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Anesthesia & Analgesia:
doi: 10.1213/ANE.0000000000000303
Critical Care, Trauma, and Resuscitation: Research Report

Hydrogen-Rich Saline Improves Survival and Neurological Outcome After Cardiac Arrest and Cardiopulmonary Resuscitation in Rats

Huo, Ting-ting MD, PhD*; Zeng, Yi MD, PhD*; Liu, Xiao-nan MD, PhD; Sun, Li MD, PhD; Han, Huan-zhi MD§; Chen, Hong-guang MD§; Lu, Zhi-hong MD, PhD*; Huang, Yi MD, PhD*; Nie, Huang MD, PhD*; Dong, Hai-long MD, PhD*; Xie, Ke-liang MD, PhD§; Xiong, Li-ze MD, PhD*

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Author Information

From the Departments of *Anesthesiology, and Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province; Department of Anesthesia and Operation Center, Chinese People’s Liberation Army General Hospital & Medical School of Chinese People’s Liberation Army, Beijing; and §Department of Anesthesiology, Tianjin Institute of Anesthesiology, General Hospital of Tianjin Medical University, Tianjin, P. R. China.

Accepted for publication February 26, 2014.

Published ahead of print June 17, 2014.

Funding: This work was supported by research grants from the National Natural Science Foundation of China (30930091 to Li-ze Xiong, 81101409 to Ke-liang Xie, 30600840 to Zhi-hong Lu, 81271195 to Yi Zeng), the Natural Science Foundation of Tianjin (13JCQNJC11400 to Ke-liang Xie), and the Foundation of Tianjin Bureau of Public Health (2011KZ108 to Ke-liang Xie).

The authors declare no conflicts of interest.

Drs. Ting-ting Huo, Yi Zeng, and Xiao-nan Liu contributed equally to this work.

Reprints will not be available from the authors.

Address correspondence to Li-ze Xiong, MD, PhD, Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, P. R. China. Address e-mail to mzkxlz@126.com.

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Abstract

BACKGROUND: Sudden cardiac arrest is a leading cause of death worldwide. Three-fourths of cardiac arrest patients die before hospital discharge or experience significant neurological damage. Hydrogen-rich saline, a portable, easily administered, and safe means of delivering hydrogen gas, can exert organ-protective effects through regulating oxidative stress, inflammation, and apoptosis. We designed this study to investigate whether hydrogen-rich saline treatment could improve survival and neurological outcome after cardiac arrest and cardiopulmonary resuscitation, and the mechanism responsible for this effect.

METHODS: Sprague-Dawley rats were subjected to 8 minutes of cardiac arrest by asphyxia. Different doses of hydrogen-rich saline or normal saline were administered IV at 1 minute before cardiopulmonary resuscitation, followed by injections at 6 and 12 hours after restoration of spontaneous circulation, respectively. We assessed survival, neurological outcome, oxidative stress, inflammation biomarkers, and apoptosis.

RESULTS: Hydrogen-rich saline treatment dose dependently improved survival and neurological function after cardiac arrest/resuscitation. Moreover, hydrogen-rich saline treatment dose dependently ameliorated brain injury after cardiac arrest/resuscitation, which was characterized by the increase of survival neurons in hippocampus CA1, reduction of brain edema in cortex and hippocampus, preservation of blood-brain barrier integrity, as well as the decrease of serum S100β and neuron-specific enolase. Furthermore, we found that the beneficial effects of hydrogen-rich saline treatment were associated with decreased levels of oxidative products (8-iso-prostaglandin F2α and malondialdehyde) and inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and high-mobility group box protein 1), as well as the increased activity of antioxidant enzymes (superoxide dismutase and catalase) in serum and brain tissues. In addition, hydrogen-rich saline treatment reduced caspase-3 activity in cortex and hippocampus after cardiac arrest/resuscitation.

CONCLUSIONS: Hydrogen-rich saline treatment improved survival and neurological outcome after cardiac arrest/resuscitation in rats, which was partially mediated by reducing oxidative stress, inflammation, and apoptosis.

Sudden cardiac arrest is one of the leading causes of death worldwide, accounting for an estimated 325,000 deaths each year in the United States.1 The initial success rate of cardiopulmonary resuscitation (CPR) is approximately 30% to 40%, but only 2% to 12% of resuscitated patients are discharged from hospital with a satisfactory outcome.1 The current outcomes of CPR are disappointing. Three-fourths of patients die after successful resuscitation or develop permanent neurological impairments.2 Although therapeutic hypothermia has proved effective in clinical studies, no drug is available to improve outcomes after cardiac arrest/resuscitation.3

Numerous basic and clinical studies have shown that oxidative stress and inflammatory response play an important role in the pathogenesis of mortality and organ damage after cardiac arrest/resuscitation.1,4,5 We and other researchers have found that hydrogen gas exerts protective effects in many diseases via regulating oxidative stress (selective reduction of hydroxyl radicals and peroxynitrite) and inflammatory response.6–15 Furthermore, some studies have found that hydrogen-rich saline or hydrogen-rich water can ameliorate multiple organ damage including brain, heart, lung, and intestine by reducing oxidative stress.16–21 Application of hydrogen-rich saline (normal saline containing a therapeutic dose of hydrogen) is an alternative mode of delivery of molecular hydrogen.22 The primary advantages of hydrogen-rich saline are that it is a portable, easily administered, and safe means of delivering hydrogen gas.22

Therefore, using a well-established model of asphyxia-induced cardiac arrest in rats,23,24 the purpose of this study was to investigate the effectiveness of different doses of hydrogen-rich saline therapy on survival and neurological outcome after cardiac arrest/resuscitation. Furthermore, we investigated the effects of hydrogen-rich saline treatment on changes of oxidative products, antioxidant enzymes, inflammatory cytokines, and apoptosis in serum and brain tissues after cardiac arrest/resuscitation.

