Original Research
Hydrogen inhalation protects hypoxic–ischemic brain damage byattenuating inflammation and apoptosis in neonatal rats
Guojiao Wu1 , Zhiheng Chen1, Peipei Wang1, Mingyi Zhao1, Masayuki Fujino2,3,
Chen Zhang1, Wenjuan Zhou1, Shin-Ichi Hirano4, Xiao-Kang Li1,2 and Lingling Zhao1
1

Department of Pediatrics, Third Xiangya Hospital, Central South University, Changsha 410013, China; 2Division of Transplantation
Immunology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan; 3AIDS Research Center, National
Institute of Infectious Diseases, Tokyo 162-8640, Japan; 4MiZ Co., Ltd, Kanagawa 247-0062, Japan
Corresponding author: Lingling Zhao. Email: llzhao2011@qq.com

Impact statement

Abstract

Hypoxic–ischemic brain damage (HIBD) is one of the leading causes of brain injury in infant
with high risk of mortality and disability; therefore, it is important to explore more feasible
and effective treatment strategies. Here, we assessed the neuroprotective effects of different hydrogen inhalation times for the treatment of HIBD. We induced hypoxia–ischemia inSprague–Dawley rats (postnatal day 7, both sexes), followed by treatment with hydrogen
inhalation for 30, 60, or 90 min. Morphological brain injury was assessed by Nissl and
TUNEL staining. Acute inflammation was evaluated by examining the expression of interleukin-1b (IL-1b) and NF-jB p65, as well as Iba-1 immunofluorescence in the brain. Neuralapoptosis was evaluated by examining the expression of P-JNK and p53 as well as NeuN
immunofluorescence. Neurobehavioral function of rats was evaluated by Morris water mazetest at 36 days after surgery. The results showed that hypoxia–ischemia injury induced theinflammatory response of microglia; however, these changes were inhibited by hydrogen
inhalation. The inhibitory effects became more apparent as the treatment duration increased(P < 0.05). Furthermore, hypoxia–ischemia induced neuronal damage and increased theexpression of the apoptotic factors, P-JNK, and p53, which were attenuated by hydrogen inhalation (P < 0.05). Hypoxia–ischemia
caused long-term spatial memory deficits during brain maturation, which were ameliorated by hydrogen inhalation (P < 0.01). In
conclusion, hypoxia–ischemia induced severe long-term damage to the brain, which could be alleviated by hydrogen inhalation ina time-dependent manner.
Oxidative stress is known to be involved in
the main pathological progression of neonatal hypoxic–ischemic brain damage(HIBD). Hydrogen (H2) is an antioxidant thatcan be used to treat HIBD; however, themechanism by which hydrogen may be
used as a promising treatment for neonates with HIBD is not very clear. This
study demonstrated that inhaled H2 isneuroprotective against HIBD in
SpragueDawley rats by inhibiting thebrain’s inflammatory response and neuronal apoptosis or damage and protecting
against spatial memory decline. Further,
this study showed that inhaled H2 haspotential as a therapeutic approach for
HIBD. This is relevant to clinical treatment
protocols when hypoxia–ischemia is suspected in neonates.

Keywords: Cerebral ischemia–hypoxia, hydrogen, spatial memory, inflammation, apoptosis
Experimental Biology and Medicine 2019; 0: 1–11. DOI: 10.1177/1535370219855399

Introduction
Hypoxic–ischemic brain damage (HIBD) refers to brain
damage caused by asphyxia due to hypoxia and ischemia
during the perinatal period, which is proved to be themain cause of mortality and disability in neonates.1,2
The global incidence of HIBD among live births isapproximately 1–8‰, reaching up to 26‰ in developing
countries and up to 60% among newborns with very low
birth weight. The mortality rate for children with HIBD is
ISSN 1535-3702
Copyright ! 2019 by the Society for Experimental Biology and Medicine

approximately 20–25% in the neonatal period, andapproximately 25% of surviving children experience permanent neurological deficits, such as mental retardation,
developmental retardation, motor or cognitive impairment, cerebral palsy, and epilepsy.2,3 Mild hypothermia
therapy can alleviate the brain damage caused by hypoxia–ischemia (HI); however, some neonatal patients exhibit
persistent neurological defects even after treatment.4–6
Therefore, it is essential to develop more feasible andExperimental Biology and Medicine 2019; 0: 1–11

