Shock, Publish Ahead of Print
DOI: 10.1097/SHK.0b013e31824ed57c
Effect of Hydrogen Gas on the Survival Rate of Mice Following Global Cerebral Ischemia
Kimihiro Nagatani1, Kojiro Wada1, Satoru Takeuchi1, Hiroaki Kobayashi1, Yoichi Uozumi1, Naoki
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Otani1, Masanori Fujita2, Shoichi Tachibana2, and Hiroshi Nawashiro1
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Department of Neurosurgery, National Defense Medical College, Saitama, Japan
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Saitama, Japan
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Corresponding author: Kimihiro Nagatani
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Division of Environmental Medicine, Research Institute, National Defense Medical College,
Department of Neurosurgery, National Defense Medical College, 3-2 Namiki, Tokorozawa,
Saitama 359-8513, Japan
Phone: +81-4-2995-1656
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Fax: +81-4-2996-5207
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E-mail: [email protected]
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Running head: Hydrogen gas and global cerebral ischemia
Disclosure of funding: This study was supported by a research grant from the General Insurance
Association of Japan (Grant no. 0001).
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Abbreviations:
8-OHdG: 8-hydroxy-2′-deoxyguanosine
BBB: blood–brain barrier
BCCAO: bilateral common carotid artery occlusion
CA1: cornu ammonis 1
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CBF: cerebral blood flow
I/R: ischemia and reperfusion
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FR: free radical
LC3: microtubule-associated protein 1 light chain 3
MDA: malondialdehyde
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P-com A: posterior communicating artery
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ROS: reactive oxygen species
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Abstract
Global cerebral ischemia and reperfusion (I/R) often result in high mortality. Free radicals (FRs)
have been reported to play an important role in global cerebral I/R and, therefore, reduction of
these might improve the outcome. Here, we investigated the effect of hydrogen gas (a strong FR
scavenger) on the survival rate of mice following global cerebral I/R. We further examined the
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histopathological outcome, and also the brain water content (as a possible determinant of
mortality). Male C57BL/6J mice were subjected to global cerebral I/R by means of 45-min
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bilateral common carotid artery occlusion (BCCAO). A total of 160 mice were divided into 3
groups: sham surgery (sham group); BCCAO without hydrogen gas (BCCAO group); and
BCCAO treated with 1.3% hydrogen gas (BCCAO + H2 group). We observed that hydrogen gas
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treatment significantly (P = 0.0232) improved the 7-day survival rate of mice, from 8.3%
(BCCAO group, n = 12) to 50% (BCCAO + H2 group, n = 10). Histopathological analysis
revealed that hydrogen gas treatment significantly attenuated neuronal injury and autophagy in the
hippocampal CA1 sector and also brain edema, after 24 hr of reperfusion. The beneficial effects of
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hydrogen gas treatment on brain injury were associated with significantly lower levels of
oxidative stress markers (8-hydroxy-2′-deoxyguanosine and malondialdehyde) in the brain tissue.
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Thus, we believe that hydrogen gas may be an effective treatment for global cerebral I/R.
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Keywords: reactive oxygen species, ischemia/reperfusion injury, bilateral common carotid artery
occlusion, cardiac arrest, brain edema, autophagy, free radical scavenger, mortality
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Introduction
Resuscitation from global cerebral ischemia associated with cardiac arrest often results in a poor
neurological outcome (1, 2). In such cases, protection of the brain from cerebral ischemia and
reperfusion (I/R) is of crucial importance. Numerous reactive oxygen species (ROS) are generated
during cerebral I/R. Recent studies have suggested that, clinically and experimentally, a surge of
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ROS may be a potential cause of high mortality rates of global cerebral I/R (3, 4). Among ROS,
hydroxyl radicals and peroxynitrite appear to play critical roles in tissue injury (5, 6). Hydrogen
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gas (H2) is known to possess potent scavenger actions against hydroxyl radicals and peroxynitrite.
Nitrogen-based high-pressure mixed gas consisting of 1.3% hydrogen and 30% oxygen is safe and
suitable for emergency use. Transient bilateral common carotid artery occlusion (BCCAO)
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induces global cerebral I/R (1, 7, 8) and is associated with a high mortality rate in male C57BL/6J
mice (1, 9, 10). We hypothesized that inhalation of hydrogen gas may improve the survival rate of
global cerebral I/R via reduction of potent ROS.
The aim of the present study was to investigate the effects of hydrogen gas inhalation on the
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survival rate of mice following global cerebral I/R. Furthermore, we examined the
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histopathological outcome and the brain water content (as a possible determinant of mortality).
Materials and Methods
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Animals
All experimental procedures used in this study were approved by the institutional animal care
committees of The National Defense Medical College. Throughout the study, extreme care was
taken to minimize pain and discomfort to animals. Male C57BL/6J mice, 9–11 weeks in age and
weighing 23–27 g (CLEA Japan Inc., Tokyo, Japan), were allowed free access to food and water
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prior to experimental use.