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METHODS

Animals

Adult male Sprague-Dawley rats weighing 300 to 350 g were provided by the Laboratory Animal Center of Fourth Military Medical University (FMMU). Animals were housed in a temperature- and humidity-controlled room that was maintained on 12-hour/12-hour light/dark cycles. Standard rat chow and water were freely available. The Institutional Animal Care and Use Committee of FMMU approved all experimental protocols, which were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All animals were housed in a nonstressful, warm environment to avoid any “preconditioning” for at least 1 week before operation. All efforts were made to minimize animal suffering in all experiments.

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Animal Preparation, Cardiac Arrest, and Resuscitation

All animals were fasted overnight except for free access to water. Cardiac arrest was induced by asphyxia as described previously.23,24 Animals were anesthetized with isoflurane (1.5%–2%), and the trachea was orally intubated with a 14-gauge plastic catheter and connected to a ventilator (Inspira ASV, Harvard Apparatus Inc., Holliston, MA). A muscle relaxant (1.0 mg/kg vecuronium) was administered IV and as needed thereafter. Mechanical ventilation was established with a frequency of 60 breaths/min. Tidal volume and inspired oxygen concentration were adjusted to control arterial pH at 7.35 to 7.45, arterial carbon dioxide pressure at 35 to 45 mm Hg, and arterial oxygen pressure at 90 to 120 mm Hg. The right femoral artery and vein were cannulated with PE-50 catheters for arterial blood pressure monitoring and drug administration, respectively. The catheters were flushed intermittently with saline solution containing 2.5 IU/mL bovine heparin. A conventional lead II electrocardiogram was continuously monitored. Body temperature was maintained at 37.0°C ± 0.5°C by a thermostatically controlled heating blanket connected to a thermometer probe in the rectum until 1 hour after restoration of spontaneous circulation.

After baseline measurements, isoflurane was washed out with pure oxygen for 3 minutes, followed by room air for 2 minutes with the tidal volume and frequency unchanged. Cardiac arrest was induced by discontinuing mechanical ventilation and clamping the endotracheal tube at the end of expiration for 8 minutes. Cardiac arrest was defined as systolic blood pressure <25 mm Hg without arterial fluctuations. After 8 minutes of apnea and airway obstruction, CPR was started by unclamping the endotracheal tube and initiating mechanical ventilation with pure oxygen. Resuscitation was continued by applying sternal compression at a rate of 200/min and giving 0.01 mg/kg epinephrine and 1 mEq/kg sodium bicarbonate IV until return of spontaneous circulation, defined as a pulsatile rhythm with spontaneous systolic aortic blood pressure >60 mm Hg for at least 5 consecutive minutes. If CPR exceeded 3 minutes, the rat was considered a failure of resuscitation. All physiological variables were continuously monitored and recorded (Siemens Sirecust 1281 and Siredoc 220; Siemens Medical Electronics Inc., Danvers, MA).

After restoration of spontaneous circulation, rats’ lungs were mechanically ventilated with pure oxygen, and the animals were monitored for 1 hour. Blood samples were drawn for blood gases, glucose, and lactate measurements at baseline and 5, 30, and 60 minutes after restoration of spontaneous circulation, and ventilator adjustments were appropriately made. Animals were then weaned from the ventilator, tracheally extubated, and placed in a chamber with 50% oxygen for another 1 hour. Thereafter, animals were subcutaneously given 20 mL/kg/d isotonic saline with 5% dextrose until they could eat and drink without assistance. Sham animals received the same operation except cardiac arrest and CPR.

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Preparation of Hydrogen-Rich Saline

Detailed information for the preparation of hydrogen-rich saline has been described in previous reports.6,16 Hydrogen gas was dissolved in normal saline for 6 hours under high pressure (0.4 MPa) to a supersaturated level. Saturated hydrogen-rich saline was sterilized by γ radiation and stored under atmospheric pressure at 4°C in an aluminum bag. The hydrogen level in the saline was measured using gas chromatography as described by Ohsawa et al.6 The average hydrogen level was 0.85 mmol/L. Hydrogen-rich saline was freshly prepared every week to ensure that the concentration of hydrogen was >0.6 mmol/L.

