 0.5 ◦ C using an infrared thermolamp until awake or 4 h after ROSC. Arterial and venous catheterization, anesthesia and endotracheal intubation were performed in the sham group. An esophageal electrode was implanted in the sham group with a length of 10 cm from the incisor, and then electrical stimulation using the same parameters was performed for 90 s to induce generalized twitching but not CA. In the three CA groups, ventricular fibrillation was induced for 5 min and then standard CPR was performed. Animals in the CA group were given an intraperitoneal injection of prewarmed physiological saline at a dose of 10 ml/kg 30 s prior to CPR; rabbits in CA + H2 group 1 and CA + H2 group 2 were given intraperitoneal injections of H2 at doses of 10 and 20 ml/kg, respectively. The first experiment was designed to investigate the effect of intraperitoneal injection of H2 on survival rate and neurological function in rabbits with CA. Forty-two New Zealand white rabbits were randomly divided into the a sham group (6 rabbits), a CA group (12 rabbits), a CA + H2 group 1 (12 rabbits) and a CA + H2 group 2 (12 rabbits). The electrocardiogram and blood pressure was monitored for 4 h after ROSC in the CA group and the two CA + H2 groups. During this period, those animals with weak spontaneous respiration underwent mechanical ventilation, but no other drugs were given. Respiration was assessed every 15 min to determine if further mechanical ventilation was necessary. Four hours later, mechanical ventilation was terminated. The tracheal tube was removed, and each rabbit was returned to its cage. The duration of mechanical ventilation after ROSC was defined as the period between the termination of CPR and the termination of mechanical ventilation. Rabbits that were still in a coma after 12 h of ROSC and could not drink water were given 5% glucose-saline at a dose of 1 ml/kg/h using a mini-pump. When the rabbits spontaneously drank water, the intravenous infusion was terminated. The survival duration after resuscitation was observed until 72 h. At 24, 48 and 72 h after ROSC, the neurological function of rabbits was scored using the 5-score evaluation method as previously described.15 Scores 1 and 2 were considered to indicate a good prognosis of neurological function, whereas score 3 and greater were considered to indicate a poor prognosis. Those surviving to the termination of the observation period were sacrificed using an overdose of anesthetic. The brain was removed and fixed in 4% paraformaldehyde. Then, 3–5 mm thick brain tissues posterior to the optic chiasma in the coronal plane were cut and embedded in paraffin for pathological examination. The second experiment was designed to assess the effect of intraperitoneal injection of H2 on oxidative stress in rabbits with CA. Twenty-four rabbits were randomly assigned to one of the four treatment groups with 6 animals in each group. The observation period was terminated 6 h after CPR. Blood samples were collected before and 6 h after CPR, and centrifuged at 4000 rpm for 10 min. The plasma was collected and stored at −80 ◦ C for subsequent analysis. After 6 h of observation, the rabbits were sacrificed using an overdose of anesthetic and perfused with 0.9% physiological saline at 4 ◦ C via the heart. The brain was removed and the hippocampal tissues were isolated on ice. The hippocampus was mixed with physiological saline and ground at 4000 rpm for 10 min. The supernatant was 10% tissue homogenate, and this was stored at −80 ◦ C for subsequent analysis.
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2.5. Nissl staining and TUNEL (TdT-mediated dUTP nick-end labeling) assay The paraffin-embedded brain tissues were cut into sections of 5 m, and Nissl staining (Beyotime Institute of Biotechnology, Nantong, China) and TUNEL assay (ROCHE, USA) were performed for in situ detection of apoptosis. Five fields of vision (magnification of 40×) in the hippocampal CA1 area were randomly sampled from each brain tissue sample, and the Nissl-stained neuronal cells were counted. For TUNEL staining, the immunohistochemical score (IHS) is determined by the percentage of positive-stained cells and the intensity of staining as previously described.16 2.6. Determination of biochemical indicators All biochemical indicators were determined with the corresponding reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturers’ instructions. The 8-hydroxydeoxyguanosine (8-OHDG) levels in plasma and brain tissues were determined with ELISA reagent kits, and the malondialdehyde (MDA) level in plasma and brain tissues was determined using the thiobarbituric acid (TBA) method. The superoxide dismutase (SOD) activity was measured using a water-soluble tetrazolium salt (WST-1) and catalase (CAT) activity was detected using ultraviolet spectrophotometry. 2.7. Statistical analysis All measurement data were expressed as mean ± standard deviation (SD), and all statistical analyses were performed using statistical software (SPSS version 13.0). The normality test showed the data were consistent with a normal distribution. A paired t test was used to compare the same parameters before and after treatment. Comparison of the same parameters among groups was done using one-way analysis of variance (ANOVA), and the difference between pairs of means was tested post hoc with Fisher’s Least Significant Difference (LSD) test. Fisher’s exact test was employed to compare the neurological scores between groups. The difference in survival rates among the various groups was compared using Kaplan–Meier survival analysis. A P-value <0.05 was considered statistically significant.
Fig. 1. Survival curves for the 4 groups. # P < 0.05 and *P < 0.01 vs. the sham group revealed by Kaplan–Meier survival analysis; P < 0.05 vs. the CA group.
3.3. Neurological scores There were significant differences in neurological scores at 24, 48 and 72 h after ROSC between the three groups with CA and the sham group (P < 0.05 or 0.01). Although a significant difference in neurological score was observed at 72 h after ROSC between the CA group and CA + H2 group 2, no other significant differences were found between the 3 groups with CA at the various time points after ROSC (Fig. 2). 3.4. Nissl staining and TUNEL assay
There were no significant differences in sex ratio, body weight, basal body temperature, breathing frequency, heart rate and mean arterial pressure of rabbits among the four groups.