Hydrogen gas (H2) treatment
For gaseous treatment, a tank with nitrogen-based high-pressure pre-mixed gases was purchased
directly from the manufacturer (Japan Fine Products Co., Kanagawa, Japan). The manufacturer
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(Japan Fine Products Co.) confirmed the concentrations of hydrogen (1.3%), oxygen (30%), and
nitrogen (68.7%). In Japan, 1.3% is the highest concentration of hydrogen gas that can be mixed
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and bottled under high pressure with 30% oxygen for clinical use. Hydrogen gas from the premixed gas tank was delivered to mice via a tight-fitting reservoir face mask. Mice without
BCCAO model
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hydrogen gas treatment inhaled oxygen (30%) and nitrogen (70%) gas (Japan Fine Products Co.).
A total of 160 mice were divided into 3 groups: sham surgery (sham group); BCCAO without
hydrogen gas (BCCAO group); and BCCAO treated with hydrogen gas (BCCAO + H2 group).
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During the surgical and postoperative periods, the rectal temperature was maintained at
approximately 37°C by means of a feedback-controlled heating pad. The mean arterial blood
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pressures, heart rates, and respiratory rates were recorded. Mice were anesthetized
intraperitoneally with 20-mg/kg sodium pentobarbital. The skull was exposed following a midline
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scalp and periosteum incision with lidocaine local anesthesia; a mild dose of sodium pentobarbital
was used to minimize the effects on cerebral blood flow (CBF). The CBF in the cortex was
measured semiquantitatively for each hemisphere, using a non-invasive and non-contact laser
Doppler blood perfusion imager (PeriScan PIM II; PeriMed, Stockholm, Sweden). BCCAO was
performed as reported previously (11), with modifications (12). Briefly, each common carotid
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artery was surgically exposed and occluded, by applying non-traumatic small aneurysm clips.
After 45 min of occlusion, the clips were carefully removed to restore the blood flow. The CBF
was measured sequentially as follows: immediately prior to BCCAO; at 5 min, 15 min, 30 min,
and 45 min during BCCAO; and at 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, and 180 min
after BCCAO. After 180 min of reperfusion, the neck incision was closed, and the mice were
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allowed to recover. In a previous anatomical study (12), we confirmed that mice without the
posterior communicating artery (P-com A) exhibited a low residual CBF (less than 30% of pre-
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occlusion values) in the same hemisphere during BCCAO. Therefore, in the present study, mice
that did not exhibit a low residual CBF (less than 30% of pre-occlusion values) in the same
hemisphere were excluded from further analysis; a total of 55 mice were excluded. In the BCCAO
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+ H2 group, mice inhaled hydrogen gas during the 225-min process (45 min of occlusion and 180
min of reperfusion). Furthermore, hydrogen gas was administered for 3 hr per day, from 1–3 days
after surgery. In the BCCAO group, mice inhaled 30% oxygen gas, instead of hydrogen gas,
during the entire process. Furthermore, 30% oxygen gas was administered for 3 hr per day, from
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1–3 days after surgery. In each group, the number of surviving mice at 7 days after surgery was
counted. Sham-operated mice did not undergo BCCAO, but were otherwise treated as for the
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BCCAO group.
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Tissue preparation
For histopathological analysis, a total of 12 mice were used. Mice were divided into 3 groups:
sham group (n = 4); BCCAO group (n = 4); and BCCAO + H2 group (n = 4). After 24 hr of
reperfusion, mice were anesthetized and transcardially perfused with 0.9% saline solution,
followed by 4% buffered paraformaldehyde. After fixation for 24 hr at 4°C, the brains were
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removed and embedded in paraffin. Three series of 5-μm thick coronal sections were cut at a level
of 1.94 mm posterior to the bregma. Each series of sections was used for hematoxylin and eosin
staining, Nissl staining, and immunostaining against 8-hydroxy-2′-deoxyguanosine (8-OHdG).
Hematoxylin and eosin staining, and Nissl staining
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Serial coronal sections were stained with hematoxylin and eosin. For Nissl staining, the sections
were stained with 0.2% cresyl violet. The cornu ammonis 1 (CA1) area of the hippocampus from
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each mouse was captured using a microscope (Axio Imager.A1, Carl Zeiss, Jena, Germany),
equipped with a digital camera system (Axio Cam MRc 5, Carl Zeiss, Jena, Germany).
Quantitative analysis of cell numbers was performed with AxioVision 4.5 software (Carl Zeiss,
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Jena, Germany). Histological changes in the hippocampal CA1 sections were evaluated by an
independent rater. For each section, 3 fields of view (×400) were sequentially selected, and the
numbers of pyramid cells inside were counted.
