Neurochemistry International 65 (2014) 23–29

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Neurochemistry International
journal homepage: www.elsevier.com/locate/nci

Effects of acupuncture on declined cerebral blood flow, impaired

mitochondrial respiratory function and oxidative stress in multi-infarct
dementia rats
Xuezhu Zhang 1, Bangqi Wu 1, Kun Nie, Yujie Jia, Jianchun Yu ⇑
First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

a r t i c l e i n f o a b s t r a c t

Article history: Brain energy disorders and oxidative stress due to chronic hypoperfusion were considered to be the
Received 10 October 2013 major risk factors in the pathogenesis of dementia. In previous studies, we have demonstrated that acu-
Received in revised form 5 December 2013 puncture treatment improved cognitive function of VaD patients and multi-infarct dementia (MID) rats.
Accepted 12 December 2013
Acupuncture therapy also increased the activities of glycometabolic enzymes in the brain. But it is not
Available online 19 December 2013
clear whether acupuncture treatment compensates neuronal energy deficit after cerebral ischemic
through enhancing the activities of glucose metabolic enzymes and preserving mitochondrial function,
Keywords:
and whether acupuncture neuroprotective effect is associated with activations of mitochondrial antiox-
Acupuncture
Vascular dementia
idative defense system. So, the effect of acupuncture therapy on cognitive function, cerebral blood flow
Cerebral blood flow (CBF), mitochondrial respiratory function and oxidative stress in the brain of MID rats was investigated in
Mitochondrial respiration this study. The results showed that acupuncture treatment significantly improved cognitive abilities and
Oxidative stress increased regional CBF of MID rats. Acupuncture elevated the activities of total SOD, CuZnSOD and
MnSOD, decreased the level of malondialdehyde (MDA) and superoxide anion, regulated the ratio of
reduced glutathione (GSH) and oxidized glutathione (GSSG) in mitochondria, and raised the level of
the respiratory control index (RCI) and P/O ratio and the activities of mitochondrial respiratory enzymes
of MID rats. These results indicated that acupuncture treatment improved cognitive function of MID rats;
and this improvement might be due to increased CBF, which ameliorated mitochondrial dysfunction
induced by ischemia and endogenous oxidative stress system of brain.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Brain energy disorders and oxidative stress due to chronic

Vascular dementia (VaD) is one of the most common types of
dementia in older adults, characterized by a progressive worsening
of memory and other cognitive functions due to chronic, reduced
blood flow in the brain. Besides cognitive impairment, patients
with severe VaD present with disruption in their personal, social
and vocational functioning (Roman, 2002). In recent years, the inci-
dence of VaD rises exponentially for patients with cerebrovascular
risk factors such as hypertension, cardiac disease, diabetes, smok-
ing, alcoholism and hyperlipidemia. In the US and Europe, the ratio
of VaD to AD (Alzheimer’s disease) is thought to be 1:5, and
dementia following stroke is thought to occur in one quarter to
one third of cases of stroke. VaD is also a growing problem in China,
with a prevalence of about 1–3%, similar to prevalence rates in the
US and Europe (Dubois and Hebert, 2001).
⇑ Corresponding author. Fax: +86 22 27432227.
E-mail address: zhangxuezhu1999@126.com (J. Yu).
1 et al., 2009). Enhanced GLUT1 expression has neuroprotective
These authors contributed equally to this work.
hypoperfusion were considered to be the major risk factors in
the pathogenesis of dementia. Hypoperfusion cause impairment
of bioenergetics, which appears early and progresses rapidly, con-
sistent with the primary defect. In addition, ischemia induces
chronic hypoxic state, which causes the formation of a large
amount of reactive oxygen species (ROS). Increased ROS produc-
tion disrupts antioxidant defense and directly impairs mitochon-
drial homeostasis and energy production, which also appear to
be a key factor in the development of VaD (Piantadosi and Zhang,
1996).