Nissl staining revealed a loose structure of the brain tissue in the CA group, which exhibited vacuolar degeneration. The number of neurons was significantly reduced, and the nucleus was compressed; a Nissl body was not found in the cytoplasm. In the two CA + H2 groups, many neurons were found, the nucleus was normal and there were many Nissl bodies observed in the cytoplasm. Compared with the sham group, the number of neurons in other three groups was significantly reduced (P < 0.01), whereas the numbers of neurons in the two CA + H2 groups were significantly greater than that in the CA group (P < 0.05). TUNEL staining showed many strongly TUNEL-positive apoptotic cells in the CA group, and a few weakly positive cells in the two CA + H2 groups. However, no TUNEL-positive cells were found in the sham group. Compared with the sham group, the TUNEL staining immunohistochemistry scores were significantly increased in the other three groups (P < 0.01). However, the scores in the two CA + H2 groups were significantly lower than that in the CA group (P < 0.05) (Fig. 3).
3.2. Survival of rabbits
3.5. Biochemical indicators in blood samples
Ventricular fibrillation was successfully induced in all rabbits in the CA group and the two CA + H2 groups. In the first experiment, one rabbit in the CA group could not be resuscitated, whereas the others all had successful resuscitation. There were no significant differences in the resuscitation parameters among the groups (P > 0.05) (Table 1). The 72-h survival rates in the sham group, CA group, CA + H2 group 1 and CA + H2 group 2 were 100% (6/6), 16.7% (2/12), 41.7% (5/12) and 50% (6/12), respectively. The survival rate of rabbits in the sham group was significantly different from those in the 3 groups with CA. In addition, a significant difference in the survival rate was found between the CA + H2 group 2 and the CA group (P < 0.05) (Fig. 1).
The plasma 8-OHDG and MDA levels significantly increased from before to after ROSC in all four groups, except for the plasma 8-OHDG level in the sham group. Furthermore, the SOD activity significantly decreased in all four groups (P < 0.01). The plasma 8OHDG and MDA levels in the three groups with CA at 6 h after ROSC were significantly greater than those in the sham group (P < 0.01), whereas the SOD activities in the three groups with CA were significantly lower than that in the sham group (P < 0.01). At 6 h after ROSC, the plasma MDA levels in the two CA + H2 groups were significantly lower than that in the CA group (P < 0.01), whereas the SOD activities in the two CA + H2 groups were significantly higher than that in the CA group (P < 0.05) (Fig. 4).
3. Results 3.1. General conditions of rabbits
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Table 1 Comparison of resuscitation parameters in the 3 groups with CA. Group
ROSC (n)
ICAT (s)
DOCPR (s)
DF
AF
MVT (min)
CA CA + low dose of H2 CA + high dose of H2
11 12 12
35.45 ± 14.52 37.33 ± 16.97 34.58 ± 13.08
221.73 ± 89.64 213.50 ± 102.50 208.30 ± 96.68
0.73 ± 0.65 0.75 ± 0.62 0.33 ± 0.49
3.77 ± 1.89 3.04 ± 2.46 2.54 ± 1.31
30.18 ± 11.72 31.00 ± 13.31 24.32 ± 13.84
ROSC: restoration of spontaneous circulation; ICAT: induced CA time (s); DOCPR: duration of CPR(s); DF: defibrillation frequency; AF: administration frequency; MVT: mechanical ventilation time (min).
Fig. 2. Comparison of the neurological scores in the 4 groups. (A) 24 h, (B) 48 h, (C) 72 h. # P < 0.05 and *P < 0.01 vs. the sham group revealed by Fisher’s exact test; P < 0.05 vs. the CA group.
Fig. 3. Nissl staining and TUNEL staining of the hippocampal CA1 area in the 4 groups. (A) The pathological changes in CA1 region; (B) the number of vital neurons; (C) the HIS of TUNEL staining. *P < 0.01 vs. the sham group; P < 0.05 vs. the CA group. Bar = 50 m.
Fig. 4. The biochemical parameters in sera of rabbits from the 4 groups. The level of: (A) 8-OHGD, (B) MDA; (C) the activity of SOD. # P < 0.01 of the self-control before and after treatment; § P < 0.05 of the self-control before and after treatment; *P < 0.01 vs. the sham group at the same time point revealed by the LSD test; P < 0.05 and P < 0.01 vs. the CA group at the same time point revealed by the LSD test.
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Fig. 5. The biochemical parameters in brain tissues of rabbits from the 4 groups. The level of: (A) 8-OHGD, (B) MDA; the activity of (C) SOD, (D) CAT. *P < 0.01 vs. the sham group revealed by the LSD test; P < 0.05 and # P < 0.01 vs. the CA group.