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8-Hydroxy-2′-deoxyguanosine staining
Immunohistochemistry was performed using a Histofine MOUSESTAIN Kit (Nichirei Co.,
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Tokyo, Japan), according to the manufacturer’s instructions. Serial coronal sections were stained
overnight at 4°C with a mouse monoclonal antibody against 8-OHdG (1:100; Japan Institute for
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the Control of Aging, Shizuoka, Japan), to detect oxidative DNA damage. Immunoreactivity was
detected using a diaminobenzidine method. Images were observed and captured at ×400
magnification with a microscope (Axio Imager.A1, Carl Zeiss) equipped with a digital camera
system (Axio Cam MRc 5, Carl Zeiss). Quantitative analysis of cell numbers was performed with
AxioVision 4.5 software (Carl Zeiss). Oxidative DNA damage was quantified in a blinded manner
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by counting the number of 8-OHdG-positive cells in 3 areas of the cortex (motor, somatosensory,
and piriform) at the coronal section (1.94 mm posterior to the bregma). Each area consisted of a
rectangle measuring 350 µm × 260 µm. The mean number of 8-OHdG-positive cells was
calculated from the 3 areas in the cortex.
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Malondialdehyde detection
Intracellular malondialdehyde (MDA) is a biomarker for lipid peroxidation. For MDA analysis, a
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total of 30 mice were used. Mice were divided into 3 groups: sham group (n = 10); BCCAO group
(n = 10); and BCCAO + H2 group (n = 10). After 24 hr of reperfusion, the MDA level in each
hemisphere was measured using a Bioxytech LPO-586 assay kit (OxisResearch, Foster City, CA,
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USA), according to the manufacturer’s instructions. The intracellular MDA concentration was
calculated by measuring the absorbance at 586 nm on a spectrophotometer (U-1500, Hitachi HighTechnologies Corporation, Minato-ku, Tokyo, Japan), using N-methyl-2-phenylindole as a
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substrate.
Brain water content
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For measurement of the brain water content, a total of 19 mice were used. Mice were divided into
3 groups: sham group (n = 6); BCCAO group (n = 7); and BCCAO + H2 group (n = 6). The brain
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water content was measured using the wet/dry method. Briefly, mice were decapitated after 24 hr
of reperfusion, and the brain of each mouse was removed. The cerebral cortex of each hemisphere
was separated and weighed, after which the tissues were placed in an oven at 110°C for 48 hr and
then reweighed. The brain water content was calculated by using the following formula: (wet
weight - dry weight)/wet weight × 100%.
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Serum albumin extravasation
For the evaluation of serum albumin extravasation, a total of 12 mice were used. Mice were
divided into 3 groups: sham group (n = 4); BCCAO group (n = 4); and BCCAO + H2 group (n =
4). After 24 hr of reperfusion, mice were anesthetized and transcardially perfused with 0.9%
saline solution, followed by 4% buffered paraformaldehyde. The brains were removed and
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embedded in paraffin as described above. Five-micrometer thick coronal sections were cut at 2
levels (0.50 mm anterior and 1.94 mm posterior to the bregma) and stained overnight at 4°C with
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a rabbit polyclonal antibody against mouse serum albumin (1:10000; Rockland Immunochemicals
Inc., Gilbertsville, PA, USA), to detect extravasated serum albumin. Immunoreactivity was
detected using a diaminobenzidine method. Images were observed and captured using a
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microscope (Axio Imager.A1, Carl Zeiss) equipped with a digital camera system (Axio Cam MRc
5, Carl Zeiss). For each section, a color image was obtained as a TIFF file, using Windows
software. Moreover, the extravasated albumin signals of the parietal cortex in the coronal section
of each hemisphere (1.94 mm posterior to the bregma) were extracted with Adobe Photoshop 4.0J
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(Adobe Systems, Tokyo, Japan), and quantified using the public domain NIH Image program
developed at NIH (USA) (13). The percentage of positive stained areas in the parietal cortex was
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calculated as described previously (14).
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Microtubule-associated protein 1 light chain 3 immunostaining
We used an antibody against microtubule-associated protein 1 light chain 3 (LC3) as a marker of
autophagosomes. For evaluating LC3 immunostaining, a total of 12 mice were used. They were
divided into 3 groups: sham group (n = 4); BCCAO group (n = 4); and BCCAO + H2 group (n =
4). After 24 hr of reperfusion, the mice were anesthetized and transcardially perfused with 0.9%
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saline solution, followed by 4% buffered paraformaldehyde. The brains were removed and
embedded in paraffin as described above. Coronal sections of 5-μm thickness were cut at a level
of 1.94 mm posterior to the bregma. Immunohistochemistry was performed using the Histofine
MOUSESTAIN Kit (Nichirei Co., Tokyo, Japan), according to the manufacturer’s instructions.
Serial coronal sections were stained for 60 min at room temperature with a rabbit anti-LC3
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antibody (1:1000; Medical and biological laboratories Co. Ltd., Nagoya, Japan).
Immunoreactivity was detected using the diaminobenzidine method. Images were observed and
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captured at ×400 and ×1000 magnification with a microscope (Axio Imager.A1, Carl Zeiss)
equipped with a digital camera system (Axio Cam MRc 5, Carl Zeiss). Quantitative analysis of
LC3-positive cell numbers was performed as reported previously (15). In brief, the number of
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LC3-positive cells was counted in every tenth section of the hippocampal CA1 sector, and at least
6 sections were counted in a blinded manner. In each section, 3 high-magnification visual fields
(0.091 mm2) with LC3-positive cells in the hippocampal CA1 sector were counted. Cell density
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was calculated from the total number of cells counted divided by the counting volume.