Glucose is the primary fuel for the brain and sufficient energy
support is essential to maintain normal brain function (Laughlin,
2004). An interruption of blood flow to the brain causes energy
shortage, resulting in neuronal injury and death, even long-term
cognitive decline. GLUT1 mainly expresses on both luminal and
abluminal faces of CNS endothelium and responsible for transport-
ing glucose from blood into the brain across the blood–brain barrier
(Dick et al., 1984). The level of GLUT1 is regulated by glucose con-
centration, ischemia and hypoxia (Baumann et al., 2002; Vemula

0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuint.2013.12.004
24 X. Zhang et al. / Neurochemistry International 65 (2014) 23–29
properties during cerebral ischemia due to transport more glucose
into the brain to meet its metabolic needs (Espinoza-Rojo et al.,
2010).
Acupuncture is a branch of traditional Chinese medicine and
has been proven effective in treating VaD. Based on his clinical
experience, Professor Jingxian Han thought that age-related de-
cline in activity of ‘‘qi’’ within Sanjiao could cause abnormal
changes in functions of many tissues and organs, which could
eventually lead to dementia (Han, 2007). Therefore, he created
the ‘‘Sanjiao’’ acupuncture method to regulate abnormal function
of qi activity within Sanjiao and the prescription included ‘‘Danz-
hong’’ (CV17), ‘‘Zhongwan’’ (CV12), ‘‘Qihai’’ (CV6), bilateral ‘‘Xue-
hai’’ (SP10) and ‘‘Zusanli’’ (ST36) five acupoints. In the past few
years, we gave acupuncture treatment to more than 200 VaD pa-
tients and most of them benefited from the therapy. Evaluated
by MMSE, HDS-R and ADL exams, these patients showed remark-
able improvements in memory, orientation, calculation and self-
care abilities (Yu et al., 2006). In our previous studies, we have
demonstrated that acupuncture therapy could improve the cogni-
tive function of multi-infarct dementia (MID) rats, and increase the
activities of glycometabolic enzymes (e.g., hexokinase, pyruvate ki-
nase, and glucose 6 phosphate dehydrogenase) in the cortex (Zhao
et al., 2011). However, it is not clear whether acupuncture treat-
ment can compensate neuronal energy deficit through enhancing
the activities of glucose metabolic enzymes and preserving mito-
chondrial function after cerebral ischemic, and whether acupunc-
ture neuroprotective effect is associated with activations of
mitochondrial antioxidative defense system. So, in this paper, the
effect of acupuncture on the cerebral blood flow, mitochondrial
respiratory function and oxidative stress of MID rats was studied
to explore its anti-dementia mechanism further.
2. Material and methods
2.1. Animals
Sixty healthy male Wistar rats (300–320 g) were randomly as-
signed into the following three groups: normal control group
(Gn, n = 10), sham-operated group (Gs, n = 10) and operated group
(n = 40). The animals were housed under standard conditions and
allowed free access to water and food. All animals were maintained
in accordance with principles of laboratory animal care (NIH publi-
cation No. 86-23, revised 1985) and Guide for the Care and Use of Lab-
oratory Animals, revised 2006 (Ministry of Science and Technology of
the People’s Republic of China), and animal protocols were approved
by the Laboratory Animal Care Committee of the Tianjin University
of Traditional Chinese Medicine.
Before the surgery, 10 mL whole blood was collected from a
Wistar rat, dried at 37 °C and ground into microemboli with diam-
eter of 100–200 lm. After anesthetized, the bifurcation of the right
common carotid and external carotid artery of rats was exposed
and a temporary clip was applied to the external carotid artery just
above its origin. The embolic insult was induced in rats in operated
group by injecting 0.3 mL of 3% microemboli saline suspension into
the internal carotid artery over 1–2 mins. Sham-operated rats re-
ceived carotid injections of 0.3 mL of physiologic saline. Rats were
allowed to recover for 1 week and three animals were dead during
the recovery period.
One week later, all the animals were given hidden platform tri-
als for five consecutive days. And the operated rats were further
randomly divided into impaired group (Gi), acupuncture group
(Ga) and sham-acupuncture group (Gsa) (ten rats in each group)
according to their performance in Morris water maze test. After
that, all the animals were handled according to their groups. The
Ga group was given treatment with the ‘‘Sanjiao’’ acupuncture
method once daily for 21 days and the location of acupoints was
shown in Table 1. One-off sterile acupuncture needles (Huatuo,
Suzhou Medical Instruments Factory, Suzhou, China), with a length
of 13 mm and diameter of 0.14 mm, were used in the trial. After
sterilization with iodine, needles were inserted horizontally 2–
3 mm into CV17; 3–4 mm perpendicularly into CV12, CV6 and
ST36; 2–3 mm obliquely into SP10, and rotated 2–3 times per sec-
ond clockwise for 30 s respectively. The Gsa rats were needled at
the two points located at the hypochondrial region of the body
for 105 s per point in the same way as the Ga group. The rats in
the other groups were grasped in the same manner and for the
same periods of time as the Ga group.