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Experimental Design
Experiment 1: Effects of Hydrogen-Rich Saline Treatment on Survival and Neurological Outcome After Cardiac Arrest/Resuscitation in Rats

Eighty animals were blindly randomized into 4 groups: sham (n = 10), control (n = 30), HS-5 (n = 20), and HS-10 (n = 20). Based on our previous and preliminary experiments, animals in the HS-5 and HS-10 groups were administered 5 or 10 mL/kg hydrogen-rich saline IV 1 minute before CPR followed by injections at 6 and 12 hours after restoration of spontaneous circulation, respectively. As a control, sham, and control group, animals were administered 10 mL/kg normal saline IV at the same time points. Neurological function scores were evaluated at 24 hours, 72 hours, and 7 days after restoration of spontaneous circulation or sham operation. Survival was measured at 12 hours and 1, 2, 3, 4, and 7 days after restoration of spontaneous circulation or sham operation. Surviving animals were anesthetized and their brains removed for histopathological examination.

In addition, we investigated whether hydrogen-rich saline could improve survival and neurological outcome if hydrogen-rich saline was administered after the start of CPR. Forty animals were blindly randomized into 2 groups (n = 20 per group): control and HS groups. All animals were subjected to cardiac arrest/resuscitation. Animals in the HS group were administered 10 mL/kg hydrogen-rich saline IV 1 minute after the start of CPR followed by injections at 6 and 12 hours after restoration of spontaneous circulation, respectively. Control animals were administered 10 mL/kg normal saline IV at the same time points. We evaluated neurological function scores at 24 hours, 72 hours, and 7 days after restoration of spontaneous circulation. Survival was measured at 12 hours and 1, 2, 3, 4, and 7 days after restoration of spontaneous circulation.

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Experiment 2: Effects of Hydrogen-Rich Saline Treatment on Brain Edema, Brain Injury, and Blood-Brain Barrier After Cardiac Arrest/Resuscitation in Rats

An additional 96 animals were used in this experiment and were blindly assigned to 4 groups (n = 24 per group). The grouping method and experimental protocols were the same as experiment 1. We measured brain edema 1 hour after restoration of spontaneous circulation or sham operation. We measured blood-brain barrier integrity and the levels of serum S100β and neuron-specific enolase 24 hours after restoration of spontaneous circulation or sham operation. The animal was euthanized and the brain was removed for histopathological examination 72 hours after restoration of spontaneous circulation or sham operation.

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Experiment 3: Effects of Hydrogen-Rich Saline Treatment on Oxidative Stress, Inflammatory Cytokines, and Apoptosis in Serum and Brain Tissues After Cardiac Arrest/Resuscitation in Rats

An additional 24 animals were used in this experiment and were assigned to 3 groups (n = 8 per group): sham, control, and HS-10 groups. Antioxidant enzymes (superoxide dismutase and catalase), oxidative products (malondialdehyde and 8-iso-prostaglandin F2α), and proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and high-mobility group box protein 1 [HMGB1]) in serum were measured at baseline (0 hour) and at 3, 12, and 24 hours after restoration of spontaneous circulation or sham operation. Moreover, these above variables in brain tissues (cortex and hippocampus) were measured at 24 hours after restoration of spontaneous circulation or sham operation. In addition, caspase-3 activity in brain tissues (cortex and hippocampus) was detected at 24 hours after restoration of spontaneous circulation or sham operation.

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Assessment of Neurological Function

All animals were evaluated before the experiment to ensure normal neurological function. Neurological function was assessed at 24 hours, 72 hours, and 7 days after restoration of spontaneous circulation or sham operation with a previously reported neurological function scoring system.25 Five variables were assessed and scored: consciousness (no reaction to pinching of tail: 0; poor response to tail pinch: 1; normal response to tail pinch: 2), corneal reflex (no blinking: 0; sluggish blinking: 1; normal blinking: 2), respirations (irregular breathing pattern: 0; decreased breathing frequency with normal pattern: 1; normal breathing frequency and pattern: 2), coordination (no movement: 0; moderate ataxia: 1; normal coordination: 2), and movement/activity (no spontaneous movement: 0; sluggish movement: 1; normal movement: 2). The total score was reported as the neurological function score (10 = normal, 0 = brain death). Neurological function evaluations were performed and confirmed by 2 investigators blinded to treatment.

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Histopathological Evaluation

At 72 hours and 7 days after restoration of spontaneous circulation or sham operation, the animals were euthanized and transcardially perfused.25,26 The brains were then removed and coronal brain blocks were embedded in paraffin; 10 μm coronal sections were cut and stained with hematoxylin and eosin. Neuronal death was diagnosed based on visual evidence of nuclear pyknosis, karyorrhexis, and karyolysis. Neurons with intact nuclei and nucleolar structures were judged to be living. Survival neuronal counts were made within the CA1 region of hippocampus at ×40 magnification by 2 investigators blinded to treatment assignment.

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Evaluation of Brain Edema

Brain water content, an indicator of brain edema, was measured with the wet-dry method at 1 hour after restoration of spontaneous circulation or sham operation.24 Animals were decapitated under anesthesia, and the entire brain was removed. The cortex and hippocampus of the brain were separated and weighed immediately after removal and then placed in a laboratory oven (100°C) for slow evaporation for 48 hours. The dried samples were weighed, and the water content (%) was calculated as (wet weight − dry weight)/(wet weight) × 100%.