Statistical analysis
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All data are presented as mean ± SEM. Differences in survival between the groups were compared
using the Mantel-Cox log-rank test. Comparisons among multiple groups were performed with
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ANOVA, followed by the Bonferroni/Dunn test and Fisher’s least significant difference test.
Comparisons between the 2 groups were made by means of the unpaired t test. A value of P <
0.05 was considered statistically significant.
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Results
Physiological parameters and cerebral blood flow
We carefully monitored several physiological parameters and found no significant differences
between the BCCAO group and the BCCAO + H2 group. In each group, the residual CBF in each
differ significantly between the 2 groups (Table 1).
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hemisphere during BCCAO was less than 30% of the pre-occlusion value; the values did not
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Effect of hydrogen gas treatment on neurological findings and survival rate in BCCAO mice
In the BCCAO group and the BCCAO + H2 group, seizures were observed in 50% (6/12) and 30%
(3/10) of mice, respectively. The 7-day survival rate in the BCCAO group was 8.3% (Fig. 1).
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Inhalation of 1.3% hydrogen gas (BCCAO + H2 group) improved the 7-day survival rate from
8.3% to 50% (P = 0.0232; Fig. 1). Additionally, all mice in the sham group survived throughout
the experiment (n = 10; data not shown). In the BCCAO group, a mouse that survived at 7 days
after surgery exhibited the disappearance of the righting reflex (suggestive of severe disturbance
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of consciousness) and torsion of the neck. In the BCCAO + H2 group, 3 of 5 mice that survived at
7 days after surgery exhibited paucity of movement (suggestive of mild disturbance of
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consciousness) and 2 of 5 mice that survived at 7 days after surgery exhibited no neurological
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deficit.
Effect of hydrogen gas treatment on neuronal injury in the hippocampal CA1 sector of BCCAO
mice
We detected no obvious neuronal injury in the sham group (Fig. 2A; a and d). By contrast, in the
BCCAO group, severe neuronal injury in the CA1 sector was observed after 24 hr of reperfusion
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(Fig. 2A; b and e). Additionally, many pyramidal neurons in the CA1 sector exhibited pyknotic,
shrunken nuclei. In the BCCAO + H2 group, fewer injured neurons in the CA1 sector were
observed after 24 hr of reperfusion (Fig. 2A; c and f). We quantitatively compared the CA1
neuronal injuries among the 3 groups (Fig. 2B). In comparison with the sham group, the BCCAO
and BCCAO + H2 groups showed a significant increase in neuronal injuries (P < 0.001).
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Moreover, the BCCAO group exhibited a significantly higher number of neuronal injuries than
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did the BCCAO + H2 group (P < 0.05).
Effect of hydrogen gas treatment on 8-OHdG immunoreactivity in BCCAO mice
We identified oxidative DNA damage by using an 8-OHdG antibody. We observed almost no
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positive staining in the sham group (Fig. 3A; a). By contrast, after 24 hr of reperfusion, we
detected strong 8-OHdG immunoreactivity in the nuclei of neurons located in the cortex of the
BCCAO group (Fig. 3A; b). In the BCCAO + H2 group, the nuclei of neurons exhibited weakly
positive immunoreactivity for 8-OHdG after 24 hr of reperfusion (Fig. 3A; c). We quantitatively
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compared the 8-OHdG-positive neurons among the 3 groups (Fig. 3B). In comparison with the
sham group, the BCCAO and BCCAO + H2 groups showed a significant increase in 8-OHdG-
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positive neurons (P < 0.001, P < 0.05, respectively). Moreover, the BCCAO + H2 group exhibited
a significantly lower number of 8-OHdG-positive neurons than did the BCCAO group (P <
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0.001).
Effect of hydrogen gas treatment on brain MDA levels in BCCAO mice
We assessed oxidative stress by measuring the concentration of MDA in the brain tissue (Fig. 4).
In comparison with the sham group, the BCCAO group showed a significant increase in the MDA
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level after 24 hr of reperfusion (P < 0.001). By contrast, in the BCCAO + H2 group, the MDA
level increased, but did not differ significantly from that of the sham group. Moreover, the
BCCAO + H2 group exhibited a significantly lower MDA level than did the BCCAO group (P <
0.01).
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Effect of hydrogen gas treatment on the brain water content in BCCAO mice
In comparison with the sham group, the BCCAO group showed a significant increase in brain
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water content after 24 hr of reperfusion (78.7 ± 0.3% vs. 78.2 ± 0.1%; P < 0.05; Fig. 5).
Meanwhile, the BCCAO + H2 group exhibited a significantly lower brain water content than did
the BCCAO group (78.3 ± 0.3% vs. 78.7 ± 0.3%; P < 0.05; Fig. 5). The brain water contents of
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the BCCAO + H2 and sham groups did not differ significantly.