At the end of the treatment, all the animals were trained in
Morris water maze again to evaluate their cognitive function,
including two hidden platform trials per day for 2 days and a probe
trial. The details of surgical procedure, Morris water maze test, the
anatomical location of acupoints and acupuncture treatment have
been described in our previous studies (Liu et al., 2006; Yu et al.,
2005).
2.2. Determination of regional cerebral blood flow
After Morris water maze test, all the rats were initially anesthe-
tized with isoflurane in a mixture of 70% N2 and 30% O2 (5% induc-
tion, 2% maintenance). Anesthesia depth was assessed by testing
the corneal reflexes and motor responses to the tail pinch. Femoral
artery was catheterized for arterial pressure and heart rate (Power-
Lab, ADInstruments) and for blood sampling. Animals were artifi-
cially ventilated with an O2–N2 mixture and the O2 concentration
in the mixture was adjusted to provide an arterial PO2 of
120–150 mm Hg. End-tidal CO2, monitored by a CO2 analyzer (Cap-
star-100, CWI), was controlled at 33–35 mm Hg. A heating lamp
thermostatically controlled by a rectal probe was used to maintain
the rat body temperature at 37 ± 0.5 °C. Throughout the experi-
ment, two samples (50 lL) of arterial blood were collected for
blood gas analysis.
Regional CBF was measured with a DRT4 laser Doppler blood
flow and temperature monitor (Moor Instruments Inc). A small
area of skull approximately 2 mm posterior to the bregma and
2 mm lateral to the sagittal suture was thinned to allow placement
of the laser-Doppler probe. The tip of the probe (U 2 mm) was sta-
bilized to the thin-skull window using a tissue adhesive (Aron Al-
pha; Toa, Tokyo, Japan). After achieving a stable baseline, the CBF
was continuously measured for 10 min. Zero value for CBF was
determined by the end of the experiment, when the heart was
stopped with an overdose of isoflurane.
2.3. Studies in isolated mitochondria
After the measurement of CBF, the brain was removed from rats,
washed and minced in isolation buffer (25 mM sucrose, 75 mM
mannitol, 1 mM EGTA) before being homogenized. Mitochondria
were obtained by differential centrifugation, and the protein con-
tent was measured by the BCA method. Markers of oxidative dam-
age and activity of antioxidant enzymes in isolated mitochondria
were determined using the respective kits (Nanjing Jiancheng, Chi-
na). Oxygen consumption was measured using a Clark type oxygen
electrode (YSI 5300, Yellow Springs, Ohio, USA). State 4 respiration
was evaluated in the presence of succinate plus rotenone or with
sodium glutamate and sodium malate. State 3 respiration was stim-
ulated by the addition of ADP. Respiratory control index (RCI) was
calculated as the ratio of state 3/state 4. The P/O ratio was calcu-
lated from the added amount of ADP and total amount of oxygen
consumed during state 3. The activities of mitochondrial respira-
tory chain enzyme complexes [complex I (NADH-ubiquione oxido-
reductase), complex II (succinate-ubiquinone oxidoreductase), and
 X. Zhang et al. / Neurochemistry International 65 (2014) 23–29
Table 1
Name, anatomical position and innervation of acupuncture points.
Theraphic method Points Local innervation
CV17 The anterior cutaneous branch of the fourth intercostal
nerve
CV12 The anterior cutaneous branch of the eighth intercostal
nerve
‘‘Sanjiao’’ acupuncture method CV6 The anterior cutaneous branch of the eleventh
intercostal nerve
SP10 The anterior femoral cutaneous nerve
ST36 The lateral sural cutaneous nerve and the cutaneous
branch of the saphenous nerve, deeper, the deep
peroneal nerve
Sham-acupuncture
complex IV (cytochrome c oxidase)] were studied by a method
modified from that described by Rafael (Rafael, 1983).
