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Neuroprotective effects of electroacupuncture on hypoxic-ischemic

encephalopathy in newborn rats association with increased
expression of mTOR
Tao Xu1, Na Zhao1, Jing-Dong Liu1, Mo Xiao-Mei1, Hui Zhang2, Peng-Fei Qin1,
Xiang-Chun Feng1 and Qing Sun1
1
Qingdao Women and Children Hospital, Qingdao, Shangdong, 266 000, P.R. China; 2Qingdao University Medical
College, Qingdao, Shangdong, 266 000, P.R. China.

Article Info Abstract

Received: 16 January 2016 In this study, we observed the therapeutic effects of acupuncture and investi-
Accepted: 21 February 2016 gated the underlying molecular mechanisms by constructed a hypoxic-
Available Online: 6 March 2016 ischemic encephalopathy (HIE) animal model. In the electroacupuncture
DOI: 10.3329/bjp.v11iS1.26418 group, mTOR expression increased since 1d, and continued to rise till the 21 st
day. All of the differences were significantly (p<0.05 vs the model group).
Cite this article: Meanwhile, mTOR expression was analyzed by Western blotting. There was
Xu T, Zhao N, Liu J, Mei M X, Zhang statistical significance between the model group and the electroacupuncture
H, Qin P, Feng X, Sun Q. Neuropro-
group in the four time periods (p<0.05). The results provide evidence that
tective effects of electroacupuncture
on hypoxic-ischemic encephalopathy electroacupuncture treatment protected cortical neurons against HIE-induced
in newborn rats association with in- neuronal damage and degenerative changes in rats, which is in association
creased expression of mTOR. with activation of mTOR both at the mRNA level and protein level. Therefore,
Bangladesh J Pharmacol. 2016; 11: S98 electroacupuncture may become a potential therapeutic strategy for HIE of
-S105. newborn.

Introduction ischemia brain damage through regulating the expre-

ssion of some relative protein (Fahling, 2009). PI3-K/
Hypoxic-ischemic encephalopathy (HIE) is an impor- Akt is an important pathway related to cell fate deter-
tant cause of brain injury in the newborn and can result mination.
in long-term devastating consequences. There has been
The mammalian target of rapamycin (mTOR), an
amounting research progress in HIE over the last two
atypical Ser/Thr protein kinase, is a main down-stream
decades and many target molecules have been found.
of PI3K/AKT signaling pathway (Nakamura et al.,
Despite all these advances, therapeutic interventions
2006; Gunn and Hailes, 2008; Kang et al., 2008).
are still limited (Lai and Yang, 2011; Fatemi et al.,
Previous study found that mTOR as a key regulator of
2009).
autophagy, could regulate cell growth, cell apoptosis,
After neonatal hypoxic-ischemic disease happening, energy metabolism and inhibition of autophagy after
there was a significant increase in neural autophagy in activation. It plays as a role in brain protection. When
a short time (Koike et al., 2008; Rudov et al., 2014; nutritional status, growth circumstance, hormone
Bellot et al., 2009). Autophagy is reported to play a levels, and other conditions changes, mTOR has special
predominant role in the pathological progress of residues phosphorylation, and changes its activity.
hypoxiaischemia brain damage (Li et al., 2007). Thus, it can play an important role in the processes of
Previous studies found that the phosphatidylinositol 3- many diseases (Mazure and Pouyssgur, 2010; Blagosk-
kinase/protein kinase B (PI3K/AKT) signaling path- lonny, 2013).
way was involved in the survival process after hypoxia As an alternative medicine methodology originating in
 Bangladesh J Pharmacol 2016; 11: S98-S105 S99