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Evaluation of Blood-Brain Barrier Integrity

Blood-brain barrier integrity was determined by Evans blue extravasation at 24 hours after restoration of spontaneous circulation or sham operation.4 At 23 hours after restoration of spontaneous circulation or sham operation, 2% Evans blue was injected IV 2 mL/kg. Animals were anesthetized at 24 hours and perfused with saline to remove intravascular Evans blue dye. Animals were then decapitated; the brain tissues (right cerebral cortex) were dissected. Each tissue sample was weighed, homogenized in 2 mL 50% trichloroacetic acid (w/v), and centrifuged at 10,000g for 20 minutes. The supernatant was then diluted with solvent (one part 50% trichloroacetic acid to 3 parts ethanol). Tissue levels of Evans blue dye were quantitated using a spectrofluorometer at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. Sample values were compared with those of Evans blue dye standards mixed with the solvent (100–1000 ng/mL).

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Measurement of Serum S100β and Neuron-Specific Enolase

Blood samples for S100β and neuron-specific enolase were collected at 24 hours after restoration of spontaneous circulation or sham operation.27 Blood was allowed to clot for 30 minutes at room temperature and then centrifuged at 3000g for 15 minutes and stored at −80°C. Serum S100β and neuron-specific enolase were quantified with an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. All assays were conducted in triplicate.

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Measurement of Antioxidant Enzymes

Blood was collected at baseline (0 hour) and 3, 12, and 24 hours after restoration of spontaneous circulation or sham operation. Serum was separated by centrifugation at 3000g for 15 minutes at 4°C, aliquoted, and stored at −80°C until assayed. At 24 hours after restoration of spontaneous circulation or sham operation, animals were euthanized under anesthesia, and then perfused transcardially with 200 mL chilled phosphate-buffered saline (0.1 M, pH 7.4). The cortex and hippocampus of brain were dissected and homogenized in chilled phosphate-buffered saline, and then centrifuged at 10,000g for 10 minutes at 4°C. The supernatants were collected, aliquoted, and stored at −80°C until the following analysis.

The activity of superoxide dismutase and catalase was measured using commercial kits purchased from Cayman Chemical Company (Ann Arbor, MI) with a microplate reader (Molecular Devices, Sunnyvale, CA). The tissue protein concentration was determined using a standard commercial kit (Bio-Rad Laboratories, Hercules, CA). All assays were conducted in triplicate.

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Measurement of Oxidative Products

The specimens obtained were also used for detecting the levels of oxidative products (8-iso-prostaglandin F2α and malondialdehyde). The levels of 8-iso-prostaglandin F2α and malondialdehyde were detected by commercial kits (Cayman Chemical Company) using a microplate reader (Molecular Devices). The tissue protein concentration was determined by a standard commercial kit (Bio-Rad Laboratories). All assays were conducted in triplicate.

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Measurement of Inflammatory Cytokines

The specimens obtained were also used for detecting the levels of inflammatory cytokines (TNF-α, IL-1β, and HMGB1). The levels of inflammatory cytokines were detected by specific ELISA kits (TNF-α and IL-1β, R&D Systems Inc.; HMGB1, IBL, Hamburg, Germany) with a microplate reader (Molecular Devices). The tissue protein concentration was determined by a standard commercial kit (Bio-Rad Laboratories). All assays were conducted in triplicate.

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Measurement of Caspase-3 Activity

The brain samples obtained were also used in this test. The activity of caspase-3 was measured with caspase-3/CPP32 Fluorometric Assay Kit (BioVision Research Products, Mountain View, CA). All assays were run in duplicate.

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Statistical Analysis

To determine the sample size, a power analysis was performed based on the results of previously performed research28 and a preliminary experiment. The survival rate of the control group was 40% and the HS-10 group was 90%. Assuming an α error of 0.05 with a power of 0.90, we calculated a necessary sample size of 17 to show a significant effect. Based on this calculation and to ensure reasonable data, we increased the sample size to 20.

All normally distributed values, except for neurological function scores and survival, were presented as mean ± SD and analyzed by 1-way analysis of variance with equal variances. Between-group differences were assessed using the Tukey post hoc test (Tukey-corrected P values). Survival was expressed as a percentage and tested by the Fisher exact probability method. We used a Bonferroni correction for 3 comparisons when comparing the HS-5 group, the HS-10 group, and the control group. We calculated confidence intervals for the difference of percentages using Wilson’s procedure without continuity correction (http://vassarstats.net/prop2_ind.html). Neurological function scores were analyzed by a nonparametric method (Kruskal-Wallis test) followed by the Mann-Whitney U test with Bonferroni correction. Statistical analysis was performed with SPSS 16.0 software (SPSS Inc., Chicago, IL), and a 5% probability of type I errors was used to determine statistical significance. We considered a P value of <0.05 to be statistically significant.

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RESULTS

Physiological Variables

Physiological variables were measured at baseline and 5, 30, and 60 minutes after restoration of spontaneous circulation or sham operation. There were no significant differences in mean arterial blood pressure, heart rate, and arterial pH, PaO2, PaCO2, glucose, and lactate among the 4 groups at baseline (Table 1). Compared with the sham group, mean arterial blood pressure and heart rate in the control, HS-5, and HS-10 groups were higher at 5 and 30 minutes after restoration of spontaneous circulation (P = 0.0017, Table 1). When compared with the sham group, arterial pH in the control, HS-5, and HS-10 groups was decreased at 5 minutes after restoration of spontaneous circulation (P < 0.0001, Table 1), while PaCO2 was increased at 5 minutes after restoration of spontaneous circulation (P = 0.013, Table 1). Moreover, blood glucose and lactate in the control, HS-5, and HS-10 groups were significantly higher at 5, 30, and 60 minutes after restoration of spontaneous circulation (P < 0.0001 versus sham group, Table 1).