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effective therapeutic strategies for reducing brain damage
caused by HI in neonates.
Hydrogen (H2) is a colorless, odorless, non-toxic antioxidant that is protective against diseases mediated by oxidative stress, including ischemia–reperfusion injury in theadult rat brain, heart, liver, kidney, small intestine, spinal
cord, and other organs.7–12 In 2008, Kajiyama et al.13 investigated H2 in clinical trials, observing that drinking H2-richwater could effectively improve the symptoms in patientswith diabetes. At present, there are three primary forms of
H2 administration: H2 inhalation, injection of H2-richsaline, and oral ingestion of H2-saturated water.14,15 One
study reported that oral H2-rich water may result in harmful alterations to some biochemical indices.16 Oral ingestion
or injection of H2-rich water or saline, respectively, canincrease the allostatic load. In addition, it is difficult to control the concentration of H2 using these methods. Moreover,the curative effect associated with these treatments arelimited by short exposure times and other factors, and thecomplex protocols required are not suitable for newborns.17,18 In contrast, H2 inhalation treatment is safe forthe human body at concentrations < 4%, is not associatedwith a risk of explosion when exposed to air, and the concentration is easy to control.19,20 H2 has a low molecularweight and strong permeability, and it easily penetrates
the biofilm and diffuses into the cytoplasm, mitochondria,
and nucleus.12 Thus, researchers have focused more heavily on this method of administration.
Cai et al.21 demonstrated that inhalation of H2 has a neuroprotective effect in HIBD rats; however, Matchett et al.22
reported that H2 inhalation does not protect against HIBD
and aggravates brain damage in rats. To identify a safe andeffective method, we examined the effect of 3% H2 inhalation for different durations in HIBD rats. Furthermore, thepotential mechanisms underlying the neuroprotective
effect of H2 inhalation will be assessed as well.

Materials and methods
Animals
Seven-day-old female and male Sprague–Dawley (SD) rats
(body weight range from 9 to13 g) were purchased from
Slac Corporation (Changsha Slac Animal Corporation,
Hunan, China). The rats’ pups were housed in cages with
their mothers. All experiments were approved by theCenter for Medical Ethics of Central South University andwere performed according to the Guide for the Care andUse of Laboratory Animals of the National Institute of
Health (eight edition).
Hypoxia–ischemia model in neonatal rats
23

We induced HIBD in rats using the Rice–Vannucci model.
The pups were anesthetized with isoflurane, and the leftcommon carotid artery (CCA) was carefully exposed.
Afterwards, the CCA was double-ligated using a 6–0 surgical silk tie, which was cut between sutures. The skin incision was then sutured, and pups were permitted to recoverfor 2 h with their dam in their home cages. Following this,rats were placed in a glass anoxic chamber (constanttemperature of 37 C; 8% O2þ 92% N2) for 100 min to
induce brain injury.
H2 inhalation and experimental design
H2 was generated via water electrolysis in a hydrogen gas
supply device (MiZ Co. Ltd, Kanagawa, Japan). Following
HI injury, the rat pups were treated with mixed gas (3%hydrogen and 97% air) in a device. The device was
30  13  12.5 cm in size, white, and transparent, allowing
us to observe the activity of the rats. A total of 140 rats weredivided randomly into seven groups. The four experimental groups were classified according to the length of H2
inhalation. The rats divided in the first three groups listed
below were interfered with H2 inhalation immediately afterHI, while the rats in the last group were started at 2 h after
HI: (1) 30-min (n ¼ 22; female: 12, male: 10); (2) 60 min(n ¼ 22; female: 11, male: 11); (3) 90 min (n ¼ 22; female:
10, male: 12); (4) 90 min (start at 2 h after HI, n ¼ 8;
female: 4, male: 4). The three control groups were classified
as follows: (1) naive (no processing) (n ¼ 22; female: 10,
male: 12); (2) sham-surgery (left common carotid artery
was separated without ligation then sutured, and no hypoxia) (n ¼ 22; female: 13, male: 9); and (3) HIBD (carotid
ligation and hypoxia) (n ¼ 22; female: 11, male: 11). Rats
were interfered with H2 inhalation twice daily then euthanized at 24 h or 72 h after surgery for 2,3,5- triphenyltetrazolium chloride (TTC) staining or immunoblot andhistological staining, respectively. Twelve rats per groupwere used for pathology and molecular biology experiments, and ten rats per group were used to evaluate neurobehavioral function.
Infarct volume
TTC (Sigma, St. Louis, MO, USA) staining was used to evaluate cerebral infarcted area in rats. At 24 h post-surgery,rats were sacrificed via decapitation, and brains were sliced
into five sequential coronal sections (thickness, 2 mm), followed by immersed in the 1% TTC solution for 30 min at
37 C and turned every 5 min. The sections were sequentially photographed (Sony, Tokyo, Japan) and the cerebral
infarction/non-infarction volume was calculated by
Image J software. The calculation of the percentage of
infarct volume was the same as Cai et al. described.21
The percentage of infarct volume (%) ¼ the infarct
volume/the total volume of the sections.
Nissl staining
Rats’ pups were deeply anaesthetized and the brains wereobtained following perfusion and fixed. Then, the fixed
brain tissue was paraffin embedded, followed by serial sectioning into 5-lm slices. After deparaffinization, the slices
were immersed in 2% Nissl working solution for 5 min.
Afterwards, the slices were rinsed in differentiation solution until the Nissl body was clarified (Wellbio, Beijing,
China). The slices were photographed under a microscope(magnification 200; Nikon, Tokyo, Japan).