3.6. Biochemical indicators in brain tissues The 8-OHDG and MDA levels in hippocampal tissues were significantly greater in the three groups with CA than those in the sham group (P < 0.01). However, significantly lower SOD and CAT activities were detected in the three groups with CA than those in the sham group (P < 0.01). Compared with the CA group, the 8OHDG and MDA levels in the hippocampal tissues in the two CA + H2 groups were significantly reduced (P < 0.05 or 0.01), whereas the SOD and CAT activities were significantly increased (P < 0.05 or 0.01) (Fig. 5). 4. Discussion Sudden cardiac death is mainly caused by malignant arrhythmias in clinical practice, and the arrhythmia first recorded in 75–80% of the patients with CA is ventricular fibrillation.17 This arrhythmia is well simulated by electric simulation of the heart in experimental animals. Compared with alternating current delivered to the right ventricular endocardium,18 open-chest stimulation of the epicardium 19 and transcutaneous electrical epicardial stimulation,20 ventricular fibrillation induced by stimulation between the esophagus and chest wall electrodes in the present study was easier to perform, less harmful and more reproducible. Previous studies have investigated different methods of H2 administration such as inhalation of H2 , or intravenous or intraperitoneal injection of H2 -rich saline. However, the flammability and easy explosion of H2 is a serious risk with inhalation of H2 . Furthermore, H2 -rich saline used for intraperitoneal injection must be prepared in advance, which is not beneficial for long-term preservation. Direct intraperitoneal injection of H2 is a novel method of H2 administration that we developed. According to the principle of pneumoperitoneum in laparoscopic operation, direct intraperitoneal injection of H2 should not lead to a significant change of abdominal pressure due to the small injection volume at a dose of 10–20 ml/kg. Our previous study demonstrated that intraperitoneal injection of H2 at a dose of 10 ml/kg rapidly increases the concentration of expired H2 in rabbits; the half-life was more than 2 h and the concentration of H2 was effectively sustained for about 6 h. The present study found that intraperitoneal injection of H2 prior to CPR improved the survival rate and neurological function in a rabbit model of CA. The major pathological change after global ischemia is delayed neuronal cell death, and histological evidence is usually present 2–3 days after global ischemia.21 The hippocampus is the most sensitive region to cerebral ischemia, which is closely related to cognitive dysfunction after ischemia-reperfusion injury. In the present study, therefore, the hippocampal CA1 area of the rabbits surviving until 72 h was selected for pathological examination. Nissl staining and TUNEL assay revealed various degrees of neuronal degeneration, necrosis and apoptosis in rabbits after 5 min of CA, and the degree of pathological changes was consistent
with the general neurological scores. The intraperitoneal injection of H2 reduced neuronal damages induced by 5 min of CA, and few apoptotic cells were detected. Currently, the protection mechanism of H2 against ischemiareperfusion injury is not fully clear. H2 has been shown to selectively neutralize the hydroxyl radical (• OH), the most cytotoxic of reactive oxygen species (ROS), and peroxynitrite (ONOO− ), but does not react with superoxide anion (O2 • ).5 The present study demonstrated that systemic hypoxia, ischemia and reperfusion after 5 min of CA caused severe oxidative damage to rabbits, and the plasma MDA and 8-OHDG levels significantly increased in the three groups with CA at 6 h after ROSC. Although CA was not induced in the sham group, the electric stimulation for 90 s caused generalized twitching and significantly elevated MDA levels compared with that before stimulation. In addition, the MDA and 8-OHDG levels either in plasma or brain tissues of rabbits with CA were significantly lower than those in the sham group at 6 h after ROSC, suggesting that administration of H2 reduced systemic and cerebral oxidative damage after CA. Since H2 exhibits non-antioxidant signal regulation 22 and affects gene expression to some extent,23 we cannot exclude an effect of H2 on the endogenous protection pathway against ischemia-reperfusion injury. In the current study, the activities of two important antioxidant enzymes, SOD and CAT, were determined. The antioxidant enzymes are converted into nontoxic or low-toxicity products through catalyzing free radicals and through increasing the water solubility of their products for excretion, and this is important to maintain redox equilibrium.24 The SOD activity in each of the three groups with CA was reduced compared with the basal level, indicating that there was consumption of SOD. The SOD activities in the plasma and hippocampal tissues and the CAT activity in the hippocampal tissues after intraperitoneal injection of H2 were significantly greater in the two CA + H2 groups than those in the CA group, which indicates that there was less of a reduction in the consumption of antioxidant enzymes due to neutralization of free radicals by H2 . In addition, H2 may increase the expression of the Nrf2 (Nuclear factor-E2 related factor 2) downstream protective genes through activation of the Nrf2 signaling pathway. Oxidative stress induced by reperfusion after CA leads to the exhaustion of cerebral antioxidant reserves and causes severe oxidative damage. However, the activity of free radicals after ischemia is characterized by a sudden onset and a short duration of action, and the cerebral antioxidant reserves are restored within 2 h after reperfusion.25 Therefore, antioxidants should be introduced into tissues to treat ischemia-reperfusion injury when reperfusion is performed. Many antioxidants have been applied in experimental studies and show active protection against ischemia-reperfusion injury.26,27 Unfortunately, antioxidants are difficult to deliver when CPR is performed at the scene of resuscitation. In addition, nonselective antioxidants may lead to oxidant/antioxidant imbalance and interfere with the normal physiological effects of oxygen free radicals. Intraperitoneal injection of H2 is a novel, safe, fast and effective method to administer H2 to treat ischemia-reperfusion
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injury and may be potentially useful in the clinical setting. Acute myocardial infarction (AMI) is an important cause of CA. Thrombolytic therapy is an effective treatment for CA caused by AMI after ROSC.28 In this study, intraperitoneal injection of H2 prior to CPR did not cause coagulation disorders and should not be a contraindication of thrombolytic therapy. However, further studies are needed to investigate the optimal dose of H2 and its duration of use. Conflict of interest The authors declare no conflict of interest. Acknowledgements This study was supported by funding of the NSFC (81071030), Science and Technology Foundation of Guangdong Province, China (2011B080701006) and Young Teacher Foundation of Sun Yat-sen University (80000-3171914). The authors express their gratitude to the staff of Pathophysiology laboratory of Sun Yat-sen University for their excellent technical help and constructive criticism. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.resuscitation.2012.10.018. References 1. Peberdy MA, Kaye W, Ornato JP, et al. Cardiopulmonary resuscitation of adults in the hospital: a report of 14720 cardiac arrests from the National registry of cardiopulmonary resuscitation. Resuscitation 2003;58:297–308. 2. Nichol G, Thomas E, Callaway CW, et al. Regional variation in out-of-hospital cardiac arrest incidence and outcome. JAMA 2008;300:1423–31. 3. Brooks SC, Schmicker RH, Rea TD, et al. Out-of-hospital cardiac arrest frequency and survival: evidence for temporal variability. Resuscitation 2010;81:175–81. 4. Zhang N, Komine-Kobayashi M, Tanaka R, et al. Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 2005;36:2220–5. 5. Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007;13:688–94. 6. Hayashida K, Sano M, Ohsawa I, et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun 2008;373:30–5. 7. Ohsawa I, Nishimaki K, Yamagata K, et al. Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice. Biochem Biophys Res Commun 2008;377:1195–8. 8. Wang F, Yu G, Liu SY, et al. Hydrogen-rich saline protects against renal ischemia/reperfusion injury in rats. J Surg Res 2011;167:e339–44. 9. Buchholz BM, Kaczorowski DJ, Sugimoto R, et al. Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury. Am J Transplant 2008;8:2015–24.