Table 1
Table 1
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In addition, asphyxial cardiac arrest and resuscitation were similar among the control, HS-5, and HS-10 groups (Table 2). However, hydrogen-rich saline treatment increased the rate of successful resuscitation (Table 2). These data demonstrate that we successfully produced a rat model of cardiac arrest/resuscitation and hydrogen-rich saline treatment that had no significant effect on the physiological variables of this model.

Table 2
Table 2
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Survival

As shown in Fig. 1, 7-day survival of control animals was approximately 40% (P = 0.0042 vs 100% in the sham group), while hydrogen-rich saline treatment significantly increased the 7-day survival of animals to 70% in the HS-5 group (P = 0.021 versus control group) and 90% in the HS-10 group (P = 0.0081 versus control group), respectively. In addition, 7-day survival of animals in the HS-10 group was higher than the HS-5 group (P = 0.023). This result shows that hydrogen-rich saline treatment can dose dependently increase the survival of cardiac arrest/resuscitation rats.

Figure 1
Figure 1
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Neurological Function Scores

Neurological function scores evaluated at 24 hours, 72 hours, and 7 days after restoration of spontaneous circulation or sham operation in all groups are shown in Figure 2. Control animals showed neurological deficit at 24 hours, 72 hours, and 7 days after restoration of spontaneous circulation (P = 0.0089, P = 0.0022, P < 0.0001 versus sham group, Fig. 2), which was dose dependently attenuated by hydrogen-rich saline treatment (P = 0.0095, P = 0.0053, P < 0.0001 versus control group, Fig. 2). In addition, all groups had equivalent baseline neurological function (data not shown). These results indicate that hydrogen-rich saline treatment can dose dependently improve neurological outcome after cardiac arrest/resuscitation in rats.

Figure 2
Figure 2
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In addition, hydrogen-rich saline treatment administered after the start of CPR significantly improved neurological outcome and survival after cardiac arrest/resuscitation (Fig. 3).

Figure 3
Figure 3
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Histopathology and Neuronal Counts

Hippocampal CA1, known to be selectively vulnerable to global ischemia, showed significant histopathological injury at 72 hours and 7 days after restoration of spontaneous circulation (Fig. 4). Representative micrographs of hematoxylin and eosin staining in hippocampal CA1 at 72 hours after restoration of spontaneous circulation or sham operation are shown in Figure 4, A–D. Survival neuron counts of hippocampal CA1 at 72 hours and 7 days after restoration of spontaneous circulation or sham operation were determined in all groups (Fig. 4, E and F). Control animals showed significant decreases in the number of survival neurons at 72 hours and 7 days after restoration of spontaneous circulation (P = 0.0083, P = 0.0069 versus sham group), which were markedly ameliorated by 5 and 10 mL/kg hydrogen-rich saline treatment (P = 0.0092, P = 0.0066 versus control group). Moreover, the number of survival neurons in the 10 mL/kg hydrogen-rich saline treatment group was significantly increased when compared with the 5 mL/kg hydrogen-rich saline treatment group at 72 hours and 7 days after restoration of spontaneous circulation (P = 0.0073, P = 0.0081). The above results indicate that hydrogen-rich saline treatment can dose dependently improve histopathological injury in hippocampal CA1 after cardiac arrest/resuscitation in rats.

Figure 4
Figure 4
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Brain Edema

Water content of cortex and hippocampus was measured at 1 hour after restoration of spontaneous circulation or sham operation (Fig. 5). Eight animals in the sham group, 7 in the control group, 8 in the HS-5 group, and 8 in the HS-10 group were analyzed. Water content of cortex and hippocampus increased in the control group when compared with the sham group (P = 0.0095, P = 0.0083). However, hydrogen-rich saline treatment markedly reduced the water content of cortex and hippocampus (P = 0.022, P = 0.015; P = 0.023, P = 0.018 versus control group). This result indicates that hydrogen-rich saline treatment can ameliorate brain edema after cardiac arrest/resuscitation in rats.

Figure 5
Figure 5
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Blood-Brain Barrier Integrity

Blood-brain barrier integrity was detected by Evans blue extravasation at 24 hours after restoration of spontaneous circulation or sham operation (Fig. 5). Eight animals in the sham group, 6 in the control group, 7 in the HS-5 group, and 8 in the HS-10 group were analyzed. Evans blue extravasation of brain tissue was increased in the control group when compared with the sham group (P = 0.0008). However, hydrogen-rich saline treatment markedly reduced Evans blue extravasation of brain tissue (P = 0.0081, P < 0.0001 versus control group). Moreover, Evans blue extravasation of brain tissue in the 10 mL/kg hydrogen-rich saline treatment group was significantly reduced when compared with the 5 mL/kg hydrogen-rich saline treatment group (P = 0.0036). This result indicates that hydrogen-rich saline treatment can preserve blood-brain barrier integrity after cardiac arrest/resuscitation in rats.

