ORIGINAL PAPER

Hydrogen-Rich Saline is Cerebroprotective in a Rat Modelof Deep Hypothermic Circulatory Arrest

Li Shen • Jun Wang • Kun Liu • Chunzhang Wang •

Changtian Wang • Haiwei Wu • Qiang Sun •

Xuejun Sun • Hua Jing

Accepted: 11 April 2011 / Published online: 22 April 2011

� Springer Science+Business Media, LLC 2011

Abstract Deep hypothermic circulatory arrest (DHCA)

has been widely used in the operations involving the aortic

arch and brain aneurysm since 1950s; but prolonged

DHCA contributes significantly to neurological deficit

which remains a major cause of postoperative morbidity

and mortality. It has been reported that hydrogen exerts a

therapeutic antioxidant activity by selectively reducing

hydroxyl radical. In this study, DHCA treated rats devel-

oped a significant oxidative stress, inflammatory reaction

and apoptosis. The administration of HRS resulted in a

significant decrease in the brain injury, together with lower

production of IL-1b, TNF-a, 8-OHdG and MDA as well as

decreased activity of NOS while increased activity of SOD.

The apoptotic index as well as the expressions of caspase-3

in brain tissue was significantly decreased after treatment.

HRS administration significantly attenuated the severity of

DHCA induced brain injury by mechanisms involving

amelioration of oxidative stress, down-regulation of

inflammatory factors and reduction of apoptosis.

Keywords Cerebral protection � Deep hypothermic

circulatory arrest � Hydrogen � Oxidative stress � Rat

Abbreviation

DHCA Deep hypothermic circulatory arrest

8-OH-dG 8-Hydroxydeoxyguanosine

ELISA Enzyme-linked immunosorbent assay

EMSA Electromobility Shift Analysis

HRS Hydrogen-rich saline

IL-1b Interleukin-1bMDA Malondialdehyde

NF-jB Nuclear factor-jB

NOS Nitric oxide synthase

RNS Reactive nitrogen species

ROS Reactive oxygen species

SIRS Systemic inflammatory response syndrome

SOD Superoxide dismutase

TNF-a Tumor necrosis factor-a

Introduction

Deep hypothermia circulation arrest (DHCA) has been

used in the surgical repair of complex congenital heart

malformations and the operations involving the aortic arch

and brain aneurysm since invented in 1950s [1]. Such

technique has been widely used as it can provide a quiet,

dry and motionless surgical field for a perfect operation [2].

However, the long duration of DHCA results in some

severe neurological complications which contribute sub-

stantially to postoperative mortality and morbidity [3]. It is

reported that about 25% of patients undergoing a tempo-

rary exclusion of cerebral circulation suffer from tempo-

rary neurological dysfunction [4], and 55% of patients

undergoing DHCA demonstrated a neuropsychological

deficit 12 weeks after the operation [5]. How to limit the

neurological complications of DHCA is a critical issue that

must be solved practically. The pathophysiology of DHCA

L. Shen � J. Wang � K. Liu � C. Wang � C. Wang � H. Wu �H. Jing (&)

Department of Cardiothoracic Surgery, Jinling Hospital, Clinical

Medicine School of Nanjing University, 305 East Zhongshan

Road, Nanjing 210002, People’s Republic of China

e-mail: drhuajing@yahoo.cn

Q. Sun � X. Sun (&)

Department of Diving Medicine, Faculty of Naval Medicine,

Second Military Medical University, Shanghai 200433,

People’s Republic of China

e-mail: sunxjk@hotmail.com

123

Neurochem Res (2011) 36:1501–1511

DOI 10.1007/s11064-011-0476-4

mainly consists of the systemic inflammatory response

syndrome (SIRS) caused by cardiopulmonary bypass and

the ischemia–reperfusion (I/R) injury associated with cir-

culatory arrest. It is believed that mechanisms of I/R-

induced damage are multifactorial and interdependent,

involving hypoxia, inflammatory responses and free radical

damage [6, 7]. The major free radicals causing severe tis-

sue damage include reactive oxygen species (ROS) and

reactive nitrogen species (RNS). Oxidative stress repre-

sents an imbalance between the production of free radicals

and the activity of antioxidant defense systems [8].

Therefore, in theory, agent with anti-inflammatory or anti-

oxidation activity may be cerebroprotective alleviating the

damage induced by DHCA.