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Terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) staining
TUNEL staining assay kit (Roche, Basel, Switzerland) wasapplied to detect apoptosis. Briefly, the slices were incubated with TUNEL reaction solution for 1 h. Afterwards,
washed the slices with PBS and counterstained the nucleus
with DAPI for 10 min. The sections were visualized under afluorescence microscope (Nikon) with 400 magnification.
Four slide fields within the left frontal and parietal cortex
and hippocampal CA1 were randomly examined and theresults are expressed as the percentage of TUNEL-positive
cells in selected field (TUNEL-positive cells/total numberof nuclei).
Immunofluorescence
After deparaffinization, sections were under antigen
retrieval process. Followed by blocking with 5% BSA for
1 h, the slices were immersed overnight at 4 C in primary
antibodies. The antibodies examined in the experiment arelisted below: IL-1b (1:100; Proteintech, Rosemont,
Maryland, USA), NF-jB p65 (1:100; Abcam, Cambridge,
CA, USA), P-JNK (1:100; Abcam), p53 (1:200; Proteintech),Iba-1 (1:500; Abcam) and NeuN (1:500; Millipore, Billerica,
MA, USA). Next day, the sections were rinsed with PBST
three times, and visualized with Alexa Fluor-labeled secondary antibodies (Alexa Fluor 594, 1:800; Thermo
Scientific, Rockford, IL, USA; Alexa Fluor 488, 1:800;
Thermo Scientific, Rockford, IL, USA) for 2 h. DAPI wereused for counterstaining the nucleus. The micro- andmacro-images from the frontal and parietal cortex region
or hippocampal CA1 were acquired under a microscope(magnification 400; Nikon), and merged using Image J.
Western blot analysis
The tissue proteins of the frontal and parietal cortex region
were extracted at 72 h after surgery and quantified with a
BCA kit (CWBIO, Beijing, China). Protein samples wereseparated by using SDS-PAGE gels under different concentrations range from 8% to 12%, and then transferred onto
PVDF membranes (Millipore, Billerica, MA, USA). After
blocking with 5% skimmed milk in PBS for 2 h, membranes
were incubated overnight at 4 C with the following primary antibodies: IL-1b (1:500, Bioss, Beijing, China), NF-jB
p65 (1:500, Abcam, Cambridge, CA, USA), P-JNK (1:500,
Abcam), p53 (1:1000, Proteintech, Rosemont, Maryland,USA), b-actin (1:1000, Proteintech), and GAPDH (1:1000,
Proteintech). The next day, membranes were washed with
PBST. Afterwards, membranes were immersed in the secondary antibody (IRDye 800CW, 1:10,000). Immunoblotting
bands were automatically photographed by the OdysseyCLX infrared imaging system (Li-CORVR , USA). The relative
intensities of the bands were analyzed using Image J.
Morris water maze
The MWM test was performed during adolescence (postnatal days 43–47) to assess neurobehavioral function of rats
mainly related to the abilities including the spatial memory
and learning.24 The experiment was carried out in acylindrical pool (Panlab, Spain), and water temperature of
22  1 C. The pool included four equal quadrants: southeast, southwest, northeast, and northwest. A platform was
hidden in the middle of the northeast quadrant. Visual reference objects were located around the pool for spatial
localization, such as fixed experimental items, lights, andcards of different shapes, which were unchanged throughout the experiment.
The experiment included two parts: (1) Examination of
spatial learning ability (location–navigation test). All rats
underwent four trials per day, lasting four days. In a trial,
animals were required to find the hidden platform in lessthan 1 min. An experimenter recorded the time to reach theplatform, which was defined as the “escape latency.” If therats could not reach the hidden platform in less than 1 min,the experimenter guided them to the hidden platform, andrecorded the escape latency for 1 min. Each rat was required
to stay on the hidden platform for 20 s and allowed a 20 min
intertrial interval. The escape latency was automaticallytracked and analyzed using tracking software (SMART 3.0
video-tracking system, Panlab, Spain). The average escapelatency recorded for each of the four trials was taken as thelearning achievement of the rats on the same day, and thetrial was repeated for four days. (2) Examination of spatial
memory (space exploration test). In probe trials, the platform
was taken away, and each rat was placed in the same selected quadrant (opposite quadrant to the original hidden platform). The tracking software recorded the time (residencetime) to stay in the probe quadrant and swimming pathover 1 min. The residence time was used as a measure of
spatial memory.
Statistical analysis
The quantified data were shown as means  SD. One-way
analyses of variance (ANOVA) followed by Tukey’s post hoc
tests were used for multiple comparisons of different
groups. The univariate repeated measures ANOVA wasused to analyze the MWM results for independent groupcomparisons. Statistical analysis was performed using SPSS
22.0 Software and the bar graphs were drawn by GraphPad
Prism 6 software. Differences with P < 0.05 were defined asstatistically significant. In the Nissl staining experiment,
TTC staining, and immunofluorescence tests, no significantly differences were obtained between the naive groupand the sham-surgery group. So, the results of the experiment in the naive group are not shown in the picture.