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S100β and Neuron-Specific Enolase

The levels of serum S100β and neuron-specific enolase, as sensitive markers of brain injury, were detected at 24 hours after restoration of spontaneous circulation or sham operation (Fig. 6). Eight animals in the sham group, 6 in the control group, 7 in the HS-5 group, and 8 in the HS-10 group were analyzed. Serum S100β and neuron-specific enolase levels were significantly increased in the control group when compared with the sham group (P < 0.0001, P = 0.0004). However, hydrogen-rich saline treatment markedly reduced serum S100β and neuron-specific enolase levels in a dose-dependent manner (P = 0.0091, P = 0.0089 versus control group). This result further indicates that hydrogen-rich saline treatment can improve brain injury after cardiac arrest/resuscitation in rats.

Figure 6
Figure 6
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Oxidative Products and Antioxidant Enzymes

To investigate the possible mechanisms underlying the neuroprotective effect of hydrogen-rich saline treatment for cardiac arrest and CPR, we first detected the levels of oxidative products malondialdehyde and 8-iso-prostaglandin F2α, as well as the activity of antioxidant enzymes superoxide dismutase and catalase in serum and brain tissues at baseline (0 hour) and 3, 12, and 24 hours after restoration of spontaneous circulation or sham operation (Figs. 7 and 8). Eight animals in the sham group, 6 in the control group, 7 in the HS-5 group, and 8 in the HS-10 group were analyzed. Serum malondialdehyde and 8-iso-prostaglandin F2α levels were significantly elevated from 3 to 24 hours after restoration of spontaneous circulation (P = 0.0044, P = 0.0073, control group versus sham group, Fig. 7). Serum superoxide dismutase and catalase activity may have been slightly increased at 3 hours after restoration of spontaneous circulation (P = 0.078, control group versus sham group), but significantly decreased at 12 and 24 hours after restoration of spontaneous circulation (P = 0.0031, control group versus sham group, Fig. 7). Hydrogen-rich saline treatment significantly mitigated the increase in serum malondialdehyde and 8-iso-prostaglandin F2α levels, as well as the decrease in serum superoxide dismutase and catalase activity after restoration of spontaneous circulation (P = 0.0084 versus control group, Fig. 7).

Figure 7
Figure 7
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Figure 8
Figure 8
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Moreover, control animals showed a significant increase in malondialdehyde and 8-iso-prostaglandin F2α levels in cortex and hippocampus at 24 hours after restoration of spontaneous circulation (P = 0.0008 versus sham group, Fig. 8), while superoxide dismutase and catalase activity in cortex and hippocampus of control animals was markedly decreased at 24 hours after restoration of spontaneous circulation (P = 0.0057 versus sham group, Fig. 8). However, hydrogen-rich saline treatment significantly ameliorated the increase of malondialdehyde and 8-iso-prostaglandin F2α levels, as well as the decrease of superoxide dismutase and catalase activity in cortex and hippocampus (P = 0.0025 versus control group). The above results suggest that hydrogen-rich saline treatment attenuates oxidative stress conditions in serum and brain tissues after cardiac arrest/resuscitation.

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Inflammatory Cytokines

In addition, we detected TNF-α, IL-1β, and HMGB1 levels in serum and brain tissues at baseline (0 hour) and 3, 12, and 24 hours after restoration of spontaneous circulation or sham operation (Figs. 9 and 10). Eight animals in the sham group, 6 in the control group, 7 in the HS-5 group, and 8 in the HS-10 group were analyzed. The data showed that serum TNF-α, IL-1β, and HMGB1 levels were increased from 3 to 24 hours after restoration of spontaneous circulation (P = 0.0003, control group versus sham group, Fig. 9), which were significantly ameliorated by hydrogen-rich saline treatment (P = 0.0023 versus control group, Fig. 9). Moreover, TNF-α, IL-1β, and HMGB1 levels in cortex and hippocampus were significantly elevated at 24 hours after restoration of spontaneous circulation in control animals (P = 0.0032 versus sham group, Fig. 10), which were attenuated by hydrogen-rich saline treatment (P = 0.0049 versus control group, Fig. 10). The above results suggest that hydrogen-rich saline treatment reduces inflammatory cytokines in serum and brain tissues after cardiac arrest/resuscitation.

Figure 9
Figure 9
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Figure 10
Figure 10
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Caspase-3 Activity

Caspase-3 activation, widely accepted as a reliable indicator for cell apoptosis, was measured at 24 hours after restoration of spontaneous circulation or sham operation (Fig. 11). Eight animals in the sham group, 6 in the control group, 7 in the HS-5 group, and 8 in the HS-10 group were analyzed. Control animals showed a significant increase of caspase-3 activity in cortex and hippocampus (P = 0.0003 versus sham group), which was reduced by hydrogen-rich saline treatment (P = 0.0008 versus control group). This result suggests that hydrogen-rich saline treatment ameliorates neuron apoptosis after cardiac arrest/resuscitation.