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Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Experimental paper
The neuroprotective effects of intraperitoneal injection of hydrogen in rabbits with cardiac arrest夽,夽夽 Guoqing Huang a,b,d , Jun Zhou c,d , Wei Zhan a , Yan Xiong a , Chunlin Hu a , Xiangmin Li b , Xin Li a , Yingqing Li a , Xiaoxing Liao a,∗ a
Department of Emergency, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China Department of Emergency, Xiangya Hospital of Central South University, Changsha 410008, China c Department of Anesthesiology, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China b
a r t i c l e
i n f o
Article history: Received 9 July 2012 Received in revised form 11 October 2012 Accepted 18 October 2012
Keywords: Cardiac arrest Cardiopulmonary resuscitation Oxidative stress Hydrogen
a b s t r a c t Objective: The purpose of this study was to investigate the neuroprotective effects of intraperitoneal injection of hydrogen (H2 ) in rabbits with cardiac arrest (CA). Methods: A rabbit model of CA was established by the delivery of alternating current between the esophagus and chest wall to induce ventricular fibrillation. Before CA, the animals were randomly divided into four groups: a sham group (no CA), a CA group, a CA + low dose (10 ml/kg) H2 group (CA + H2 group 1), and a CA + high dose (20 ml/kg) H2 group (CA + H2 group 2). In the first experiment, animals were observed for 72 h after the restoration of spontaneous circulation (ROSC). The neurological scores were assessed at 24, 48 and 72 h after ROSC. The rabbits that survived until 72 h were sacrificed using an overdose of anesthetic, and the brain tissues were collected and Nissl-stained to observe nerve cell damage in the hippocampal CA1 area. In addition, TUNEL assay was performed to detect apoptosis. In the second experiment, animals were observed for 6 h after ROSC. Blood samples and brain hippocampal tissues were collected, and differences in oxidative stress indicators were compared among the four groups. Results: Intraperitoneal injection of H2 improved the 72-h survival rate and neurological scores, reduced neuronal injury and inhibited neuronal apoptosis. Intraperitoneal injection of H2 reduced oxidative stress indicators in the plasma and hippocampal tissues and enhanced antioxidant enzyme activity. No significant difference was observed between the two CA groups treated with different doses of H2 . Conclusions: Intraperitoneal injection of H2 is a novel hydrogen administration method and can reduce cerebral ischemia-reperfusion injury and improve the prognosis of cardiopulmonary cerebral resuscitation in a rabbit model of CA. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: H2 , hydrogen; CA, cardiac arrest; ROSC, restoration of spontaneous circulation; CPR, cardiopulmonary resuscitation; HIS, immunohistochemical score; TUNEL, TdT-mediated dUTP nick-end labeling; 8-OHDG, 8-hydroxydeoxyguanosine; MDA, malondialdehyde; TBA, thiobarbituric acid; WST1, water-soluble tetrazolium salt; CAT, catalase; SD, standard deviation; ANOVA, analysis of variance; LSD, least significant difference; • OH, hydroxyl radical; ROS, reactive oxygen species; ONOO-, peroxynitrite; O2 • , superoxide anion; Nrf2, Nuclear factor-E2 related factor 2; AMI, acute myocardial infarction. 夽 A Spanish translated version of the summary of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2012.10.018. 夽夽 Institutional protocol number: [2012]146. ∗ Corresponding author. Tel.: +86 20 87755766 8500/13922132259; fax: +86 020 87750632. E-mail addresses: [email protected], [email protected] (X. Liao). d These two authors contributed equally to this work as co-first authors. 0300-9572/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.resuscitation.2012.10.018
Cardiac arrest (CA) is a medical emergency that is a serious threat to human life. Recently, with the advances in cardiopulmonary resuscitation (CPR) techniques, a continuous rise in the rate of restoration of spontaneous circulation (ROSC) has been achieved in patients with CA.1 However, the final survival rate remains very low, since only 7.9–8.5% of out-of-hospital patients with CA survive to hospital discharge.2,3 Furthermore, about 50% of patients that survive to hospital discharge are complicated by moderate or severe neurological dysfunction. Therefore, enhancement of the success rate of CPR and reduction of impairment of cerebral function is still a hot topic for research in emergency medicine. Systemic ischemia-reperfusion injury is the most important pathophysiological process after CA, while oxidative stress and inflammation are the leading causes of systemic ischemia-reperfusion injury.4 In 2007, inhalation of hydrogen (H2 ) was shown to reduce cerebral infarct area and improve prognosis in a rat model of stroke,
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and H2 was considered to act as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals.5 Since then, H2 has been demonstrated to protect tissues and organs from ischemiareperfusion injury in several other animal models.6–10 In addition to inhalation, some other administration techniques of H2 have been developed, such as intravenous injection of H2 -rich saline,10,11 intraperitoneal injection of H2 -rich saline8,12 and drinking H2 containing water.7,13 In the present study, a rabbit model of CA was treated with intraperitoneal injection of H2 to investigate the effect of H2 on the prognosis of cardiopulmonary cerebral resuscitation. 2. Materials and methods 2.1. Animals All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Sun Yat-Sen University (Institutional protocol number: [2012]146). New Zealand white rabbits were purchased from the Laboratory Animal Center of Sun Yat-Sen University and raised in individual cages. 