Recently, Ohsawa et al. demonstrated that hydrogen

(H2) ameliorates focal ischemic injury in adult rats, the

mechanisms may be related to direct consumption of free

radical such as •OH by H2 [9]. It is also reported that H2

saturated water could attenuate superoxide formation in ex

vivo mouse brain slices with diminished endogenous free

radical buffering capacity [10]. Similar result has been

observed in chronically restrained mice [11] and the rat

model of carbon monoxide encephalopathy [12]. Addi-

tionally, Cai et al. found that H2 therapy after a mild

(90 min hypoxia) hypoxia–ischemia in neonatal rats

decreases infarction volume and apoptotic cell death of the

brain [13]. Further studies carried out in recent years also

indicated that H2 could effectively reduce the injury in the

experimental intestinal I/R model [14, 15, 16], myocardial

ischemia model [17, 18], contused spinal cord injury model

[19], chronic liver inflammation [20] and arteriosclerosis

patients [21].

Accumulated data suggested that the H2 could therapy

I/R lesions by mechanisms involving anti-inflammation

and anti-oxidation. However, the effects of H2 on brain

injury induced by DHCA have not yet been reported.

Prompted by the previous findings, we hypothesized that

the H2 was able to effectively ameliorate the brain injury

induced by DHCA by mechanisms involving anti-inflam-

mation and/or anti-oxidation. Our results provided evi-

dence that H2 is cerebroprotective in a rat model of DHCA.

Experimental Procedure

Animals and Protocol

This study was conformed to the Guide for the Care and

Use of Laboratory Animals published by the US National

Institute of Health (NIH Publication No. 85–23, revised

1996) and was approved by the Animal Care and Use

Committee of Nanjing University. Male Sprague–Dawley

rats (250 ± 10 g) were housed with free access to food and

water in 12 h day/night cycle. All rats were acclimated for

7 days before any experimental procedures, and were fas-

ted for 12 h with water ad libitum before operation. Ani-

mals were randomly divided into the following three

groups: (1) Sham-operated group; (2) DHCA group; (3)

H2-treated DHCA group.

The hydrogen-rich saline (HRS) was prepared as

described by Cai et al. [22]. In brief, H2 was dissolved in

normal saline for 2 h under high pressure (0.4 MPa) to the

supersaturated level using a self-designed hydrogen-rich

water producing apparatus. The saturated H2 saline was

stored under atmospheric pressure at 4�C in an aluminum

bag without dead volume, sterilized by gamma radiation.

HRS was freshly prepared every week to ensure a constant

concentration more than 0.6 mM. Gas chromatography

(Biogas Analyzer Systems-1000, Mitleben, Japan) was

used to confirm the content of H2 in saline by the method

described by Ohsawa et al. [9].

The rats were anesthetized with 10% chloral hydrate

(0.4 ml/100 g body weight, i.p.), and the 4-vessel occlu-

sion (4-VO) rat global I/R model [23] was set up with some

modification [24]. Briefly, the anesthetized rat was incised

on the middle posterior neck, and the bilateral vertebral

arteries were dissected and electrocauterized carefully.

Subsequently, the rats were positioned supine, and the

bilateral common carotid arteries (CCA) were carefully

explored, isolated from the adjacent nerve tissue. Next,

bilateral CCA were snared loosely with a 7# thread, fol-

lowed by saturation of the incision. The rats were still in an

anesthetized state following these procedures. Subse-

quently, the rats were put in an ice nest with a thermometer

inserted into rectum about 5–6 cm-deep near sub-dia-

phragm to monitor the core temperature. The temperature

decreased slowly by no more than 3�C per 10 min. When

the core temperature reached 20�C, bilateral CCAs were

occluded for 45 min while the core temperature was

maintained between 20 ± 1�C. The normal saline (in

DHCA group) or HRS (in DHCA ? H2 group) were

intraperitoneally administered (5 ml/kg body weight)

10 min before the opening of bilateral CCAs. The rats were

then moved out if the ice nest and rewarmed to normal

body temperature at a rate less than 2�C per 10 min. After

rewarming, the rats were reared in the cage. We checked

blood flow signal of 4 vessels with the color Doppler

ultrasonography (Philips iE33, Sector array probe S12-4).