Effect of hydrogen gas treatment on serum albumin extravasation in BCCAO mice
We detected no obvious serum albumin extravasation in the sham group (Fig. 6A; a and d). By
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contrast, in the BCCAO group, we observed massive extravasation of serum albumin in the
striatum (Fig. 6A; b) and parietal cortex (Fig. 6A; e) after 24 hr of reperfusion. The BCCAO + H2
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group exhibited less extravasation of serum albumin in the striatum and parietal cortex after 24 hr
of reperfusion (Fig. 6A; c and f). We quantitatively compared the serum albumin extravasation in
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the parietal cortex between the BCCAO group and the BCCAO + H2 group (Fig. 6B). In
comparison with the BCCAO group, the BCCAO + H2 group exhibited a significant reduction in
serum albumin extravasation (7.8 ± 0.1% vs. 13.8 ± 0.3%; P < 0.0001).
Effect of hydrogen gas treatment on LC3 immunoreactivity in the hippocampal CA1 sector of
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BCCAO mice
Autophagy was evaluated using an anti-LC3 antibody. We observed almost no positive staining in
the sham group (Fig. 7A; a and d). In contrast, after 24 hr of reperfusion, we detected strong LC3
immunoreactivity in the cytoplasm of neurons located in the hippocampal CA1 sector of the
BCCAO group mice (Fig. 7A; b and e). In the BCCAO + H2 group, the cytoplasm of neurons
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exhibited weakly positive LC3 immunoreactivity after 24 hr of reperfusion (Fig. 7A; c and f). We
quantitatively compared the LC3-positive neurons among the 3 groups (Fig. 7B). In comparison
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with the sham group, the BCCAO and BCCAO + H2 groups showed a significant increase in LC3positive neurons (P < 0.001, P < 0.001, respectively). Moreover, the BCCAO + H2 group
exhibited a significantly lower number of LC3-positive neurons than did the BCCAO group (P <
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0.001).
Discussion
In the present study, treatment with 1.3% hydrogen gas significantly improved the 7-day survival
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rate of male C57BL/6J mice subjected to 45 min of BCCAO. Moreover, it significantly attenuated
neuronal injury and autophagy in the hippocampal CA1 sector and also brain edema, after 24 hr of
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reperfusion. The beneficial effects of hydrogen gas were associated with reduced levels of
oxidative DNA damage (as assessed by 8-OHdG) and lipid peroxidation (as assessed by MDA) in
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the brain tissues.
Hydrogen gas reacts with oxidizing elements such as chlorine, fluorine, and hydroxyl radicals
(16), and this may explain its anti-oxidative effect following ischemic brain injury (5). Inhalation
of hydrogen gas has been reported to reduce infarct size in the rat model of myocardial, intestinal,
and hepatic I/R injuries (17–19). In all of these injuries, the dominant ROS (hydroxyl radicals and
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peroxynitrite) indiscriminately react with nucleic acids, lipids, and proteins, resulting in DNA
fragmentation, lipid peroxidation, and protein inactivation (20, 21). Numerous ROS are also
generated during cerebral I/R. Among these, hydroxyl radicals and peroxynitrite appear to play a
critical role in tissue injury via the above-mentioned reaction with nucleic acids, lipids, and
proteins (5, 6). The human body has no endogenous detoxification system for hydroxyl radicals
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and peroxynitrite (5). Recently, Ohsawa et al. revealed that hydrogen gas selectively scavenged
hydroxyl radicals and peroxynitrite in vitro, thereby exerting a therapeutic antioxidant activity in a
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focal cerebral I/R rat model (5). They reported that hydrogen gas was more effective than
edaravone (approved in Japan as an ROS scavenger for the treatment of cerebral infarction) and as
effective as FK506 (used in the USA in clinical trials for cerebral infarction) in alleviating
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oxidative injury (5); wherefore, we used this strong FR scavenger for the treatment of global
cerebral I/R in this study. In the present study, treatment with 1.3% hydrogen gas significantly
reduced the levels of oxidative DNA damage and lipid peroxidation in the brain tissues. This
neuroprotective effect may be at least partly attributed to the ability of hydrogen gas to scavenge
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hydroxyl radicals and peroxynitrite.
Hydrogen is a flammable gas. In the presence of specific catalysts or heat, it is highly reactive
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with oxygen and other oxidants (20). However, a concentration of <4.6% hydrogen gas in air is
reported safe by volume (20). In the present study, we selected a hydrogen gas concentration of
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1.3% because, in Japan, this is the highest concentration that can be mixed and bottled under high
pressure with 30% oxygen for clinical use. Ohsawa et al. used concentrations of 1–4% in an in
vivo rat study and demonstrated that, even at 1%, hydrogen gas could reduce the infarct volume in
a focal cerebral I/R rat model (5). Furthermore, a concentration of 4% was less effective than was
a concentration of 2%. Thus, the effects of hydrogen gas inhalation do not appear to be positively
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correlated with concentration (5, 22). We believe that the standard mixed concentration of 1.3%
hydrogen gas used in the present study was reasonable for estimating the effects in the clinical
setting. In addition, Ohsawa et al. reported that hydrogen gas exerted its strongest effect on
reducing infarct volume in a focal cerebral I/R rat model when it was inhaled during whole I/R;
infarct volume was significantly less decreased when hydrogen gas was inhaled during
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reperfusion only (5). In a clinical setting, there is a time lag before successful reperfusion therapy
for global cerebral I/R, and we believe that it is reasonable to start hydrogen gas treatment in order
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to reduce the cytotoxic ROS as early as possible. Further studies are required to elucidate the
precise mechanism by which hydrogen gas reduces oxidative stress and to optimize the duration
of the hydrogen gas treatment for global cerebral I/R.