Results
H2 inhalation improves neuronal injury and cerebral
infarction volume
In the present study, Nissl staining was used to observe
neuronal damage in the left frontal and parietal cortex
and hippocampal CA1 of rats. In the sham-surgery
group, neurons had a complete morphological structure,large cell bodies, and abundant cytoplasm and Nissl
bodies; however, the HIBD group exhibited extensive neuronal damage, such as karyopyknosis, dark-blue staining of
nuclei, neuronal structure and Nissl’s body disappeared.

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Figure 1. Nissl and TTC staining following H2 inhalation in rats with HIBD. (a) Representative images of Nissl staining in the left frontal and parietal cortex andhippocampal CA1 region of the sham-surgery, HIBD, 30-min H2, 60-min H2, and 90-min H2 groups at 72 h after surgery. Scale bar ¼ 100 lm. n ¼ 4 per group. (b)Representative images of TTC staining on the first day after surgery from coronal brain sections of the sham-surgery, HIBD, 30-min H2, 60-min H2 and 90-min H2
groups at 24 h after surgery. (c) The percentage of the cerebral infarct volume in the ipsilateral hemisphere of different groups. n ¼ 4 per group. **P < 0.001 vs. HIBD
group, ††P < 0.001.

Interestingly, this neuronal damage was obviously alleviated after H2 inhalation, especially in the 90-min H2 group(Figure 1(a)).
In addition, TTC staining was used to analyze cerebral
infarction volume. There was a dramatically higher
cerebral infarction rate in HIBD rats than those in shamsurgery rats (Figure 1(a) and (b)). Infarction rates (8.96 0.08%, 7.50  0.02%, and 4.22  0.04%, respectively)
were significantly lower in the 30-min H2, 60-min H2, and90-min H2 groups than in the HIBD group (16.16  0.16%,
P < 0.001; Figure 1(c)). These results indicate that H2 inhalation ameliorated neuronal injury and reduced cerebral
infarction volume, which was most evident after 90-minof H2 inhalation (30-min H2 vs. 60-min H2, P < 0.001; 60min H2 vs. 90-min H2, P < 0.001). And we found that theeffects of inhaling H2 immediately after HI were better thanthat inhalation of H2 at 2 h after HI (P < 0.05,
Supplementary Figure 1).
H2 inhalation inhibits microglial activation andinflammation
Microglia are immune cells. Iba-1 is a marker of resting andactivated microglia. At 72 h after surgery, we examined Iba1 fluorescence in the left frontal and parietal cortex and left

hippocampal CA1 regions to investigate microglial activation. In the sham-surgery group, microglia were dendritic,
and few positive cells were observed, indicative of resting
microglia. However, in the HIBD group, microglia wereoval or amoeboid in shape, and the number of positive
cells had increased significantly, indicative of activatedmicroglia (Figure 2(a) and (b)). Immunofluorescence staining showed that H2 inhalation remarkably reduced the activation of microglia after HI (Cortex, 30-min H2 vs. HIBD,
P ¼ 0.055; 60-min H2 vs. HIBD, P < 0.001; 90-min H2 vs.
HIBD, P < 0.001. Hippocampal CA1, 30-min H2, 60-min
H2 or 90-min H2 vs. HIBD, P < 0.001), and decreases in
Iba-1 expression became more obvious as the duration of
H2 inhalation increased (Cortex, P < 0.01; Hippocampal
CA1, P < 0.001) (Figure 2(a) and (b)). These findings indicate that H2 inhalation significantly inhibited the activationand proliferation of microglia induced by hypoxia–
ischemia, and this inhibition was most pronounced in the
90-min H2 group.
Furthermore, to verify whether H2 can inhibit the
inflammatory response of microglia, we examined theexpression of Iba-1, IL-1b, and NF-rB p65 in the left frontal
and parietal cortex. There were dramatically increased
Iba-1þ/IL-1bþ and Iba-1þ/NF-rB p65þ cells at 72 h after
surgery, which suggested that HI induced microglial