Rats with the vertebral artery blood flow signal were

excluded from the experiment. The sham-operated group

was only subjected to anesthesia and the procedures to

isolate four vessels without occlusion.

The rats were sacrificed 24 h after treatment for sample

collection. Blood was taken from the left ventricle, and the

separated serum was stored at -80�C immediately for

future assay. The rat brain was fast perfused with cold

1502 Neurochem Res (2011) 36:1501–1511

123

normal saline (4�C) through ascending aortic artery till the

backflow fluid in the right atrium was clear, then the brain

was removed quickly and stored at -80�C.

For histopathology analysis and TUNEL staining, the

rats were sacrificed 72 h after the treatment, and the brain

were perfused with 150 ml 4% paraformaldehyde/PB (PH

7.4) for 30 min after normal saline perfusion, followed by

immersion in the 4% paraformaldehyde/PB.

Histopathology Analysis

Brain tissues were sectioned (5 lm thick) and stained with

hematoxylin and eosin (HE) using standard methods. The

neuronal damage of the hippocampus CA1 region was eval-

uated qualitatively by an experienced pathologist blinded to

the identity of the samples with light microscopy. Normal

appearing neurons and neurons showing features of ischemic

cell death (shrunken cell bodies, triangulated, pyknotic

nuclei, and eosinophilic cytoplasm) were counted using a

defined rectangular field area with magnification 9200. Cell

death, expressed as neuropathological score, was assessed

using a 0–4 grading scale as described by Hua et al. (0: no

damage; 1:[0, B12.5% damage; 2:[12.5%, B25% damage;

3:[25%, B50% damage and 4:[50% damage) [25].

ELISA Assay

The brain tissues were homogenized on ice in 1 ml normal

saline (4�C) and centrifuged at 12,000 g at 4�C for 20 min.

The protein content in the brain was determined by Com-

massie blue assay. The levels of IL-1b, TNF-a and

8-OHdG in serum and brain were quantified using enzyme-

linked immunosorbent assay (ELISA) kits specific for rat

according to the manufacturers’ instructions (R&D sys-

tems). The concentrations of IL-10 were detected by

commercial ELISA kits (Biosource). The concentrations in

the brain samples were expressed as per milligram protein.

Colorimetric Assay

MDA concentration, nitric oxide synthase (NOS) and

superoxide dismutase (SOD) activity in the serum or brain

samples were measured using colorimetric kits according

to the manufacturers’ instructions (Nanjing Jiancheng

Institute of Bio-engineering, China).

TUNEL Staining for Apoptosis

The fixed brain samples were dehydrated and embedded in

paraffin, sectioned at 5 lm, and examined with TUNEL

staining according to the manufacturer’s instructions

(Roche). In brief, the sections were dewaxed and rehy-

drated by heating the slides at 60�C, followed by digestion

with proteinase K at 20 lg/ml for 15 min at room tem-

perature. The slides were rinsed three times with PBS and

then incubated in the TUNEL reaction mixture for 1 h at

37�C. Dried area around sample and added Converter-AP

on samples for 1 h at 37�C. After rinsing with PBS (5 min,

3 times), sections were colourated in dark with nitroblue

tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylpho-

sphate (BCIP). Six visual fields (0.6 mm2) of the CA1 area

of hippocampus were photographed in each section. The

number of stained cells in each field was counted (9400

magnification). One hundred cells were counted in each

field. The data were presented as the apoptosis index (AI).

Nuclear Protein Extract and EMSA for NF-jB

Nuclear protein was extracted and quantified as described

by Zhou ML, et al. [26]. Briefly, frozen brain samples were

homogenized in 0.8 ml ice-cold buffer A composed of

10 mmol/l HEPES pH 7.9, 10 mmol/l KCl, 2 mmol/l

MgCl2, 0.1 mmol/l EDTA, 1 mmol/l dithiothreitol (DTT),

and 0.5 mmol/l phenylmethylsulfonyl fluoride (PMSF) (all

from Sigma Chemical Co., St. Louis, Mo, USA). The

homogenates were incubated on ice for 30 min and vor-

texed for 30 s after addition of 50 ll 10% NP-40 (Sigma

Chemical Co., Mo, USA). The mixture was then centri-

fuged for 10 min (5,000 g, 4�C). The pellet was suspended

in 100 lL ice-cold buffer B composed of 50 mmol/l

HEPES pH 7.9, 50 mmol/l KCl, 300 mM NaCl, 0.1 mmol/

l EDTA, 1 mmol/l DTT, and 0.5 mmol/l PMSF, and 10%

(v/v) glycerol and incubated on ice 30 min with frequent

mixing. After centrifugation (12,000 g, 4�C) for 15 min,

the supernatants were collected as nuclear extracts and

stored at -70�C for further use. Protein concentration was

determined using a bicinchoninic acid assay kit with

bovine serum albumin as the standard (Pierce Biochemi-

cals, Rockford, Ill, USA).