2.2. Establishment of a CA animal model A rabbit model of CA was established by the delivery of alternating current between the esophagus and chest wall to induce ventricular fibrillation. All animals were fasted but given free access to water on the night prior to the experiment. A 24-guage venous needle was used for right auricular vein puncture, and pentobarbital sodium (Sigma, USA) at a dose of 30 mg/kg was injected intravenously for anesthesia. The left ear artery was punctured using a 22-gauge venous needle to monitor arterial blood pressure. A BL-420s multichannel physiological signal recording system (Chengdu Taimeng Science and Technology Co., Ltd., Chengdu, China) was used to record the electrocardiogram and arterial blood pressure. A tracheal tube with internal diameter of 3.0 mm was inserted via the mouth using a blind endotracheal intubation method and fixed at a distance of 11 cm between the distal end of the tube and the incisor. A 30-guage acupuncture needle (0.30 mm × 2 mm) was inserted subcutaneously into the precordial region where the apical pulse was strongest, and esophageal pacing electrodes were implanted in the esophagus. The distance from the location of the metal ring of the electrodes to the incisor was 16 cm. We induced ventricular fibrillation with 35-mA constant current at a frequency of 50 Hz. CA was identified using the following criteria: (1) the systolic arterial pressure after electrical stimulation gradually fell to below 25 mmHg; and (2) pulsations in the arterial pressure waveform disappeared. After reaching the criteria for CA, electrical stimulation was performed for 1 min followed by 4 min of observation without treatment. After 5 min of CA, CPR was performed as previously described.14 The indicators of ROSC included recovery of a supraventricular rhythm and a mean arterial pressure ≥60 mmHg that was sustained for >10 min. Animals without ROSC following standard cardiopulmonary resuscitation (CPR) for 15 min were defined as resuscitation failures. 2.3. Preparation of H2 A M177021 hydrogen generator (Beijing Midwest Yuanda Technology Co., Ltd., Beijing, China) was used to produce pure H2 (99.999% purity) by electrolysis of water. The H2 produced was stored in aseptic soft plastic infusion bags and was used the same day when they were prepared.
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2.4. Experimental design Sixty-six New Zealand white rabbits were randomly assigned to one of four treatment groups: a sham group (no CA), a CA group, a CA + H2 group 1, and a CA + H2 group 2. The core temperature of the rabbits was continuously measured with a rectal temperature probe, which was maintained at 39 ± 0.5 ◦ C using an infrared thermolamp until awake or 4 h after ROSC. Arterial and venous catheterization, anesthesia and endotracheal intubation were performed in the sham group. An esophageal electrode was implanted in the sham group with a length of 10 cm from the incisor, and then electrical stimulation using the same parameters was performed for 90 s to induce generalized twitching but not CA. In the three CA groups, ventricular fibrillation was induced for 5 min and then standard CPR was performed. Animals in the CA group were given an intraperitoneal injection of prewarmed physiological saline at a dose of 10 ml/kg 30 s prior to CPR; rabbits in CA + H2 group 1 and CA + H2 group 2 were given intraperitoneal injections of H2 at doses of 10 and 20 ml/kg, respectively. The first experiment was designed to investigate the effect of intraperitoneal injection of H2 on survival rate and neurological function in rabbits with CA. Forty-two New Zealand white rabbits were randomly divided into the a sham group (6 rabbits), a CA group (12 rabbits), a CA + H2 group 1 (12 rabbits) and a CA + H2 group 2 (12 rabbits). The electrocardiogram and blood pressure was monitored for 4 h after ROSC in the CA group and the two CA + H2 groups. During this period, those animals with weak spontaneous respiration underwent mechanical ventilation, but no other drugs were given. Respiration was assessed every 15 min to determine if further mechanical ventilation was necessary. Four hours later, mechanical ventilation was terminated. The tracheal tube was removed, and each rabbit was returned to its cage. The duration of mechanical ventilation after ROSC was defined as the period between the termination of CPR and the termination of mechanical ventilation. Rabbits that were still in a coma after 12 h of ROSC and could not drink water were given 5% glucose-saline at a dose of 1 ml/kg/h using a mini-pump. When the rabbits spontaneously drank water, the intravenous infusion was terminated. The survival duration after resuscitation was observed until 72 h. At 24, 48 and 72 h after ROSC, the neurological function of rabbits was scored using the 5-score evaluation method as previously described.15 Scores 1 and 2 were considered to indicate a good prognosis of neurological function, whereas score 3 and greater were considered to indicate a poor prognosis. Those surviving to the termination of the observation period were sacrificed using an overdose of anesthetic. The brain was removed and fixed in 4% paraformaldehyde. Then, 3–5 mm thick brain tissues posterior to the optic chiasma in the coronal plane were cut and embedded in paraffin for pathological examination. The second experiment was designed to assess the effect of intraperitoneal injection of H2 on oxidative stress in rabbits with CA. Twenty-four rabbits were randomly assigned to one of the four treatment groups with 6 animals in each group. The observation period was terminated 6 h after CPR. Blood samples were collected before and 6 h after CPR, and centrifuged at 4000 rpm for 10 min. The plasma was collected and stored at −80 ◦ C for subsequent analysis. After 6 h of observation, the rabbits were sacrificed using an overdose of anesthetic and perfused with 0.9% physiological saline at 4 ◦ C via the heart. The brain was removed and the hippocampal tissues were isolated on ice. The hippocampus was mixed with physiological saline and ground at 4000 rpm for 10 min. The supernatant was 10% tissue homogenate, and this was stored at −80 ◦ C for subsequent analysis.