ancient China, acupuncture has been used for more suture, and then the incision was sewed up. After a two
than 1,000 years as a treatment or as an adjuvant -hour restoration, the rat was put in a sealed trans-
modality for patients with stroke. It has been frequently parent vessel and set in the warm bathe with the
used in Asian countries and has become increasingly temperature of 37C. Then the vessel was passed into
popular in the western world (MacPherson and gas with low content of oxygen (including 8% of
Hammerschlag, 2012; Fang et al., 2012; Ji and Zhang, oxygen and 92% of nitrogen) at the velocity of 1 L/min
2009; Zhou et al., 2009). for 2.5 hours. The survivors were kept warm for
another one hour and then received behavioral tests.
Several animal studies have indicated neuroprotective
For the sham-surgery group, after anesthesia, the LCCA
effect of acupunture on hypoxic-ischemic injury. It was
was separated without ligation and the incision was
shown to play an important role in functional
sewed up without any hypoxic treatment. The rat died
reorganization and cerebral compensation (Wang et al.,
or not awake 2 hours after surgery were excluded.
2005; Yi et al, 2006; Yang et al., 2005; Li et al., 2012;
Behavioral tests were made four hours later, and rats in
Gao et al., 2011). The possible mechanisms involved
this group showed no behavioral abnormality.
have attracted more and more attentions. So, using a
HIE rat model, in the present study we evaluated the Electroacupuncture treatment
in vivo therapeutic efficacy of electroacupuncture and
The bilateral common carotid arteries were separated,
investigated the underlying molecular mechanisms,
without ligation, and the rats were without hypoxia
especially the role of mTOR in the process.
after incisions sutured in the sham surgery group. The
bilateral common carotid arteries were separated and
ligated, and incisions were sutured, without any other
Materials and Methods
treatment in model group.
This study was performed at the Experimental Center,
In the electroacupuncture group, four acupoints inclu-
Medical College of Qingdao University Affiliated
ding Baihui (GV20), Dazhui (GV14), Quchi (LI11) and
Hospital, from September 2013 to May 2014.
Yongquan (KI1) were chosen, positioned according to
Animal the ordinary acupoints for acupuncture in rats. Locali-
zation of the acupoints was based on the International
A total of 236 seven-day-old SD rats (belonging to 11
Standard Scheme for acupoint names of acupuncture
broods with the mothers included, 10 to 11 newborn
drafted by the experimental acupuncture branch of the
rats per brood), male or female, breast-feeding, specific
China Acupuncture Academy. Baihui (GV20) acupoint
pathogen-free (SPF) grade, weighing 10.5 to 15 g
is located in the center of the parietal bone. Dazhui
(average weight, 112.8 1.6 g), were provided by
(GV14) is located between the seventh cervical vertebra
Qingdao Drug Detection Institute (No. SCXK (Lu) 2013-
and the first thoracic vertebra, just in the center of the
0001). They were maintained in a temperature-
back. Quchi (LI11) is in the midpoint of the line
controlled (20 to 25C) facility with a 12 hours light/12
between the outer end of the elbow stripes and the
hours dark cycle. Among which four weighing less than
epicondyle of the humerus, and Yongquan (KI1) is
11 g were excluded. In each brood, it is randomly
located in the plantar anterior third (toes excluded).
assigned into two groups, sham-surgery group and HIE
Needles 0.5 inch in length were horizontally inserted
model group. Totally there were 25 rats in the former
backwards into Baihui (GV20) for 0.2 inch deep,
group and 211 rats in the latter group.
perpendicularly inserted into Dazhui (GV14) for 0.2
Finally 176 rats were successfully established as the HIE inch deep, perpendicularly inserted into Quchi (LI11)
model with the successful rate of 83.4%. Then the for 0.5 inch deep and rapidly inserted into Yongquan
successfully established rats were randomly divided (KI1) without leaving the needle, respectively. The two
into the HIE model group, sham-acpuncture group, acupoints Baihui (GV20) and Quchi (LI11) were
acupuncture group, antagonists group, and connected with G-6805 electric acupuncture apparatus
acupuncture and antagonists group, with 35 rats in each (Shanghai Huayi Medical Instrument Factory, China),
group. All groups were subdivided into four subgroups and receive electroacupuncture for 10 min with the
in terms of four time periods of 1d, 3d, 7d and 21d. continuous wave at the frequency of 5 to 10 Hz with the
local tissue shivering slightly, and with the voltage
Establishment of HIE animal model
between 3 to 5V. The acupuncture therapy was given
The HIE model was constructed according to the LCCA once a day for consecutive day 1, 3, 7 and 21
ligation method in combination with hypoxic treatment respectively. The positions were from Baihui (GV20),
described by Rice et al. (1981). Briefly, animals were Dazhui (GV14), Quchi (LI11) and Yongquan (KI1) by 1
anesthetized by inhaling ether. In the supine position, a cm. In the antagonists group the HIE model rats were
midline ventral incision was made to expose the LCCA, injected wortmannin 5 uL (20 ug/kg wortmannin,
which was carefully separated from the vagus nerve. dissolved in DMSO) to the left ventricle by stereo
The LCCA was separated, ligated using a 5/0 silk positioning instrument. The acupuncture and anta-
S100 Bangladesh J Pharmacol 2016; 11: S98-S105