2.4. Western blots analysis of GLUT1 expression
The tissue from cerebral cortex was homogenized in ice-cold
RIPA buffer (Tris–HCl, 50 mM; NaCl, 150 mM; EDTA, 5 mM; and
1% Triton X-100, pH 7.5) containing complete EDTA-free proteinase
inhibitors (Roche). The homogenate was centrifuged at 12,000  g
for 30 min, and the supernatant was isolated for Western blot anal-
yses after protein assay by the BCA method. The samples were
loaded onto 10% SDS–PAGE, transferred to a PVDF membrane,
blocked with 5% nonfat milk in TBS, and blotted with rabbit anti-
GLUT1 (1:2000, Millipore) or rabbit anti-b-actin (1:1000, Santa
Cruz) antibodies. Immunoreactive signals were detected with a
Chemi Dos XRS system (Bio-Rad) after the reaction with Immobi-
lon Western chemiluminescent HRP substrate (Millipore). Three
rats from each group were used to extract protein and each sample
was analyzed three times, and then the relative expression of pro-
tein was averaged to provide a mean value for each group.
2.5. Statistical methods
Mean and standard deviation (SD) were calculated for all re-
sults. The differences between groups were assessed by two-way
ANOVA followed by LSD (equal variances assumed) or Dunnett’s
T3 (equal variances not assumed). For the Morris water maze tests,
escape latency times in the hidden platform trial were analyzed
with two-way ANOVA of repeated measures. A significance level
of P < 0.05 was used for all comparisons. All data analysis was per-
formed using SPSS (version 13.0).
3. Results
3.1. Cognitive impairment in rats
In this study, only hidden platform trial and probe trail were
performed. Both the Gn and Gs rats exhibited significantly im-
proved performance during the hidden platform trials (Fig. 1),
and they found their targets in much shorter time than the Gi rats.
The escape latencies of the Ga rats were much lower than that of
the Gi and Gsa groups, but there were no significant differences be-
tween the Ga and Gn groups.
Marginal, random, tendency and linear searching strategies
were searching strategies employed by animals to locate the plat-
form. Almost all the rats failed to locate the platform on the first
25
Location
On the anterior median line of the chest, at the level of
the fourth intercostal space, at the midpoint between
the two nipples.
On the anterior median line of the upper abdomen,
10 mm below the xiphisternal synchondroses.
On the anterior median line of the lower abdomen,
4 mm below the umbilicus.
On the medial aspect of the thigh when the knee is
flexed, 4 mm above the mediosuperior border of the
patella, on the bulge of the medial portion of M.
quadriceps femoris.
Three millimeters below head of fibula under knee joint,
and 1 mm from the anterior crest of the tibia.
On the hypochondrium, 3 mm above iliac crest.
Fig. 1. Escape latencies to find the hidden platform in Morris water maze test in
MID rats. Hidden platform trial was applied after acupuncture treatment, and the
arithmetic means for continuous 5 days of training achievement twice in a day were
recorded as performance records for statistical analysis. Data are expressed as
means ± S.D.
training day. Most of them swam around the tank wall and found
the submerge platform by marginal or random searching strate-
gies. Even though some rats successfully found the platform, they
were more likely to complete by chance. The Gn and Gs rats grad-
ually changed their search patterns to the tendency or linear
searching strategies during the training days, making their search-
ing process more spatially precise and efficient, whereas most of
the Gi rats still swam in marginal or random searching patterns.
Acupuncture treatment could make the Ga rats find their targets
quickly and efficiently, and their performance was similar to the
Gs and Ga rats, but it was much better than the Gi and Gsa groups.
In the probe trail, some rats in the Gn, Gs and Ga groups were
able to get to the former platform area quickly, so the latencies
of first target site crossover for them were significantly shorter
than the other two groups (P < 0.01) (Fig. 2A). They also spent sig-
nificantly more time in the middle annulus (a circular zone with a
inner diameter of 20 mm and outer diameter of 40 mm, centered
where the center of the escape platform had been in the hidden
platform trial) than the other two groups (P < 0.01) (Fig. 2B). In
addition, the number of crossings over the former platform loca-
tion for the Gn, Gs and Ga rats was obviously larger (P < 0.01)
(Fig. 2C).