Figure 11
Figure 11
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DISCUSSION

In the present study, we investigated the protective effects of hydrogen-rich saline treatment in a rat model of asphyxia-induced cardiac arrest and its associated mechanisms. Here, we found that IV injection of hydrogen-rich saline dose dependently improves survival and neurological function after cardiac arrest/resuscitation. Moreover, hydrogen-rich saline treatment significantly ameliorated brain injury after cardiac arrest/resuscitation. Furthermore, we found that the beneficial effects of hydrogen-rich saline treatment were associated with decreased levels of oxidative products (8-iso-prostaglandin F2α and malondialdehyde) and inflammatory cytokines (TNF-α, IL-1β, and HMGB1), as well as the increased activity of antioxidant enzymes (superoxide dismutase and catalase) in serum and brain tissues. In addition, hydrogen-rich saline treatment reduced caspase-3 activity in cortex and hippocampus after cardiac arrest/resuscitation.

Asphyxia-induced cardiac arrest is a well-characterized model to study cellular and molecular changes after cardiac arrest/resuscitation.23,24,27 In this study, using this model, we found that cardiac arrest/resuscitation-challenged rats had significant mortality and brain injury, which is consistent with previous studies.23,24,27 Nevertheless, hydrogen-rich saline treatment increased the rate of successful resuscitation. Hydrogen gas improved resuscitation perhaps through regulating heart and brain function, or nervous reflex. However, detailed mechanisms of this effect are still unclear.

Cardiac arrest/resuscitation results in significant mortality after initial resuscitation due in most cases to ischemia and reperfusion-induced brain injury and to a lesser degree myocardial dysfunction.1 The rate of oxidative metabolism in brain is high. Reactive oxygen species are massively produced in the brain after ischemia, and oxidative stress has been regarded as a fundamental mechanism of brain damage after cardiac arrest/resuscitation.4 Reactive oxygen species include many types such as superoxide anion, hydroxyl radicals, hydrogen peroxide, and so on. Despite their cytotoxic effects, superoxide anion and hydrogen peroxide have an important physiological role at low concentration, which function as regulatory signaling molecules that are involved in numerous signal transduction pathways and regulate biological processes such as apoptosis and cell proliferation.29 However, hydroxyl radicals are the strongest reactive oxygen species and react indiscriminately with nucleic acids, lipids, and proteins.6 More importantly, there is no known detoxification system for hydroxyl radicals in vivo. Therefore, scavenging hydroxyl radicals is a critical antioxidant process, which may be a good method for improving the survival and neurological outcome after cardiac arrest/resuscitation.

Interestingly, many studies have demonstrated that hydrogen gas exerts therapeutic antioxidant activity by selectively reducing hydroxyl radicals and effectively protecting against multiple organ injury such as transient and permanent cerebral ischemia, neonatal cerebral hypoxia-ischemia, as well as kidney, intestine, liver, lung, and myocardial injuries induced by ischemia and reperfusion, suggesting that hydrogen gas has a potential role as an antioxidant for preventive and therapeutic applications.6–15,30 Recently, 1 study further suggested that intraperitoneal injection of hydrogen gas is a favorable strategy to improve the prognosis of cardiopulmonary cerebral resuscitation in a rabbit model of cardiac arrest.28 However, hydrogen-rich saline treatment may be more portable, easily administered, and a safe means of delivering hydrogen gas.22 In our study, we found that hydrogen-rich saline treatment dose dependently improved survival and neurological outcome after cardiac arrest/resuscitation in rats. Moreover, hydrogen-rich saline treatment significantly ameliorated brain injury after cardiac arrest/resuscitation, including improvement of survival neurons in hippocampus CA1, brain edema in cortex and hippocampus, blood-brain barrier integrity, as well as the reduction of serum S100β and neuron-specific enolase. These results support that hydrogen-rich saline treatment is an effective method for cardiac arrest/resuscitation. Oxidative stress and mitochondrial dysfunction have an important role in cardiac arrest-induced blood-brain barrier injury and brain edema. Hydrogen gas might improve brain edema and injury by reducing oxidative stress and improving mitochondrial function.

Cardiac arrest-induced brain injury involves several mechanisms, including initiation of acute oxidative stress, and release of many immune mediators such as interleukins and chemotactic factors.4 To investigate the underlying mechanisms of hydrogen-rich saline treatment in cardiac arrest/resuscitation, we studied changes in the oxidant/antioxidant system in serum and brain tissues after cardiac arrest/resuscitation. Malondialdehyde is a commonly measured indicator of lipid peroxidation induced by reactive oxygen species, and the level of malondialdehyde correlates with the extent of damage caused by reactive oxygen species.12 Moreover, testing 8-iso-prostaglandin F2α, a reactive oxygen species-catalyzed product of arachidonic acid, is a reliable approach for quantitative measurement of oxidative stress status in vivo.12 Both malondialdehyde and 8-iso-prostaglandin F2α have been widely used to estimate the overall status of oxidative stress.12 The detrimental effects of reactive oxygen species can be countered by the antioxidant enzymes superoxide dismutase and catalase. In the present study, we found decreased activity of superoxide dismutase and catalase, as well as increased levels of malondialdehyde and 8-iso-prostaglandin F2α in serum and brain tissues, suggesting that cardiac arrest/resuscitation creates an environment favorable for oxidative stress. We further found that hydrogen-rich saline treatment better preserved superoxide dismutase and catalase activity, as well as decreased malondialdehyde and 8-iso-prostaglandin F2α levels. Hydrogen gas directly reduces reactive oxygen species in brain or indirectly reduce reactive oxygen species through the increase in antioxidant enzymatic activity.6,12 These results suggest that the decrease in oxidative damage and the increase in endogenous antioxidant enzymatic activity in serum and brain tissues may be attributed to protection of hydrogen-rich saline treatment in cardiac arrest/resuscitation, which is similar to the results observed in our previous studies.11,12