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In order to develop a highly reproducible BCCAO model, it is crucial to confirm the patency of Pcom A, which connects the posterior circulation of the brain from the vertebral arteries with the
anterior circulation from the carotid arteries in the circle of Willis (9, 12, 23). In a previous
anatomical study (12), we confirmed that mice without the P-com A exhibited a low residual CBF
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(less than 30% of pre-occlusion values) in the same hemisphere during BCCAO. Therefore, mice
that exhibited low residual CBF in the same hemisphere were included in the present study. The
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development of a highly reproducible BCCAO model is also dependent on adequate ischemic
duration (8, 24). In rats and gerbils, brief periods of BCCAO have been shown to induce
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consistent hippocampal CA1 injury; however, in C57BL/6J mice, longer ischemic duration may
be needed (8, 23). The required duration for C57BL/6J mice is reported to range from 10-60 min,
and many researchers have tended to adopt a relatively prolonged ischemic duration (25, 26). In
the present study, we selected 45 min as the ischemic duration for BCCAO in C57BL/6J mice. We
demonstrated that this consistently induced severe neuronal injury in the hippocampal CA1 sector.
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In the present study, we revealed that inhalation of 1.3% hydrogen gas significantly reduced
neuronal injury in the hippocampal CA1 sector of BCCAO mice. This observation is in
accordance with previous findings that inhalation of 1–4% hydrogen gas reduced the infarct size
in a focal cerebral I/R rat model (5), and that treatment with hydrogen saline significantly
increased the number of surviving neurons, and reduced the infarct size and caspase activity, in
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neonatal cerebral hypoxia-ischemia rats (6). In order to study the effect of hydrogen gas on a postI/R inflammatory response, we also investigated microglial cell activation in the hippocampus
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after 24 hr of reperfusion using anti-ionized calcium-binding adaptor molecule 1 antibody;
however, we could not detect any microglial cell activation in the hippocampus after 24 hr of
reperfusion in both the BCCAO and BCCAO + H2 groups (data not shown). A recent study
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reported that activated microglial cells in the hippocampus began to be detected after 48 hr of
reperfusion in a global cerebral I/R rat model (27). Therefore, we believe that our investigation of
microglial cell activation in the hippocampus in global cerebral I/R mice was premature, and
hence, we could not detect any activated microglial cells. In addition, we investigated the effect of
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hydrogen gas on post-ischemic autophagy in the hippocampal CA1 sector of BCCAO mice, using
an anti-LC3 antibody. We revealed that inhalation of 1.3% hydrogen gas significantly attenuated
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post-ischemic autophagy in the hippocampal CA1 sector of BCCAO mice. Currently, there is
increasing evidence that autophagy may be involved in mediating neuronal death in cerebral
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ischemia (28–30), and Nitatori et al. particularly reported that autophagy is highly induced in CA1
pyramidal neurons in gerbil hippocampus after brief BCCAO (28). Furthermore, several
investigators have reported that ROS generation, including that of hydroxyl radicals, may increase
autophagy and lead to neuronal cell death in murine model of focal cerebral I/R and cerebral
hypoxia-ischemia (31–33). Therefore, we speculate that treatment with hydrogen gas attenuated
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autophagy by reducing ROS, which might have led to the attenuation of neuronal injury in the
hippocampal CA1 sector. Further investigations of the relationship between hydrogen gas
treatment and autophagy in global cerebral I/R are required.
Cerebral I/R results in disruption of the blood–brain barrier (BBB), and causes brain edema (34).
The BCCAO model was previously reported to induce endothelial injury and breakdown of the
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BBB (35, 36). I/R injury is known to give rise to a surge of superoxide anions in endothelial cells,
immediately after the onset of reperfusion. Superoxide anions are either transformed to H2O2 by
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superoxide dismutase catalyzing or to hydroxyl radicals via the Haber-Weiss reaction (37, 38).