EMSA was performed using a commercial kit (Gel Shift

Assay System; Promega, Madison, Wis, USA) following the

methods in our laboratory [26]. Consensus oligonucleotide

probe (5-AGTTGAGGGGACTTTCCCAGGC-3) was end-

labeled with [c-32P] ATP (Free Biotech., Beijing, China)

with T4-polynucleotide kinase. Nuclear protein (10 lg) was

preincubated in a total volume of 9 ll in a binding buffer,

consisting of 10 mmol/l Tris–HCl (pH 7.5), 4% glycerol,

1 mmol/lMgCl2, 0.5 mmol/l MEDTA, 0.5 mmol/l DTT,

0.5 mmol/l NaCl, and 0.05 g/l poly-(deoxyinosinicdeox-

ycytidylic acid) for 15 min at room temperature. Then 1 ll

32P-labled oligonucleotide probe was added, and also incu-

bated for 20 min at room temperature. Finally 1 ll gel

loading buffer was added to stop the reaction and the mixture

was subjected to nondenaturing 4% polyacrylamide gel

electrophoresis in 0.5 9 TBE buffer (Trisborate-EDTA).

After electrophoresis was conducted at 390 V for 1 h, the gel

Neurochem Res (2011) 36:1501–1511 1503

123

was vacuum-dried and exposed to X-ray film (Fuji Hyper-

film, Tokyo, Japan) at -70�C. Levels of NF-jB DNA binding

activity were quantified by computer-assisted densitometric

analysis.

Western Blotting to Determine Caspase-3 Activity

The tissue samples were homogenized in 10% (w/v) Radio-

Immunoprecipitation Assay buffer (50 mM Tris, pH 7.4,

150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and

0.1% SDS) containing complete protease inhibitors (Sigma,

St. Louis, MO) and centrifuged at 16,000 g for 20 min. The

supernatants were collected and quantified for protein con-

centration by bicinchoninic acid assay method (Bio-Rad

Laboratories, Mississauga, Ontario, Canada). Prepared pro-

tein samples (375 lg/well) were separated on 8–16% SDS–

PAGE and transferred to a polyvinylidene fluoride mem-

brane (Millipore, Billerica, MA). The membranes were

blocked with 5% nonfat milk in 0.01 M PBS with 0.1%

Tween-20 (pH 7.4) at room temperature for 1 h. Then, the

membrane was incubated at 4�C overnight with cleaved

caspase-3 antibodies(Cell Signaling, Danvers, MA; 1:500,

diluted with 5% nonfat milk in 0.01 M PBS with 0.1%

Tween-20). After three washes in 0.01 M PBS with 0.1%

Tween-20, the membranes were incubated with horseradish

peroxidase-conjugated goat anti-rabbit (Santa Cruz Bio-

technology, Inc., Santa Cruz, CA) diluted to 1:2000 in PBS

with 0.1% Tween-20 for 1 h at room temperature. After three

washes in 0.01 M PBS with 0.1% Tween-20 again, the blots

were detected with Pierce ECL Western Blotting Substrate

(Thermo Fisher Scientific, Rockford, IL). To prove equal

loading, the blots were analyzed for b-actin expression using

an anti–b-actin antibody (Santa Cruz Biotechnology, Inc.).

Statistical Analysis

Data were expressed as means ± standard deviation(S.D.).

Statistical analysis was carried out with the SPSS Statistics

13.0 (SPSS Inc., Chicago, IL, USA) by using one-way

analysis of variance (ANOVA) to establish whether the

difference was statistically significant. A v2 test was per-

formed to analyze mortality. The level of significance was

set at P \ 0.05.