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Figure 2. Microglial activation changes following H2 inhalation in rats with HIBD. (a) Representative immunofluorescence staining Iba-1 (green, activated microglia)
and DAPI (blue, all nuclei) in the left frontal and parietal cortex and hippocampal CA1 region of rats in sham-surgery, HIBD, 30-min H2, 60-min H2, and 90-min H2 groups
at 72 h after surgery. Scale bar ¼ 50lm. (b) Differences in the percentage of Iba-1-positive cells in the cortex and hippocampal CA1 between groups. n ¼ 4 per group.
**P < 0.01 vs. HIBD group, ††P < 0.01, †P < 0.05.

inflammatory responses. However, immediate inhalation of
H2 after HI injury notably reversed these changes, and theeffects were most pronounced in the 90-min H2 group(P < 0.05, Figure 3(a) to (c)). The patterns of IL-1b and NFrB p65 expression observed via Western blot analysis wereconsistent with those detected via fluorescence (Figure 3(d)and (e)). The expression of IL-1b and NF-rB p65 was dramatically increased after HI injury (P < 0.001), and significantly decreased by H2 inhalation when compared with
HIBD rats at 72 h post-surgery (IL-1b, 30-min H2 vs.
HIBD, P ¼ 0.0026; 60-min H2, 90-min H2 vs. HIBD,
P < 0.0001. NF-rB p65, P < 0.0001). Decreases became
more obvious as the duration of H2 inhalation increased(IL-1b, 30-min H2 vs. 60-min H2, P ¼ 0.0019; 60-min H2 vs.
90-min H2, P < 0.001. NF-rB p65, 30-min H2 vs. 60-min H2,
P ¼ 0.9710; 60-min H2 vs. 90-min H2, P < 0.001).
H2 inhalation inhibits neuronal apoptosis in HIBD rats
TUNEL staining assay was applied to examine the inhibition effect of apoptosis by H2 inhalation. The results illustrated that numbers of TUNEL-positive cells were
obviously raised after HI, while they were reversed by
H2 inhalation (P < 0.001, Figure 4(a) and (b)), especially inthe 90-min H2 group (Cortex, 30-min H2 vs. 60-min H2,
P ¼ 0.0258, 60-min H2 vs. 90-min H2, P ¼ 0.0381.
Hippocampal CA1, 30-min H2 vs. 60-min H2, P ¼ 0.0058,
60-min H2 vs. 90-min H2, P ¼ 0.0148). However, 30 min of
H2 inhalation did not reduce the number of TUNELpositive cells in HI condition in the hippocampal CA1
region (30-min H2 vs. HIBD, P ¼ 0.1617), indicating that30 min of H2 inhalation had no obvious effect on neuronal

apoptosis induced by HI injury. Nonetheless, the inhibitory
effects of H2 inhalation on apoptosis were obvious in the 90min H2 group. And we also found that the effects of inhaling H2 immediately after HI injury were better than that
inhalation of H2 started at 2 h after HI (P < 0.05,
Supplementary Figure 2(a) and (b)).
H2 inhalation inhibits upregulation of P-JNK/p53
signaling in HIBD rats
C-Jun N-terminal kinase (JNK) activates transcription factor
p53 and induces apoptosis of the mitochondrial pathway.
NeuN is neuronal-specific. In this study, we examined the
double immunofluorescence of NeuN and P-JNK or p53
in the left frontal and parietal cortex. Doubleimmunofluorescence staining revealed that NeuNþ/P-JNKþ and NeuNþ/p53þ cells were notably raised in the
HIBD rats, indicating that P-JNK and p53 expression in neurons increased after HI injury, thereby inducing apoptosis.
However, hydrogen inhalation reversed this increase, andthese effects were most pronounced in the 90-min H2
group (P < 0.05, Figure 5(a) to (c)). Similar results wereobserved via immunoblot. The expression of P-JNK andp53 protein was remarkably increased by HI injury whencompared with naive and sham-surgery rats at 72 h after
surgery (P < 0.001; Figure 5(d) and (e)). However, H2 inhalation attenuated these increases (P-JNK, 30-min H2 vs.
HIBD, P ¼ 0. 6215, 60-min H2 vs. HIBD, P ¼ 0. 002, 90-min
H2 vs. HIBD, P < 0.001. p53, 30-min H2, 60-min H2 or 90-min
H2 vs. HIBD, P < 0.001), and such attenuation became more
obvious as the duration of H2 inhalation increased (P < 0.05).
The result of this section illustrated that H2 inhalation