gonists group had the operations of antagonists group primary antibodies: mTOR monoclonal antibody (Cell
and electroacupuncture group at the same time. Signaling, USA). Anti-GAPDH antibody (Abcam, USA).
GAPDH was used as an internal loading control. After
Hematoxylin-eosin (HE) staining for determination of
being washed and incubated with the goat anti rabbit
neuronal damage
secondary antibody (Maixin Company, China), the
Rats were anesthetized by inhaling ether at the immune complexes were visualized with a chemilumi-
indicated time. The left ventricle was cannulated and nescence reagent. Western blots were quantified
perfused with phosphate-buffered saline (PBS) densitometrically with Quantity One software (Bio-Rad,
(preheated at 37C), and then perfused and fixed with USA), and the intensity values were normalized to
4% (w/v) paraformaldehyde (in 0.1 M PBS (pH 7.4), GAPDH.
precooled at 4C) firstly at full speed till convulsion of
Statistical analysis
the limbs ceased, then perfusion was kept at the
velocity of 1 mL/min. Then, the brain was quickly All data were analyzed using SPSS 13.0 software (SPSS,
separated with the cerebellum and the brainstem Chicago, IL, USA). Measurement data were expressed
removed and placed in 4% paraformaldehyde for 24 as mean SD. Comparisons among several groups
hours at 4C. The specimens were dehydrated with 20% were made using one-way analysis of variance under
sucrose and frozen. The frozen sections were then equal variances and Kruskal-wallis H analysis under
serially cut into 20 m thick coronal slices. HE staining unequal variances. Comparisons between groups were
was performed according to the standard protocol The made using SNK-q test under equal variances and
sections were observed under a light microscope Dunnetts T3 test under unequal variances. p<0.05 (two
(Nikon, Japan) using a magnification of 200, and -tail) was considered statistically significant.
photographed.

Detection of RET mRNA expression in the injured Results

cortex by real time RT-PCR
Electroacupuncture treatment significantly ameliorates
Total RNA was extracted from the injured cortex (about neuronal damage caused by hypoxic-ischemic injury
1 mm 1 mm 1 mm ) of rats using Trizol (Invitrogen,
In the sham-surgery group, HE staining showed normal
USA) according to the manufacturers instructions. Two
structures of cortex with clear organizational structure,
micrograms of total RNA were used to synthesize first-
normal cell outline, clear nucleolus and cell nucleus
strand cDNA with M-MuLV reverse transcriptase
located in the center (Figure 1- F). In the control model
(Fermentas, USA) using random primers. Real time
group, hypoxic-ischemic injury for 1d to 3d induced
PCR was performed using the ABI 7500 real time PCR
neuronal damage with obvious signs of necrosis and
detection system (ABI, USA) with SYBR green
degeneration of neurons, such as mesh-like structure,
(Fermentas, USA). Primer sequences for specific genes
irregular arrangements of neurons, concentrated
are presented as follows. Forward primer for mTOR: 5-
cytoplasm, vacuolation of cytoplasm, karyopyknosis,
CGCTGATCCCTTTATCG-3 ; Reverse primer for
and nucleolus loss (Figure 1-A1, A3). 7d to 21d after the
mTOR: 5-ATGCTCAAACACCTCCACC-3(168bp).
operation, severe neuron loss along with proliferation
Forward primer for GAPDH: 5-GAAGGTGAAGGTC-
of surrounding astrocytes was observed (Figure 1-A7,
GGAGT-3; Reverse primer for GAPDH: 5-GAAGATG-
A21). The pathological changes of nerve cells in the
GTGATGGGATTTC-3 (193bp). GAPDH was used as
sham acupuncture group (Figure 1-B1, B3, B7, B21)
an internal control.
were similar to that in the model group. Electro-
Detection of mTOR protein expression in the injured acupuncture for 3d showed swelling and degeneration
cortex by western blot analysis of neurons but less severe than the control model group
(Figure 1-C3). Electroacupuncture treatment for 7d to
Total protein extracts were prepared from rat brains as
21d showed less signs of necrosis and degeneration of
previously described. In brief, rats were anesthetized
neurons, that is relatively clear organizational structure,
with ether and rapidly decapitated. Brain was quickly
regular arrangements of neurons, normal cell outline
separated with the cerebellum and the brainstem
and relatively clear nucleolus (Figure 1-C7, C21). In
removed and placed on ice in 10 volumes of cold
acupuncture and antagonists group, pathological
homogenization buffer (50 mmol/L Tris, 120 mmol/L
changes of nerve cells also had a trend to reduce, but
NaCl, pH 7.4) to which protease inhibitors (Sigma,
inferior to the acupuncture group (Figure 1-E3, E7, E21).
USA) had been added. The tissue was then homoge-
In antagonists group, more obvious nerve cell necrosis,
nized and stored at -80C. Protein concentrations were
cell shrinkage, nuclear condensations were seen (Figure
determined using the Bradford method (Bio-Rad, USA).
1-D1, D3, D7, D21).
Equal amounts of protein (50 g/lane) were separated
by SDS-PAGE and transferred onto polyvinylidene Expression changes of mTOR mRNA in the cerebral
difluoride membranes (Millipore, USA). After being cortex of rats from different time sessions of
blocked, the filters were incubated with the following experimental groups
 Bangladesh J Pharmacol 2016; 11: S98-S105 S101