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2.5. Nissl staining and TUNEL (TdT-mediated dUTP nick-end labeling) assay The paraffin-embedded brain tissues were cut into sections of 5 m, and Nissl staining (Beyotime Institute of Biotechnology, Nantong, China) and TUNEL assay (ROCHE, USA) were performed for in situ detection of apoptosis. Five fields of vision (magnification of 40×) in the hippocampal CA1 area were randomly sampled from each brain tissue sample, and the Nissl-stained neuronal cells were counted. For TUNEL staining, the immunohistochemical score (IHS) is determined by the percentage of positive-stained cells and the intensity of staining as previously described.16 2.6. Determination of biochemical indicators All biochemical indicators were determined with the corresponding reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturers’ instructions. The 8-hydroxydeoxyguanosine (8-OHDG) levels in plasma and brain tissues were determined with ELISA reagent kits, and the malondialdehyde (MDA) level in plasma and brain tissues was determined using the thiobarbituric acid (TBA) method. The superoxide dismutase (SOD) activity was measured using a water-soluble tetrazolium salt (WST-1) and catalase (CAT) activity was detected using ultraviolet spectrophotometry. 2.7. Statistical analysis All measurement data were expressed as mean ± standard deviation (SD), and all statistical analyses were performed using statistical software (SPSS version 13.0). The normality test showed the data were consistent with a normal distribution. A paired t test was used to compare the same parameters before and after treatment. Comparison of the same parameters among groups was done using one-way analysis of variance (ANOVA), and the difference between pairs of means was tested post hoc with Fisher’s Least Significant Difference (LSD) test. Fisher’s exact test was employed to compare the neurological scores between groups. The difference in survival rates among the various groups was compared using Kaplan–Meier survival analysis. A P-value <0.05 was considered statistically significant.
Fig. 1. Survival curves for the 4 groups. # P < 0.05 and *P < 0.01 vs. the sham group revealed by Kaplan–Meier survival analysis; P < 0.05 vs. the CA group.
3.3. Neurological scores There were significant differences in neurological scores at 24, 48 and 72 h after ROSC between the three groups with CA and the sham group (P < 0.05 or 0.01). Although a significant difference in neurological score was observed at 72 h after ROSC between the CA group and CA + H2 group 2, no other significant differences were found between the 3 groups with CA at the various time points after ROSC (Fig. 2). 3.4. Nissl staining and TUNEL assay
There were no significant differences in sex ratio, body weight, basal body temperature, breathing frequency, heart rate and mean arterial pressure of rabbits among the four groups.
Nissl staining revealed a loose structure of the brain tissue in the CA group, which exhibited vacuolar degeneration. The number of neurons was significantly reduced, and the nucleus was compressed; a Nissl body was not found in the cytoplasm. In the two CA + H2 groups, many neurons were found, the nucleus was normal and there were many Nissl bodies observed in the cytoplasm. Compared with the sham group, the number of neurons in other three groups was significantly reduced (P < 0.01), whereas the numbers of neurons in the two CA + H2 groups were significantly greater than that in the CA group (P < 0.05). TUNEL staining showed many strongly TUNEL-positive apoptotic cells in the CA group, and a few weakly positive cells in the two CA + H2 groups. However, no TUNEL-positive cells were found in the sham group. Compared with the sham group, the TUNEL staining immunohistochemistry scores were significantly increased in the other three groups (P < 0.01). However, the scores in the two CA + H2 groups were significantly lower than that in the CA group (P < 0.05) (Fig. 3).
3.2. Survival of rabbits
3.5. Biochemical indicators in blood samples
Ventricular fibrillation was successfully induced in all rabbits in the CA group and the two CA + H2 groups. In the first experiment, one rabbit in the CA group could not be resuscitated, whereas the others all had successful resuscitation. There were no significant differences in the resuscitation parameters among the groups (P > 0.05) (Table 1). The 72-h survival rates in the sham group, CA group, CA + H2 group 1 and CA + H2 group 2 were 100% (6/6), 16.7% (2/12), 41.7% (5/12) and 50% (6/12), respectively. The survival rate of rabbits in the sham group was significantly different from those in the 3 groups with CA. In addition, a significant difference in the survival rate was found between the CA + H2 group 2 and the CA group (P < 0.05) (Fig. 1).