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Figure 3. Inflammatory changes following H2 inhalation in rats with HIBD. (a, b) Representative images of IL-1b (red) or NF-jB p65 (red) and Iba-1 (green) immunofluorescence staining, and merged immunofluorescence in the left frontal and parietal cortex of rats from sham-surgery, HIBD, 30-min H2, 60-min H2, and 90-min H2
groups. Scale bar ¼ 50 lm. (c) Quantitative analyses of Iba-1þ/IL-1bþ and Iba-1þ/NF-jB p65þ cells. n ¼ 4 per group. (d) Representative western blot bands of IL-1b and
NF-jB p65 in the cortex of rats from the naive, sham-surgery, HIBD, 30-min H2, 60-min H2, 90-min H2 groups. b-actin protein levels were used as internal loading
controls. (e) Data represent the mean  SD of the normalized densitometry values of IL-1b and NF-jB p65. Quantitative protein levels were normalized with b-actin.
n ¼ 4 per group. **P < 0.01 vs. HIBD group, ††P < 0.01, †P < 0.05.

inhibited the P-JNK/p53 signaling in a time-dependent
manner, thus, it inhibited neuronal apoptosis.
H2 inhalation improves long-term neurobehavioral
deficits in HIBD rats
To further assess the neuroprotective effects of H2 inhalation, the long-term neurobehavioral function was examined by MWM experiment in HI rats during adolescence(P43–47). From the first to fourth day, the mean escapelatency gradually decreased in all groups. On the firstday, H2 inhalation could not shorten the escape latency

after HI (P > 0.05). However, from days 2 to 4, H2 inhalation
significantly shortened the mean escape latency (maineffects of group, P < 0.001; main effects of time, P < 0.001;
the interaction between group and time, P < 0.001; days
2–4, P < 0. 001) (Figure 6(b)), compared with the HIBD
group. Furthermore, the mean escape latency was shortest
in the 90-min H2 group. These results indicated that all rats
showed the abilities of learning and remembering the location of the platform; however, this learning ability was
impaired because of hypoxia and ischemia. Furthermore,
the findings suggest that H2 inhalation rescued spatial
learning in HIBD rats. On the fifth day, the swimming

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Figure 4. H2 inhalation reduces neuronal apoptosis in the cortex and hippocampal CA1 of rats with HIBD. (a) Representative TUNEL staining in the left frontal andparietal cortex and hippocampal CA1 region of rats from the sham-surgery, HIBD, 30-min H2, 60-min H2, and 90-min H2 groups at 72 h after surgery (green, TUNELpositive cells; blue, cell nuclei). Scale bar ¼ 50 lm. (b) The percentage of TUNEL-positive staining in each group. n ¼ 4 per group. **P < 0.01 vs. HIBD group, ††P < 0.01.

path was located mainly around the initial quadrant(Figure 6(a)) in the HIBD group, which also exhibited theshortest time in the target quadrant (Figure 6(c)). Thesefindings suggested that HI damage during the neonatal
period resulted in memory impairment in adolescent rats.
However, H2 inhalation obviously improved the duration
spent in the platform quadrant (30-min H2 vs. HIBD,
P ¼ 0.0231, 60-min H2 or 90-min H2 vs. HIBD, P < 0.0001),
which was longest in the 90-min H2 group (Figure 6(c)).
These results suggested that H2 inhalation effectively
improved memory impairments induced by HI.