Shame-surgery group

A1 A3 A7 A21

HIE model group

B1 B3 B7 B21

Shame acupuncture

group

C1 C3 C7 C21

Acupuncture group

D1 D3 D7 D21

Antagonist group

E1 E3 E7 E21

Antagonist and
acupuncture group

Day 1 Day 3 Day 7 Day 21

Figure 1: Effect of electroacupuncture treatment on neuronal damage in the injured cortex of rat with HIE (Hematoxylin-eosin
staining , optical microscope, 200).

MTOR mRNA expression levels were detected with the differences were significant compared to that of
realtime RT-PCR. As was shown in Figure 2, compared model group (p<0.05). Table I showed the changes of
with that of the sham-surgery group, expression levels mTOR mRNA in each group at different times.
of mTOR mRNA in the cerebral cortex with hypoxic-
Expression changes of mTOR protein in the cerebral
ischemic injury from the model group increased at the
cortex of rats from different time sessions and different
first day post-surgery (p<0.05), continued increasing
groups
three days later and reached the peak at the seventh
day (p<0.05). To 21 days, the expression of mTOR MTOR expression was analyzed by western blotting. In
mRNA in model group was not significantly increased, comparison with the sham-surgery group, mTOR
but compared with the sham operation group, and the expression at the protein level was significantly
difference was also significantly (p<0.05). The increased both in model group (p<0.05) and in the
expression in the sham-acupuncture group was close to electroacupuncture group (p<0.05) at the 1st, 3rd, 7th,
that of the model group with sustained rising in 1d, 3d, 21th day. Furthermore, mTOR expression was increa-
7d and 21d, and the differences were not significant sed both in the model group and in electroacupuncture
(p>0.05). In the electroacupuncture group, mTOR group. There was statistical significance between the
expression also increased since the first day, and model group and the electroacupuncture group in the
continued to rise till the 21st day. All of the differences four time periods (p<0.05). MTOR expressions in
were significantly (p<0.05 vs the sham-surgery group). electroacupuncture and antagonists group were not
The expressions of mTOR in the acupuncture and significant compared to model group at 1st, 3rd, 7th
antagonists group were significantly different from that day (p>0.05), but the difference was significant at 21 rd
of the sham-surgery group in 1d, 3d and 7d, and to 21d, day (p<0.05) (Figure 3).
S102 Bangladesh J Pharmacol 2016; 11: S98-S105

4.0

3.5

3.0
mTOR mRNA expression

Shame-surgery
Model
2.5
Shame-Acupuncture
Acupuncture
2.0 Antagonists
Antagonist + Acupuncture
1.5

1.0

0.5
1 3 7 21

Time (Day)

Figure 2: Effect of electroacupuncture treatment on mTOR mRNA expression in the injured cortex of rat with HIE (real time RT-
PCR)