These ROS directly destroy lipids, proteins, and nucleic acids, resulting in damage to vascular
endothelial cells and the basement membrane (39). Thus, the inhibition of ROS is an effective
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therapeutic intervention for improving the integrity of the BBB during I/R injury (40). Chen et al.
reported that hydrogen gas reduced hemorrhagic transformation in a focal cerebral I/R rat model,
and that the reduction of oxidative agents might contribute to increased survival of endothelial
cells, neurons, and glial cells (41). To our knowledge, the present study is the first to demonstrate
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that treatment with hydrogen gas attenuates serum albumin extravasation and brain edema in
BCCAO mice. We propose that the attenuation of serum albumin extravasation may arise from
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the protection of endothelial cells, via reduction of oxidative stress by hydrogen gas. Further
studies are required to elucidate the precise mechanism by which hydrogen gas acts on the BBB.
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In the present study, treatment with 1.3% hydrogen gas significantly improved the 7-day survival
rate of male C57BL/6J mice subjected to 45 min of BCCAO. We observed that inhalation of
hydrogen gas significantly attenuated oxidative stress, neuronal injury and autophagy in the
hippocampal CA1 sector, and brain edema in BCCAO mice. Thus, we speculate that attenuation
of neuronal cell death in the central nervous system via the reduction of potent ROS and the
18
Copyright © 2012 by the Shock Society. Unauthorized reproduction of this article is prohibited.
reduction of brain edema via potent ROS reduction may be at least partly responsible for the
improvement in survival rate. However, the precise mechanism by which the hydrogen gas
improves the survival rate is unclear. Although the mechanisms by which mice die following a
relatively long period of BCCAO remain to be clarified (42), various factors such as body
temperature, anesthesia, and seizure susceptibility have been identified as influencing the outcome
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(10, 43). In the present study, we detected a trend for decreasing seizure following inhalation of
hydrogen gas, from 6/12 (50%) in the BCCAO group to 3/10 (30%) in the BCCAO + H2 group.
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Increasing numbers of ROS may be a cause and/or consequence of seizures (44). Thus, we believe
that the trend for decreasing seizure rates following inhalation of hydrogen gas may also
contribute to the improvement in survival rate. Further studies are required to elucidate the precise
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mechanism by which hydrogen gas ameliorates mortality. On the behavioral aspects of the
surviving mice at 7 days after surgery, our data suggested that hydrogen gas inhalation attenuated
a disturbance of consciousness, which may be related with attenuation of the neuronal injury and
brain edema. Moreover, we did not observe any surviving mice that exhibited torsion of the neck
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in the BCCAO + H2 group. This finding may be related with attenuation of the neuronal injury in
the hippocampal CA1 sector, because neck torsion and rolling fits were reported as typical
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ischemic signs in C57BL/6J mice that exhibited neuronal death in the hippocampus and/or
caudoputamen after BCCAO (23).
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The present study had a number of limitations. Firstly, we were unable to obtain data regarding
the long-term neurological outcome, long-term histopathological outcome, and detailed functional
outcome because 45 min of BCCAO was too severe for these outcomes to be investigated in mice.
Secondly, we were unable to obtain data using other concentrations of hydrogen gas. Further
studies involving much longer duration, behaviors, and that aim at clarifying the optimal treatment
19
Copyright © 2012 by the Shock Society. Unauthorized reproduction of this article is prohibited.
concentration are required.
In conclusion, inhalation of 1.3% hydrogen gas significantly attenuated oxidative stress, neuronal
injury and autophagy in the hippocampal CA1 sector, brain edema, and mortality rate in BCCAO
mice. Thus, we believe that early-phase administration of hydrogen gas may be an effective
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therapeutic strategy for patients suffering from global cerebral I/R associated with cardiac arrest.
Competing interests: None.
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Acknowledgments: The authors thank Ms. Akiko Yano for valuable technical assistance.
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Ethics approval: This study was conducted with the approval of the ethics committee of the
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National Defense Medical College.
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Figure legends
Figure 1: Treatment with 1.3% hydrogen gas improves the survival rate in BCCAO mice. In
the BCCAO + H2 group, mice inhaled hydrogen gas during the 225-min process (45 min of
occlusion and 180 min of reperfusion). Furthermore, hydrogen gas was administered for 3 hr per
day, from 1–3 days after surgery. In the BCCAO group, mice inhaled oxygen gas, instead of
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hydrogen gas, during the entire process. Furthermore, 30% oxygen gas was administered for 3 hr
per day, from 1–3 days after surgery. In each group, the number of mice surviving at 7 days after
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surgery was counted. Values are expressed as survival percentages (BCCAO, n = 12; BCCAO +
H2 group, n = 10). *P = 0.0232 vs. BCCAO group.
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Figure 2: Treatment with 1.3% hydrogen gas attenuates neuronal injury in the hippocampal
CA1 sector of BCCAO mice. A, Representative photomicrographs demonstrating CA1
hippocampal neurons after 24 hr of reperfusion. (a-c) Hematoxylin and eosin staining; (d-f) Nissl
staining. In the sham group, no obvious neuronal injury is evident (a and d). In the BCCAO group,
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many pyramidal neurons exhibit pyknotic, shrunken nuclei (b and e). In the BCCAO + H2 group,
fewer injured neurons are evident (c and f). Arrows indicate injured neurons. Scale bar = 50 μm.