Discussion
In summary, our present findings confirmed the neuroprotective effect of H2 inhalation in HIBD rats. H2 inhaling
reduced the activation of microglia in HIBD rats, reduced
the accumulation of IL-1b and NF-KB p65, inhibited themicroglial inflammatory response, reduced the cerebral
infarction rate, inhibited neuronal apoptosis by downregulating the expression of P-JNK and p53, reduced neuronaldamage, and improved long-term neurobehavioral deficits
induced by HI in a time-dependent manner.
The common clinical treatment methods for neonatal
HIBD include mild hypothermia, hyperbaric oxygen, andstem cell transplantation26,26; however, this effect is notconsistent.4,27,28 Previous researches have indicated thatoxidative stress, apoptosis, and inflammation are associated with neonatal HIBD.12,25 Oxidative stress is involvedin the main pathological progression of HIBD. Hypoxia–
ischemia injury induces the activation of microglia to produce oxygen free radicals, which then promote thesecretion of inflammatory mediators.29 Both H2 andhydrogen sulfide (H2S) have shown neuroprotective
effect by anti-oxidative stress and selectively scavenge
oxygen free radicals.12,30 However, the discrepancy of
H2S after brain injury needs to be pointed. For example,
Li et al.31 reported that 0.1 mmol/L protected against
ischemic brain damage in rats; however, the absolutely
opposite effect was obtained by Qu et al.32 when the concentration of H2S reached 0.18 mmol/L in the samemodel. These results indicated that the effective windowof H2S is extremely small. Considering that H2S is a poisonous gas having a pungent odor, it is not suitable fornewborns. Moreover, neither is easy to control the therapeutic concentration, nor can instrument accurately measure the concentration of H2S in cells, it is difficult totrack and analyze the effects of H2S.33 Compared to
H2S, H2 is a safe and stable molecular free radicals’ scavenger odor, which can be measured easily and accurately.
Therefore, H2 was chosen for treatment in the present study.
We performed three different durations of H2 inhalation,i.e. 30 min, 60 min, and 90 min. We compared the effects ofthree different H2 inhalation durations on HIBD and foundthat inhaling H2 for 90 min was the most effective. The therapeutic effect of inhaling H2 continuously several times wasmore effective than single short-term inhalation of H2.22
Simultaneously, we performed H2 inhalation at two different
time intervals after HI injury, i.e. immediately, and 2 h after
HI injury. We found that the effects of inhaling H2 immediately after HI were better than that inhalation of H2 start at 2 hafter HI. It seems to imply that the earlier H2 intervention is
performed, the better neuroprotective effect will be obtained.

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Figure 5. H2 inhalation decreases the protein expression of P-JNK and p53 in the cortex of rats with HIBD. (a, b) Representative images of P-JNK (red) or p53 (red) and
NeuN (green) staining, and merged immunofluorescence in the left frontal and parietal cortex of rats from the sham-surgery, HIBD, 30-min H2, 60-min H2, and 90-min
H2 groups at 72 h after surgery. Scale bar ¼ 50lm. (c) Quantitative analyses of NeuNþ/P-JNKþ and NeuNþ/p53þ cells. n ¼ 4 per group. (d) Representative Western blot
bands of P-JNK and p53 in the cortex of rats from the naive, sham-surgery, HIBD, 30-min H2, 60-min H2, and 90-min H2 groups. GAPDH protein levels were used asinternal loading controls. (e) Data represent the mean  SD of the normalized densitometry values of P-JNK and p53. Quantitative protein levels were normalized withGAPDH. n ¼ 4 per group. **P < 0.01 vs. HIBD group, ††P < 0.01.

Our MWM test results indicated that neonatal rats
exposed to HI exhibit poor neurobehavioral outcomes
during adolescence, suggesting that hippocampal andcortex injury during the stage of development impairs spatial memory and learning abilities. However, inhalation ofH2 immediately after HI injury significantly improved neurobehavioral function (Figure 6). The neuroprotective effectof H2 on HIBD appears to be associated with the antiinflammatory and anti-apoptotic effects after scavenging
oxygen-free radicals, as H2 treatment significantly

diminished the activation of microglia, in addition to
IL-1b and NF-rB p65 protein expression. In addition, we
observed that H2 inhalation significantly increased neuronal survival, decreased the infarct ratio and level of apoptosis, and reduced P-JNK and p53 activity.
Microglia are derived from the hematopoietic systemand constitute immune cells in the central nervous
system, serving immune and self-defense functions.34,35
Microglia are first activated after cerebral ischemia and produce proinflammatory cytokines, which participate in theWu et al.

Hydrogen protects against hypoxic–ischemic brain damage

9...............................................................................................................................................................

Figure 6. H2 inhalation improves long-term spatial learning and memory in rats with HIBD. Rats were trained from postnatal days 43–47. Each rat underwent four trials
per day for four consecutive days and a probe trial on the fifth day. (a) Representative swimming paths of rats on day 5 of testing in the naive, sham-surgery, HIBD,
30-min H2, 60-min H2, and 90-min H2 groups of rats. (b) Latencies to reach the hidden platform for each group of rats. (c) The time spent (%) in the target quadrant on
day 5 of testing for each group of rats. Data represent mean  SD from independent experiments. n ¼ 10 per group. ** P < 0.01 vs. HIBD group, ††P < 0.01, †P < 0.05.