Results

Hydrogen-Rich Saline Administration Reduced

the Postoperative Mortality

We evaluated the mortality of the rats 24 h post-operation.

No death was observed in the sham group. In the DHCA

group, 7 rats died with a mortality of 35% (7/20). The HRS

administration improved the mortality of 15% (3/20).

Statistical analysis showed that there was a significant

difference in the mortality between DHCA group and

DHCA ? H2 group (v2 = 9.130, P = 0.010). HRS

administration significantly decreased mortality and

improved the general conditions in rats treated with

DHCA.

Hydrogen-Rich Saline Administration Improved

Pathological Features

The HE staining of the sham group showed that the

outline of pyramid cells in the CA1 area of hippocampus

was clear and the structure was compact. Big oval-shaped

cells with abundant cytoplasm were identified. The axons

were arranged with regular full nuclei, sparsely distrib-

uted nuclear chromatins and clear nucleoli. No neuronal

injury was observed. In contrast, substantial amount of

neuronal loss and a great number of dead cells were

observed in the DHCA group, the HE staining revealed

cells in CA1 sector arranged sparsely and the cell outline

was fuzzy. The eumorphism cells were significantly

reduced instead with the deflated cells and fuzzy-arranged

axons. Interestingly, HRS dramatically improved the

morphological changes found in rats treated with DHCA

which was confirmed by the HE staining (See Fig. 1). No

evidence was observed about the effects of HRS on the

pathological features in rats of sham group (data not

shown). The statistic analysis of neuopathological score to

hippocampal CA1 area showed there had a significant

differance between DHCA and DHCA ? H2 groups (see

Table 1).

Hydrogen-Rich Saline Administration Reduced

Oxidative Stress

Oxidative stress can cause toxic effects that damage all

components of a cell including lipids, proteins and DNA.

Malondialdehyde (MDA) is one of the major products

derived from lipid peroxidation and 8-Hydroxydeoxygu-

anosine (8-OH-dG) is considered to be a DNA-damage

biomarker. Our data showed that the rats subjected to

DHCA displayed a significant increased level of MDA and

8-OHdG as well as NOS activity both in serum and brain

tissue in contrast to the sham-operated rats. In accordance

with these results, DHCA treatment resulted in a decrease

in the SOD activity. Administration of HRS to the rats

treated with DHCA resulted in a marked reduction of the

NOS activity, MDA and 8-OHdG levels compared to the

DHCA group. Additionally, HRS significantly increased

the SOD activity in rats treated with DHCA. (See Fig. 2).

1504 Neurochem Res (2011) 36:1501–1511

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Hydrogen-Rich Saline Administration Ameliorated

Inflammation Effectively

To evaluate inflammation level, we checked the concen-

trations of TNF-a and IL-1b in serum and brain as well as

brain water content. Significant elevation of these bio-

markers was observed in the DHCA group compared with

those in the sham group. HRS administration significantly

decreased the levels of TNF-a and IL-1b in serum and

brain as well as brain water content when compared with

those in DHCA group. In contrast, there were no differ-

ences in the levels of IL-10 in serum (590.83 ± 64.92 vs.

625.61 ± 88.3 P = 0.46 pg/ml, P = 0.36) or in brain tis-

sue (668.24 ± 62.77 vs. 684.11 ± 86.52 pg/g brain tissue,

P = 0.72) tested by ELISA between the DHCA and DHCA

? H2 groups. In addition, we assayed the NF-jB activity in

brain in three groups, and a similar reduction pattern was

obtained as that for TNF-a and IL-1b (See Fig. 3).

Hydrogen-Rich Saline Reduced Apoptosis

To evaluate the anti-apoptotic effects, caspase-3 activity in

brain tissue was assayed by western blotting. Moreover, we

checked the histopathology alteration with TUNEL stain-

ing in the CA1 area in the hippocampus that has been

shown to be the most ischemia-sensitive area in the brain.