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B, Quantitative evaluation of intact pyramidal neurons in the hippocampal CA1 sectors of the
sham group, BCCAO group, and BCCAO + H2 group. Values are expressed as mean ± SEM (n =
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4 per group). *P < 0.001 vs. sham group; **P < 0.05 vs. BCCAO group.
Figure 3: Treatment with 1.3% hydrogen gas attenuates oxidative DNA damage in BCCAO
mice. A, Oxidative DNA damage as assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG)
immunoreactivity. In the sham group, almost no positive staining is visible (a). After 24 hr of
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reperfusion, 8-OHdG immunoreactivity is more prominent in the BCCAO group (b) than in the
BCCAO + H2 group (c). Arrows indicate positively-stained cells. Scale bar = 50 μm. B, Numbers
of 8-OHdG-positive cells in the cortices of the sham group, BCCAO group, and BCCAO + H2
group. Values are expressed as mean ± SEM (n = 4 per group). *P < 0.001 vs. sham group; **P <
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0.05 vs. sham group; ***P < 0.001 vs. BCCAO group.
Figure 4: Treatment with 1.3% hydrogen gas reduces the level of intracellular
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malondialdehyde (MDA; a biomarker for lipid oxidation) in BCCAO mice. After 24 hr of
reperfusion, the MDA level in each hemisphere was measured in the sham group, BCCAO group,
and BCCAO + H2 group. Values are expressed as mean ± SEM (n = 10 per group). *P < 0.001 vs.
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sham group; **P < 0.01 vs. BCCAO group.
Figure 5: Treatment with 1.3% hydrogen gas attenuates brain edema in BCCAO mice. The
brain water content as an indicator of brain edema was measured after 24 hr of reperfusion in the
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sham group, BCCAO group, and BCCAO + H2 group. Values are expressed as mean ± SEM
(sham group, n = 6; BCCAO group, n = 7; BCCAO + H2 group, n = 6). *P < 0.05 vs. sham group;
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**P < 0.05 vs. BCCAO group.
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Figure 6: Treatment with 1.3% hydrogen gas improves the integrity of the blood–brain
barrier (BBB) in BCCAO mice. A, BBB integrity in the striatum (a-c) and parietal cortex (d-f)
as determined by serum albumin extravasation, after 24 hr of reperfusion in the sham group,
BCCAO group, and BCCAO + H2 group. In the sham group, no obvious serum albumin
extravasation is evident (a and d). In the BCCAO group, massive serum albumin extravasation is
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Copyright © 2012 by the Shock Society. Unauthorized reproduction of this article is prohibited.
visible in the striatum (b) and parietal cortex (e). In the BCCAO + H2 group, less serum albumin
extravasation is evident (c and f). Scale bar = 200 μm. B, Quantitative evaluation of serum
albumin extravasation in the parietal cortices of the BCCAO and BCCAO + H2 groups. Values are
expressed as mean ± SEM (n = 4 per group). *P < 0.0001 vs. BCCAO group.
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Figure 7: Treatment with 1.3% hydrogen gas attenuates post-ischemic autophagy in the
hippocampal CA1 sector of BCCAO mice. A, Autophagy in the hippocampal CA1 sector after
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24 hr of reperfusion was evaluated using an antibody against microtubule-associated protein 1
light chain 3 (LC3). (a-c) ×400 magnification; (d-f) ×1000 magnification. In the sham group,
almost no positive staining is visible (a and d). After 24 hr of reperfusion, LC3 immunoreactivity
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is more prominent in the BCCAO group (b and e) than in the BCCAO + H2 group (c and f). Scale
bar = 50 μm. B, Quantitative evaluation of LC3-positive neurons in the hippocampal CA1 sector
of mice in the sham, BCCAO, and BCCAO + H2 groups. Values are expressed as mean ± SEM (n
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= 4 per group). *P < 0.001 vs. sham group; **P < 0.001 vs. BCCAO group.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Before ischemia
BCCAO
BCCAO+H2
73.0 ± 2.7
72.2 ± 1.5
100.7 ± 5.1
103.0 ± 3.9
77.2 ± 2.9
78.1 ± 3.3
CBF (%)
163.7 ± 7.6
169.4 ± 6.8
37.16 ± 0.05
37.13 ± 0.04
100
100
539.9 ± 24.1
520.4 ± 29.9
173.0 ± 10.5
145.6 ± 13.9
37.14 ± 0.03
37.20 ± 0.04
28.9 ± 0.8
29.7 ± 1.0
578.6 ± 31.4
567.3 ± 15.9
164.7 ± 2.5
163.0 ± 1.4
37.14 ± 0.03
37.23 ± 0.04
101.5 ± 6.1
106.8 ± 7.0
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After reperfusion (180 min)
BCCAO
BCCAO+H2
Data are mean ± SEM from 7 mice.
BT (°C)
494.0 ± 23.4
440.9 ± 17.1
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During ischemia (45 min)
BCCAO
BCCAO+H2
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Table 1. Physiological parameters and CBF
MABP (mmHg) HR (bpm) RR (bpm) 37
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