Furthermore, inflammation has been suggested to contribute to late stages of brain injury and result in worsening of neurological outcome after cardiac arrest/resuscitation.4 HMGB1 can activate inflammatory pathways when released from ischemic cells.31,32 Researchers have found that HMGB1 as a late cytokine-like mediator plays a critical role in the development of postischemic cerebral damage through the amplification of an inflammatory response.31,32 Meanwhile, early and late inflammatory cytokines can interact and facilitate brain injury. In the present study, levels of TNF-α, IL-1β, and HMGB1 in serum and brain tissues were increased from 3 to 24 hours after restoration of spontaneous circulation in a time-dependent manner. However, hydrogen-rich saline treatment significantly attenuated the increase of TNF-α, IL-1β, and HMGB1 levels in serum and brain tissues. These results indicate that the decrease of early and late proinflammatory cytokines may also be attributed to the protection of hydrogen-rich saline treatment in cardiac arrest/resuscitation.

Apoptosis has been demonstrated to be an important mode of neuron death in the ischemia and reperfusion brain and plays a significant role in neurological dysfunction after cardiac arrest/resuscitation.4 At the molecular level, apoptosis is activated by the aspartate-specific cysteineprotease (caspase) cascade, including caspase-12 and -3. Caspase-3 is considered to be the most important of the executioner caspases and is activated by any of the initiator caspases.33 Capase-3 activates DNA fragmentation factor, which in turn activates endonucleases to cleave nuclear DNA and ultimately leads to cell death. In this study, we also found that caspase-3 activity in brain tissues was increased after cardiac arrest/resuscitation, which was markedly alleviated by hydrogen-rich saline treatment. Therefore, these results suggest that hydrogen-rich saline treatment significantly alleviated neuron apoptosis after cardiac arrest/resuscitation.

There were certain limitations of this study. First, the rodent brain has rheological and metabolic properties vastly different from the complex, comparatively enormous gyrencephalic human brain, and the relative importance of destructive processes may be different in rats compared with humans. Thus, the outcome of our study in a rat model of CPR remains to be demonstrated in large animal and clinical studies. Second, in this study, we used rectal temperature to servo control body and brain temperature. A thermistor inserted between the scalp and calvarium might be a better approach to maintain constant brain temperature. Third, although asphyxia-induced cardiac arrest is a well-characterized model, most cardiac arrest is due to intrinsic cardiac disease. Therefore, further investigation in animal models that more closely mimics clinical scenarios of cardiac arrest is needed.

Taken together, our findings support the potential use of hydrogen-rich saline as a new agent in the therapy for conditions associated with cardiac arrest/resuscitation and may warrant further testing in future clinical trials for human cardiac arrest/resuscitation.

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DISCLOSURES

Name: Ting-ting Huo, MD, PhD.

Contribution: This author helped conduct the study, interpret the data, and prepare the manuscript.

Attestation: Ting-ting Huo approved the final version of the manuscript.

Name: Yi Zeng, MD, PhD.

Contribution: This author helped conduct the study, interpret the data, and prepare the manuscript.

Attestation: Yi Zeng approved the final version of the manuscript.

Name: Xiao-nan Liu, MD, PhD.

Contribution: This author helped conduct the study, interpret the data, and prepare the manuscript.

Attestation: Xiao-nan Liu approved the final version of the manuscript.

Name: Li Sun, MD, PhD.

Contribution: This author helped conduct the study, interpret the data, and prepare the manuscript.

Attestation: Li Sun approved the final version of the manuscript.

Name: Huan-zhi Han, MD.

Contribution: This author helped conduct the study.

Attestation: Huan-zhi Han approved the final version of the manuscript.

Name: Hong-guang Chen, MD.

Contribution: This author helped conduct the study.

Attestation: Hong-guang Chen approved the final version of the manuscript.

Name: Zhi-hong Lu, MD, PhD.

Contribution: This author helped conduct the study.

Attestation: Zhi-hong Lu approved the final version of the manuscript.

Name: Yi Huang, MD, PhD.

Contribution: This author helped conduct the study.

Attestation: Yi Huang approved the final version of the manuscript.

Name: Huang Nie, MD, PhD.

Contribution: This author helped prepare the manuscript.

Attestation: Huang Nie approved the final version of the manuscript.

Name: Hai-long Dong, MD, PhD.

Contribution: This author helped prepare the manuscript.

Attestation: Hai-long Dong approved the final version of the manuscript.

Name: Ke-liang Xie, MD, PhD.

Contribution: This author helped design the study, analyze and interpret the data, prepare and review the manuscript.

Attestation: Ke-liang Xie approved the final version of the manuscript.

Name: Li-ze Xiong, MD, PhD.

Contribution: This author helped design the study, interpret the data, and review the manuscript.

Attestation: Li-ze Xiong approved the final version of the manuscript.

This manuscript was handled by: Steven L. Shafer, MD.

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ACKNOWLEDGMENTS

We thank Prof. Xuejun Sun for his kindly information.

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