[27]. Our TUNEL assay indicated that there were few

apoptotic cells in the sham group. However, the apoptotic

index in the DHCA group displayed markedly increased

compared with the sham group (P \ 0.01). In comparison

to the DHCA group, there were fewer TUNEL-positive

Fig. 1 Morphological features of CA1 area in rat’s hippocampus

from each group were evaluated by HE staining. (magnifications: a, b,c: 940; d, e, f: 9200; g, h, i:9400). In sham group (see a, d, g), the

outline of pyramid cells in CA1 area was clear and the structure was

compact (a, d). Cells were big with an intact oval shape and have

abundant cytoplasm. The axons were intact and lined up regular with

full nucleus, sparsely nuclear chromatins and clear nucleoli. No

necrosis cell was found (g). In DHCA group (see b, e, h), lots of

neuronal loss and dead cells were observed, the cells in CA1 sector

arranged sparsely and the cell outline was fuzzy (b, e). The

eumorphism cells were significantly reduced instead with the deflated

cells and the axons arranged fuzzy (h). In DHCA ? H2 group (see c, f,i) the morphological changes in rats were improved

Table 1 The neuopathological score of hippocampal CA1 area

Group n Neuopathological score

(mean ± S.D.)

DHCA group 10 2.3 ± 0.48

DHCA ? H2 group 10 1.2 ± 0.42�

The table shows the neuropathological score of hippocampal CA1

area in rat of DHCA group and DHCA ? H2 group. The score was

assessed by using a 0–4 grading scale (0: no damage; 1:[0, B12.5%

damage; 2:[12.5%, B25% damage; 3: [25%, B50% damage and 4:

[50% damage). There were 10 rats per group. (� P \ 0.01)

Neurochem Res (2011) 36:1501–1511 1505

123

cells and decreased apoptotic index in the DHCA rats

treated with HRS (P \ 0.01). HRS showed no effects of

numbers of TUNEL-positive cells in rats from sham group

(data not shown). The level of activated caspase-3 in the

DHCA group was significantly increased compared to the

sham group. The treatment of HRS significantly suppressed

the activation of caspase-3 in the DHCA ? H2 group which

was consistent with the results of TUNEL staining (See

Fig. 4).

Discussion

The present study demonstrated that HRS can attenuate the

severity of brain injury caused by DHCA by down-regu-

lating the expression of proinflammatory cytokines, as well

as reducing the apoptosis in the rat brain. Administration of

HRS remarkably improved the clinical symptoms 24 h

post-operation as measured by evaluating the general

condition and mortality. Histopathological assays further

Fig. 2 The levels of oxidative

stress in the serum and brain

tissues in rats from each group

(a, b, c, d serum, e, f, g, h brain

tissues). The rats subjected to

DHCA displayed a significant

increased the levels of MDA

and 8-OHdG as well as NOS

activity both in serum (b, c, d)

and brain tissues (f, g, h) when

compared with those in sham-

operated rats. In accordance to

these results, DHCA treatment

resulted in a decrease in SOD

activity (a, e). Administration of

hydrogen-rich saline resulted in

a marked reduction of the NOS

activity (b, f), MDA (c, g) and

8-OHdG (d, h) levels in rats

treated with DHCA compared to

those DHCA group.

Additionally, hydrogen-rich

saline significantly increased the

SOD activity (a, e) in rats

treated with DHCA. Data

represents mean ± S.D. (n = 10

per group). (*P \ 0.05,

**P \ 0.01, compared with

sham group; �P \ 0.05�P \ 0.01, compared with

DHCA group)

1506 Neurochem Res (2011) 36:1501–1511

123

supported the protective effects of HRS following brain

tissue injury in DHCA-treated rats. To our best knowl-

edge, this is the first study demonstrating that HRS has

potential cerebroprotective activity in a rat DHCA model

through mechanisms involving anti-oxidation and anti-

inflammation.

The pathophysiology of DHCA mainly consists of SIRS

caused by cardiopulmonary bypass and I/R injury as a

result of the circulatory arrest with the protection of

hypothermia. It was reported that the main pathophysiol-

ogy of I/R injury is the oxidative stress trigged by the

excessive production of free radicals [28]. NF-jB can be

activated by lesion-induced oxidative stress [29]. The

functional importance of NF-jB in inflammation is based

on its ability to regulate the promoters of multiple

inflammatory genes such as TNF-a, IL-1b, IL-6, and

ICAM-1 [30]. TNF-a is reported to be a major initiator of

inflammation and is released early after an inflammatory

stimulus [31] while IL-1b is regarded as the prototypic

‘‘multifunctional’’ cytokine and is induced in a multitude of

consequences of cell types [32]. The brain water content is

regarded as an index to quantify the magnitude of brain

edema 24 h after injury [33]. MDA is a commonly mea-

sured end point of free radical-induced lipid peroxidation,

and MDA concentration correlates with the extent of free

radical-induced damage [34]. 8-OHdG is formed from

deoxyguanosine in DNA by hydroxyl free radicals and

might serve as a sensitive biomarker of intracellular oxi-

dative stress in vivo [35]. SOD is one of the intracellular

enzymatic antioxidants that are responsible for disposing

Fig. 3 The inflammatory reaction in rat serum and brain tissue. (a, b,c, d is the concentrations of TNF-a, IL-1b in serum and brain tissues;