The plasma 8-OHDG and MDA levels significantly increased from before to after ROSC in all four groups, except for the plasma 8-OHDG level in the sham group. Furthermore, the SOD activity significantly decreased in all four groups (P < 0.01). The plasma 8OHDG and MDA levels in the three groups with CA at 6 h after ROSC were significantly greater than those in the sham group (P < 0.01), whereas the SOD activities in the three groups with CA were significantly lower than that in the sham group (P < 0.01). At 6 h after ROSC, the plasma MDA levels in the two CA + H2 groups were significantly lower than that in the CA group (P < 0.01), whereas the SOD activities in the two CA + H2 groups were significantly higher than that in the CA group (P < 0.05) (Fig. 4).
3. Results 3.1. General conditions of rabbits
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Table 1 Comparison of resuscitation parameters in the 3 groups with CA. Group
ROSC (n)
ICAT (s)
DOCPR (s)
DF
AF
MVT (min)
CA CA + low dose of H2 CA + high dose of H2
11 12 12
35.45 ± 14.52 37.33 ± 16.97 34.58 ± 13.08
221.73 ± 89.64 213.50 ± 102.50 208.30 ± 96.68
0.73 ± 0.65 0.75 ± 0.62 0.33 ± 0.49
3.77 ± 1.89 3.04 ± 2.46 2.54 ± 1.31
30.18 ± 11.72 31.00 ± 13.31 24.32 ± 13.84
ROSC: restoration of spontaneous circulation; ICAT: induced CA time (s); DOCPR: duration of CPR(s); DF: defibrillation frequency; AF: administration frequency; MVT: mechanical ventilation time (min).
Fig. 2. Comparison of the neurological scores in the 4 groups. (A) 24 h, (B) 48 h, (C) 72 h. # P < 0.05 and *P < 0.01 vs. the sham group revealed by Fisher’s exact test; P < 0.05 vs. the CA group.
Fig. 3. Nissl staining and TUNEL staining of the hippocampal CA1 area in the 4 groups. (A) The pathological changes in CA1 region; (B) the number of vital neurons; (C) the HIS of TUNEL staining. *P < 0.01 vs. the sham group; P < 0.05 vs. the CA group. Bar = 50 m.
Fig. 4. The biochemical parameters in sera of rabbits from the 4 groups. The level of: (A) 8-OHGD, (B) MDA; (C) the activity of SOD. # P < 0.01 of the self-control before and after treatment; § P < 0.05 of the self-control before and after treatment; *P < 0.01 vs. the sham group at the same time point revealed by the LSD test; P < 0.05 and P < 0.01 vs. the CA group at the same time point revealed by the LSD test.
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Fig. 5. The biochemical parameters in brain tissues of rabbits from the 4 groups. The level of: (A) 8-OHGD, (B) MDA; the activity of (C) SOD, (D) CAT. *P < 0.01 vs. the sham group revealed by the LSD test; P < 0.05 and # P < 0.01 vs. the CA group.
3.6. Biochemical indicators in brain tissues The 8-OHDG and MDA levels in hippocampal tissues were significantly greater in the three groups with CA than those in the sham group (P < 0.01). However, significantly lower SOD and CAT activities were detected in the three groups with CA than those in the sham group (P < 0.01). Compared with the CA group, the 8OHDG and MDA levels in the hippocampal tissues in the two CA + H2 groups were significantly reduced (P < 0.05 or 0.01), whereas the SOD and CAT activities were significantly increased (P < 0.05 or 0.01) (Fig. 5). 4. Discussion Sudden cardiac death is mainly caused by malignant arrhythmias in clinical practice, and the arrhythmia first recorded in 75–80% of the patients with CA is ventricular fibrillation.17 This arrhythmia is well simulated by electric simulation of the heart in experimental animals. Compared with alternating current delivered to the right ventricular endocardium,18 open-chest stimulation of the epicardium 19 and transcutaneous electrical epicardial stimulation,20 ventricular fibrillation induced by stimulation between the esophagus and chest wall electrodes in the present study was easier to perform, less harmful and more reproducible. Previous studies have investigated different methods of H2 administration such as inhalation of H2 , or intravenous or intraperitoneal injection of H2 -rich saline. However, the flammability and easy explosion of H2 is a serious risk with inhalation of H2 . Furthermore, H2 -rich saline used for intraperitoneal injection must be prepared in advance, which is not beneficial for long-term preservation. Direct intraperitoneal injection of H2 is a novel method of H2 administration that we developed. According to the principle of pneumoperitoneum in laparoscopic operation, direct intraperitoneal injection of H2 should not lead to a significant change of abdominal pressure due to the small injection volume at a dose of 10–20 ml/kg. Our previous study demonstrated that intraperitoneal injection of H2 at a dose of 10 ml/kg rapidly increases the concentration of expired H2 in rabbits; the half-life was more than 2 h and the concentration of H2 was effectively sustained for about 6 h. The present study found that intraperitoneal injection of H2 prior to CPR improved the survival rate and neurological function in a rabbit model of CA. The major pathological change after global ischemia is delayed neuronal cell death, and histological evidence is usually present 2–3 days after global ischemia.21 The hippocampus is the most sensitive region to cerebral ischemia, which is closely related to cognitive dysfunction after ischemia-reperfusion injury. In the present study, therefore, the hippocampal CA1 area of the rabbits surviving until 72 h was selected for pathological examination. Nissl staining and TUNEL assay revealed various degrees of neuronal degeneration, necrosis and apoptosis in rabbits after 5 min of CA, and the degree of pathological changes was consistent