Table I

The expression of mTOR mRNA in hypoxic ischemic brain tissue

Group Day 1 Day 3 Day 7 Day 21
Sham-surgery 1.0 0.1 1.0 0.1 1.0 0.3379 1.0 0.2
Model 1.3 0.0a 2.1 0.2a 2.6 0.2a 2.7 0.1a
Sham-acupuncture 1.4 0.1a 2.1 0.2a 2.5 0.1a 2.6 0.1a
Acupuncture 1.8 0.1abc 2.8 0.1abc 3.2 0.1abc 3.5 0.1abc
Antagonists 1.3 0.2 1.8 0.2 1.9 0.2 1.9 0.4
Acupuncture and antagonists 1.6 0.1a 2.2 0.2a 2.8 0.1a 3.1 0.2abc

mRNA was normalized to the internal control GAPDH. Data are expressed as mean SD; n = 6 rats per group; ap<0.05 vs the
corresponding sham-surgery group; bp<0.05 vs the corresponding model group; cp<0.05 vs the corresponding antagonists group)

Discussion different acupoints, Baihui (GV20) and Dazhui (GV14)

are the main points of the Du channel, which exhibits a
In the present study, we constructed a HIE animal combined supervising effect on the entire meridian
model to evaluate the in vivo therapeutic efficacy of system. Acupuncture at Baihui in combination with
electroacupuncture and to further investigate the role of Dazhui promoted flow of blood, resuscitated conscious-
mTOR in the process. Cerebral development of seven- ness, recuperated depleted yang, and balanced Yin and
day-old rat is similar to that of the newborn, so we Yang (Fang et al., 2012). Amounting evidence of animal
chose seven-day-old rat to construct the model. Internal experiments have shown that acupuncture at the points
carotid artery and vertebral artery of rats form Willis located in the head and the neck could improve the
ring at the undersurface of cerebrum. Therefore ligation blood circulation system of the brain, dilate brain blood
of one side of the common carotid arteries cannot vessel, improve the microcirculation, ameliorate cere-
induce ideal ischemic injury. In 1981, Rice firstly bral edema, and activate the repair function of cerebral
established a rat model of HIE by ligating the common neurons to promote functional recovery (Kim et al.,
carotid artery in combination with hypoxic treatment 2011; Chuang et al., 2007; Liu et al., 2012). Quchi (LI11)
(Rice et al., 1981). Since then, the model has been widely is the He-Sea point of the large intestine channel of
used for its simple operation, high successful rate, low Hand-Yangming. Electroacupuncture at Quchi (LI11)
mortality, high reliability and good reproducibility. and Zusanli (ST36) acupoints was reported to improve
Many animal studies have indicated neuroprotective the ischemia-associated scores of neurological deficits,
effect of acupunture on hypoxic-ischemic injury. Acu- reduce cerebral infarction, alleviate inflammatory res-
point combination is a complicated issue. Among ponses, promote neovascularization, inhibit neural cell
 Bangladesh J Pharmacol 2016; 11: S98-S105 S103