e is quantification of NF-jb DNA binding activity performed by

densitometric analysis; f is the brain water content; g is NF-jb DNA

binding activity by EMSA as shown by). The concentrations of TNF-

a (a, c), IL-1b (b, d) in serum, brain and brain water content (f)were all significantly elevated in DHCA rats when compared with

those in sham-operated group. Hydrogen-rich saline administration

significantly reduced the elevated levels of TNF-a (a, c), IL-1b (b, d)

in serum and brain as well as brain water content (f) when compared

with those in DHCA group. The bands were quantified using image

analysis software (Bandleader 3.0 software, Magnitec Ltd. Tel Aviv,

Israel). The relative intensity was determined by comparison with that

of the normal rat without any treatment. NF-jB activity in brain tissue

in DHCA group was also increased significantly compared to sham

group, while administration of hydrogen-rich saline decreased the

activity of NF-jB in DHCA treated rats (e). Data represents mean ±

S.D. (n = 6–10 per group). (*P \ 0.05, **P \ 0.01, compared with

sham group; �P \ 0.05 �P \ 0.01, compared with DHCA group)

Neurochem Res (2011) 36:1501–1511 1507

123

ROS such as H2O2 and O2••. Exposure to oxidants may

lead to enhanced expression of the enzyme NOS, resulting

in increased production of NO [36]. NOS is identified as a

source of ROS with special relevance to pathological

condition. NO has limited radical reactivity and combine

readily with O2••, and possibly H2O2, to produce the

highly oxidizing, non-radical compound, peroxynitrite

(ONOO•) [37]. Caspase 3 is considered to be the most

important subtype of the executioner caspases and is acti-

vated by any of the initiator caspases [38]. TUNEL staining

can easily differentiate apoptosis in the tissue by charac-

teristic apoptotic cells whose nucleus can be buffy col-

orated. In the present study, we found that DHCA

treatment caused significant oxidative stress as indicated by

the elevated levels of NOS, MDA and 8-OHdG but a

decreased level of SOD when compared to the sham group.

The rat model of DHCA also showed that both of the

inflammatory cytokines and cell apoptosis were signifi-

cantly increased compared with those in the sham group.

Recently, it was demonstrated that H2 is a novel anti-

oxidant with certain unique properties: (1) H2 could diffuse

freely within the body, without any side effects [20]. In other

words, H2 is permeable to cell membranes and can target

organelles, including nuclei and mitochondria, the latter

being the primary site of ROS generation and difficult

to target notoriously [39]. (2) H2 specially exclusively

Fig. 4 The evaluation of apoptosis in CA1 area in rat’s hippocampus

(a-i are the TUNEL staining, magnification: a, b, c: 940; d, e, f:9200; g, h, i: 9400; j is the caspase-3 activity in the rat brain; k is the

western blot image of cleaved caspase-3 in the rat brain, b-actin

provided as an inner control; l is the apoptotic index of TUNEL

staining.). Few apoptotic cells were observed in sham group (a, d, g).

However, the apoptotic index in DHCA group increased markedly

compared with those in sham group (P \ 0.01) (l). Compared with

those in DHCA group, there were less TUNEL-positive cells (c, f, i)

and decreased apoptotic index in DHCA rats (l) treated with

hydrogen-rich saline (P \ 0.01). The activity of caspase-3 in DHCA

group (j, k) was significantly increased compared to the sham group.