with the general neurological scores. The intraperitoneal injection of H2 reduced neuronal damages induced by 5 min of CA, and few apoptotic cells were detected. Currently, the protection mechanism of H2 against ischemiareperfusion injury is not fully clear. H2 has been shown to selectively neutralize the hydroxyl radical (• OH), the most cytotoxic of reactive oxygen species (ROS), and peroxynitrite (ONOO− ), but does not react with superoxide anion (O2 • ).5 The present study demonstrated that systemic hypoxia, ischemia and reperfusion after 5 min of CA caused severe oxidative damage to rabbits, and the plasma MDA and 8-OHDG levels significantly increased in the three groups with CA at 6 h after ROSC. Although CA was not induced in the sham group, the electric stimulation for 90 s caused generalized twitching and significantly elevated MDA levels compared with that before stimulation. In addition, the MDA and 8-OHDG levels either in plasma or brain tissues of rabbits with CA were significantly lower than those in the sham group at 6 h after ROSC, suggesting that administration of H2 reduced systemic and cerebral oxidative damage after CA. Since H2 exhibits non-antioxidant signal regulation 22 and affects gene expression to some extent,23 we cannot exclude an effect of H2 on the endogenous protection pathway against ischemia-reperfusion injury. In the current study, the activities of two important antioxidant enzymes, SOD and CAT, were determined. The antioxidant enzymes are converted into nontoxic or low-toxicity products through catalyzing free radicals and through increasing the water solubility of their products for excretion, and this is important to maintain redox equilibrium.24 The SOD activity in each of the three groups with CA was reduced compared with the basal level, indicating that there was consumption of SOD. The SOD activities in the plasma and hippocampal tissues and the CAT activity in the hippocampal tissues after intraperitoneal injection of H2 were significantly greater in the two CA + H2 groups than those in the CA group, which indicates that there was less of a reduction in the consumption of antioxidant enzymes due to neutralization of free radicals by H2 . In addition, H2 may increase the expression of the Nrf2 (Nuclear factor-E2 related factor 2) downstream protective genes through activation of the Nrf2 signaling pathway. Oxidative stress induced by reperfusion after CA leads to the exhaustion of cerebral antioxidant reserves and causes severe oxidative damage. However, the activity of free radicals after ischemia is characterized by a sudden onset and a short duration of action, and the cerebral antioxidant reserves are restored within 2 h after reperfusion.25 Therefore, antioxidants should be introduced into tissues to treat ischemia-reperfusion injury when reperfusion is performed. Many antioxidants have been applied in experimental studies and show active protection against ischemia-reperfusion injury.26,27 Unfortunately, antioxidants are difficult to deliver when CPR is performed at the scene of resuscitation. In addition, nonselective antioxidants may lead to oxidant/antioxidant imbalance and interfere with the normal physiological effects of oxygen free radicals. Intraperitoneal injection of H2 is a novel, safe, fast and effective method to administer H2 to treat ischemia-reperfusion
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injury and may be potentially useful in the clinical setting. Acute myocardial infarction (AMI) is an important cause of CA. Thrombolytic therapy is an effective treatment for CA caused by AMI after ROSC.28 In this study, intraperitoneal injection of H2 prior to CPR did not cause coagulation disorders and should not be a contraindication of thrombolytic therapy. However, further studies are needed to investigate the optimal dose of H2 and its duration of use. Conflict of interest The authors declare no conflict of interest. Acknowledgements This study was supported by funding of the NSFC (81071030), Science and Technology Foundation of Guangdong Province, China (2011B080701006) and Young Teacher Foundation of Sun Yat-sen University (80000-3171914). The authors express their gratitude to the staff of Pathophysiology laboratory of Sun Yat-sen University for their excellent technical help and constructive criticism. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.resuscitation.2012.10.018. References 1. Peberdy MA, Kaye W, Ornato JP, et al. Cardiopulmonary resuscitation of adults in the hospital: a report of 14720 cardiac arrests from the National registry of cardiopulmonary resuscitation. Resuscitation 2003;58:297–308. 2. Nichol G, Thomas E, Callaway CW, et al. Regional variation in out-of-hospital cardiac arrest incidence and outcome. JAMA 2008;300:1423–31. 3. Brooks SC, Schmicker RH, Rea TD, et al. Out-of-hospital cardiac arrest frequency and survival: evidence for temporal variability. Resuscitation 2010;81:175–81. 4. Zhang N, Komine-Kobayashi M, Tanaka R, et al. Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 2005;36:2220–5. 5. Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007;13:688–94. 6. Hayashida K, Sano M, Ohsawa I, et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury. Biochem Biophys Res Commun 2008;373:30–5. 7. Ohsawa I, Nishimaki K, Yamagata K, et al. Consumption of hydrogen water prevents atherosclerosis in apolipoprotein E knockout mice. Biochem Biophys Res Commun 2008;377:1195–8. 8. Wang F, Yu G, Liu SY, et al. Hydrogen-rich saline protects against renal ischemia/reperfusion injury in rats. J Surg Res 2011;167:e339–44. 9. Buchholz BM, Kaczorowski DJ, Sugimoto R, et al. Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury. Am J Transplant 2008;8:2015–24.
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