A 0.16 B 0.18

0.14 0.16

0.14
0.12
abc
abc 0.12 a
mTOR/GAPDH ratio

mTOR/GAPDH ratio
0.10
a 0.10
a a
0.08
a a 0.08
0.06
0.06
0.04
0.04

0.02 0.02

0.00 0.00
1 1
y

y
re

re
e

e
e e
er

er
e

e
s

s
ur

ur
od od
tu

tu
ur

ur
st

st
rg

rg
ct

ct
nc

nc
ni

ni
ct

ct
M M
su

su
un

un
go

go
un

un
u

u
e-

e-
up

up
up

up
ta

ta
up

up
am

am
Ac

Ac
Ac

Ac
An

An
Ac

Ac
Sh

Sh
e-

e-
t+

t+
am

am
s

s
Sh

Sh
ni

ni
go

go
ta

ta
An

An
mTOR mTOR

GAPDH GAPDH

C D 0.20
0.20
abc
0.18
a
0.16
0.15 a
abc a
0.14 a
mTOR/GAPDH ratio

mTOR/GAPDH ratio

0.12 a
a
0.10 0.10

0.08

0.06
0.05
0.04

0.02

0.00 0.00
1 1
y

e
e

e
er

e
e

e
s
ur
ur

er

od
e
s
ur

ur

od
ur
st
rg

ur
st
ct

rg
ct

ct

ct
ni

ct

M
su

ni

ct

M
un

un

su

un

un
go

un

go

un
e-

up

up

e-

up

up
ta

up

ta
am

up
am
Ac

Ac

An

Ac

Ac

An
Ac

Ac
Sh

e-

Sh

e-
t+
am

t+
am
s
Sh

s
ni

Sh

ni
go

go
ta

ta
An

An

mTOR mTOR

GAPDH GAPDH

Figure 3: Effect of electroacupuncture treatment on mTOR protein expression in the injured cortex of rat with HIE (Western blot)
(A)-(D) mTOR protein expression at the 1st, 3rd , 7th and 21st day respectively
Bar graphs show densitometric analysis of Western blot of mTOR protein. The densitometric quantification was normalized to the internal control
GAPDH. Data are expressed as mean SD (n = 6 rats per group, ap<0.05 vs the sham-surgery group; bp<0.05 vs the model group; cp<0.05 vs the
antagonists group)
S104 Bangladesh J Pharmacol 2016; 11: S98-S105

apoptosis and promote neurological functional changes of mTOR in the hypoxic-ischemic injured
recovery in a focal cerebral ischemia-reperfusion cerebral cortex at the 1st, 3rd, 7th and 21st day post-
injured rat model (Lan et al., 2013; Hong et al., 2013; surgery and explored the interventing effect of
Chen et al., 2012; Zhao et al., 2010). Yongquan (KI1) is electroacupuncture. Results showed that the expression
the significant Jing-Well point of the kidney channel of of mTOR in model group increased at the 1st day after
Foot-Shaoyin. Using in combination with Baihui hypoxia, and the upward trend was more obvious at
(GV20), it was shown to improve energy and the 7th day, and it was not obvious from 7th day to 21st
intelligence, clear the brain collaterals and coordinate day. This suggests that rats have self protection after
the channels and collaterals (Xue et al., 2009; Xue et al., hypoxia ischemia, but the self protective ability are
2011). Acupuncture at Quchi (LI11) and Yongquan effective only in a short period of time without
(KI1) was reported to promote functional recovery of interventions. The change of mTOR in electroacu-
the extremities after neuron injury (Ji et al., 2009; Zhou puncture group showed a continuous upward trend,
et al., 2009). Considering that Baihui (GV20), Dazhui compared to model group at 1st, 3rd, 7th, and 21rd day.
(GV14), Quchi (LI11) and Yongquan (KI1) are The differences were significant.
commonly used in clinics for patient treatment and are
easy to locate, we chose the combination of the above
four acupoints in our experiments. HE staining results Conclusion
showed that electroacupuncture treatment significantly
ameliorated neuronal damage caused by hypoxic- Electroacupuncture treatment protected cortical
ischemic injury and protected structure of neurons neurons against HIE-induced neuronal damage and
against the degenerative alterations, suggesting that degenerative changes in rats, which is in association
electroacupuncture at Baihui (GV20), Dazhui (GV14), with activation of mTOR. Therefore, electroacupuncture
Quchi (LI11) and Yongquan (KI1) exerts neuropro- may become a potential therapeutic strategy for HIE of
tective function in HIE. newborn.

The mammalian target of rapamycin (mTOR), also

termed the mechanistic target of rapamycin and FK 506-
binding protein 12-rapamycin complex-associated Ethical Issue
protein 1 (FRAP 1), is a 289 kDa protein serine/ All experimental procedures were performed in accordance
threonine protein kinase that plays a critical role in with the Guidance Suggestions for the Care and Use of
protein synthesis, cytoskeletal organization, cellular Laboratory Animals, formulated by the Ministry of Science
differentiation, development, survival, and aging and Technology of the Peoples Republic of China.
(Chong et al., 2010). A large number of studies showed
that the abnormal regulation of mTOR signaling
pathway is closely related to cell proliferation. MTOR Acknowledgement
played an important role not only in the physiological
The project was supported by the Natural Science Foundation
processes such as cell growth, protein expression,
of China (NO. 81202761).
protein degeneration, signal transduction, ribosome
synthesis, but also in the formation and development of
brain, the occurrence of neural synapse, the formation
of learning and memory (Schicknik et al., 2008). Dying References
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Author Info
Feng Xiang Chun and Sun Qing (Principal contact)
e-mail: [email protected]; [email protected]
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