Treatment with hydrogen-rich saline significantly down-regulated the

activity of caspase-3 in DHCA ? H2 group (j, k) which was in

consistent with the results of TUNEL staining. Data represents mean

± S.D. (n = 6–10 per group). (*P \ 0.05, **P \ 0.01, compared

with sham group; �P \ 0.05 �P \ 0.01, compared with DHCA group)

1508 Neurochem Res (2011) 36:1501–1511

123

quenches detrimental ROS, such as •OH and ONOO•, while

maintaining the metabolic oxidation–reduction reaction and

other less potent ROS, such as O2••, H2O2, and nitric oxide

[9, 40]. (3) H2 is electronically neutral and is oxidized into

water in the body which is not harmful to cells.

Gharib et al. reported that the inhalation of H2 can

suppress the chronic hepatic inflammation in a rat model of

schistosomiasis-associated chronic liver inflammation [20].

Kajiya et al. reported that H2 released from intestinally

colonized bacteria can suppress inflammation in liver

induced by Concanavalin A. [41]. It is also reported that H2

could attenuate DSS-induced colitis by down-regulating

the expression of proinflammatory cytokines, as well as

suppressing the infiltration of macrophages in the colon

lesion. [42]. TNF-a and IL-1b, discharged from activated

macrophages and neutrophils, exert a considerable ampli-

fying effect on the systemic inflammatory response. Study

showed that the effect of oxidation and inflammatory

response (e.g., TNF-a, IL-1b) is mediated by NF-jB acti-

vation [43]. In the present study, HRS significantly

decreased the levels of TNF-a and IL-1b in serum and

brain tissues, suggesting that the protective effect of HRS

on brain was associated with down-regulation of TNF-aand IL-1b. In addition, we found that the NF-jB activity

was decreased by HRS, implying that the NF-jB activation

may be involved in the DHCA and the subsequent

pathology of brain injury. The administration of HRS could

dampen the inflammatory reaction effectively by reducing

the activity of NF-jB.

Apoptosis is programmed cell death which is character-

ized by specific ultrastructural changes that include cell

shrinkage, nuclear condensation and DNA fragmentation

[44]. Ischemia and hypoxia are linked to excessive neuronal

stimulation and hyperactivity, initiating a cascade of cel-

lular events leading to neuronal cell death [45, 46]. Apop-

tosis plays a crucial role in neuronal cell death after DHCA.

Damaged neurons are observed as early as 8 h after reper-

fusion and as long as 72 h later. [47]. Excessive ROS can

result in DNA fragmentation, lipid peroxidation, and inac-

tivation of protein [48] leading to apoptosis or necrosis

depending on the severity of oxidative stress. Among the

ROS, •OH and ONOO• are much more reactive and react

indiscriminately with nucleic acids, lipids and proteins.

Whereas H2 can quench the detrimental ROS •OH and

ONOO• specially and exclusively. At the molecular level,

apoptosis is activated by the aspartate-specific cysteine

protease cascade, and caspase-3 is considered to be one of

the most important executioner caspases and is activated by

any of the initiator caspases [38]. Our data indicated that,

TUNEL positive staining in the DHCA ? H2 group was

significantly decreased compared to those in the DHCA

group. Concomitantly, the caspase-3 activity in DHCA

rats treated by HRS was also down-regulated significantly

compared with the untreated DHCA rats, implying that

administration of HRS could effectively reduce apoptosis

caused by DHCA.

Conclusion

In summary, our results showed that HRS could effectively

protect the brain by means of anti-oxidation and anti-

inflammation in a rat model of DHCA. Our results suggested

that HRS treatment could ameliorate structural and func-

tional damages as a result of reduced oxidative stress that

can cause inflammation and apoptosis. Application of HRS

showed promising results in this model and may become a

powerful tool in the clinical treatment of brain injury caused

by DHCA. However, there still is a significant difference

between the sham and DHCA ? H2 group, which is similar

to the results reported by Matchett et al. [49] and Cai et al.

[13], those reports showed that brain injury induced by

DHCA exceeds the therapeutic potential of short-term

administration of HRS or the treatment is time-dependent on

the duration of the administration of HRS. As such, further

studies are still needed for clarification.

Acknowledgments This study was supported by grant from the

National Natural Science Foundation of China (No. 30972969). We

sincerely thank Dr. Geng-bao Feng and Miss Kang-li Hui for their

excellent technical assistance. We also sincerely thank Dr. Bing Guan

for his assistance with pathology analysis and Dr. Yi Li for language

editing.

Conflict of interest All authors declare that they have no conflict of

interest.

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