inflammatory response after ischemia injury.36 Therefore,
we investigated inflammatory responses based on microglial activation.
IL-1b interacts with activated microglia, producing persistent inflammatory products that enhance inflammationand cause neuronal damage.37,38 NF-rB upregulates theexpression of inflammatory cytokines after activation
under various extracellular stresses, thereby initiating the
inflammatory response and apoptosis, which includes p65
and p50 subunits.39–41 The secretion of the proinflammatory cytokine IL-1b activates NF-rB, which in turn upregulates the expression of IL-1b, thus enhancing theneuroinflammatory response. In the present study, weobserved microglial activation, and a corresponding
increase in IL-1b and NF-rB p65 expression, following HI
injury, which were significantly attenuated following H2treatment in HIBD rats (Figures 2 and 3). Moreover, these
inhibitory effects became more apparent as the treatment

duration increased. Furthermore, the therapeutic effects ofH2 were possibly mediated by the inhibition of IL-1b and
NF-rB p65 activities.
Activated microglia produce proinflammatory cytokinesthat have been proven to be critical mediators of neurodevelopment.42 However, over-reactive microglia and inflammatory mediators may cause neuronal damage.38 In thepresent study, the loss of Nissl bodies following HI injury
was associated with vacuole formation, and there wasa remarkable rise in infarction rates and apoptosis(Figures 1 and 4). These findings indicate that the increased
inflammatory response results in neuronal damage; however, inhalation of H2 after HI injury obviously attenuatedneuronal damage. Taken together, these data further indicate that inhalation of H2 attenuates neuronal damage
induced by HI.
The JNK signaling pathway can be activated by proinflammatory cytokines, and associated with oxidative stress,

10

Experimental Biology and Medicine...............................................................................................................................................................
inflammation, and apoptosis.43,44 The JNK pathway mediates apoptosis via the death receptor, mitochondrial
apoptosis, and endoplasmic reticulum stress apoptotic
pathways. At the early stage of apoptosis, calcium ions in
the cytoplasm continuously increase the activation of JNK[activated JNK (P-JNK)] in the nucleus, further activatingtranscription factors, such as c-Jun or p53, and regulating
the activity of the Bcl-2 family of proteins in the cytoplasm.45 This results in an increase in mitochondrial membrane permeability, which activates the mitochondrial
apoptosis pathway. P53, a tumor suppressor protein, acts
as a transcription factor to induce cell cycle arrest, DNAdamage and repair, and cell senescence.46 Therefore, wechose JNK as the major indicator, which was validated by
the JNK/p53 pathway in our experiments. Our results indicate that HI injury activated the JNK signaling pathway
and increased P-JNK expression, promoting the expressionof the downstream pro-apoptotic protein p53, which
induced apoptosis (Figure 5).
Our findings demonstrated that inhalation of 3% H2could reduce the activation of microglia. Furthermore, inhibition of the JNK/p53 pathway significantly improved
learning and memory functions in HIBD rats. However,there are some limitations to our study that must be
acknowledged. For example, we focused on microglia inthis study. Astrocytes, other immune cells, and otherbrain regions should be assessed in future studies to further
our understanding of the protective mechanism of H2. We
can further study the therapeutic effect of hydrogen on different sexes of rats.
Taken together, our study demonstrated that cerebral HI
activates microglia, promotes the secretion of IL-1b and NFrB p65, induces neuronal damage, upregulates P-JNK/p53
signal transduction, and induces long-term spatial memory
defects. Inhalation of 3% H2 immediately after HI injury
reduced inflammation and inhibited cell apoptosis. In summary, H2 may play a neuroprotective role and improve
long-term spatial memory in HIBD rats by reducing inflammation and inhibiting apoptosis. Therefore, H2 may be aneffective method for the treatment of HIBD.
Authors’ contributions: The designation of the experiments
as well as the interpretation and analysis of the data was contributed by all authors. The manuscript was drafted by Wu andreviewed and approved by all authors; GW, ZC, PW, MZ, and
WZ conducted the experiments; MF and SH supplied critical reagents.
ACKNOWLEDGMENTS

We are grateful to the staff from the Medical Center
Laboratory of the Third Xiangya Hospital for their excellent
technical assistance.
DECLARATION OF CONFLICTING INTERESTS

The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication ofthis article.

FUNDING

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of thisarticle: This study was funded by the Project for Xiangya
famous doctor in Central South University (2014[68]), New
xiangya talent of the third Xiangya hospital (2014[97]), andthe Natural Science Foundation of Hunan Province of China(2018 JJ 2620).
ORCID iD

Guojiao Wu

https://orcid.org/0000-0001-6925-7312

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