The results of Morris water maze test suggested that acupunc-
ture could obviously improve the spatial learning and memory
abilities of MID rats, and showed point specificity of acupuncture.
26 X. Zhang et al. / Neurochemistry International 65 (2014) 23–29
Fig. 2. Performances of the MID rats in the probe trial. (A) Latency to first target-site crossover. (B) Percentage of time spent in the middle annulus. (C) Number of crossings
over the platform position in the target quadrant. Data are expressed as means ± S.D. ⁄P < 0.05 and ⁄⁄P < 0.01, compared as indicated.
3.2. Regional CBF
The CBF declined 20.3% and 10.3% for the Gi rats than the Gn
and Gs groups respectively (Fig. 3) (P < 0.01). The CBF of the Ga rats
increased by over 20% compared with the Gi rats (P < 0.01),
whereas no differences were seen between the Ga and Gn groups.
The sham-acupuncture stimulation did not affect any outcome of
the CBF, and there were obvious differences between the Ga and
Gsa rats (P < 0.01).
3.3. Mitochondrial respiratory function and oxidative stress
The activities of total SOD, CuZnSOD and MnSOD in the
mitochondria of the Gi group decreased 28.3%, 39.1% and 56.2%
compared to the Gn rats (Fig. 4A). After acupuncture treatment,
the activities of these enzymes rose 17.7%, 43.9% and 74.6% respec-
tively, compared to the Gi group. There were no differences
between the Gi and Gsa rats.
The decreased reduced glutathione (GSH) and increased oxi-
dized glutathione (GSSG) were seen in the Gi group compared to
the Gn rats (Fig. 4B). Notable higher GSH and lower GSSG were de-
tected in the Ga rats than the Gi group. The value for the Gsa group
did not differ from the Gi group.
Fig. 3. Regional cerebral blood flow in MID rats. After anesthetized, the regional CBF
of MID rats was measured with a laser Doppler blood flow monitor under the
conditions of artificial ventilation and continuous arterial pressure and heart rate
recording. A small area of skull approximately 2 mm posterior to the bregma and
2 mm lateral to the sagittal suture was thinned and the laser-Doppler probe was
placed to the thin-skull window. The CBF was continuously measured for 10 mins
after a stable baseline was got. Data are expressed as means ± S.D. ⁄P < 0.05 and
⁄⁄
P < 0.01, compared as indicated.
The level of malondialdehyde (MDA) and superoxide anion
(O2 ) in the Gi group was significantly higher than the Gn group
(P < 0.01). The decreased MDA and O2 levels were found after acu-
puncture treatment compared with the Gi group. There were no
differences between the Gi and Gsa rats (Figs. 4C and D).
Compared with the Gn group, the declined RCI and P/O ratio
were observed in the Gi rats (P < 0.05). The level of the RCI and
P/O ratio was much higher in the Ga group than the Gi group
(P < 0.05). No differences were detected between the Gi and Gsa
groups (Fig. 5).
The activities of mitochondrial respiratory complex enzymes
(complex I, II and IV) were declined sharply in the Gi group com-
pared to the Gn rats (P < 0.01). The activities of complex I and IV
rose significantly after acupuncture treatment (12–15%, P < 0.05),
however, the activity of complex II did not show obvious differ-
ences. The sham-acupuncture stimulation did not produce any
changes in the activities of the three enzymes (Fig. 6).
3.4. Expression of the GLUT1 in the cortex
The expression of GLUT1 in the cortex of the Gi group was slight
increased compared with the Gn rats, but the differences did not
reach statistical significance. There were no obvious differences
among the Gn, Gs, Gi, Ga and Gsa groups (P > 0.05).
4. Discussion
Based on his clinical experience, Professor Jingxian Han thought
that the age-related decline in activity of qi within Sanjiao might
be the key cause for aging and dementia (Han, 2007). According
to the theory of traditional Chinese Medicine, Sanjiao is not only
the source of qi, but also the pathway for the transportation and
distribution of qi. With the help of qi within Sanjiao, the five-zang
organs can work together to maintain normal physiological activi-
ties. As the commander of qi activities, Sanjiao controls functional
activities of five zang- and six fu-organs. Therefore, the disruption
of qi activities within Sanjiao was thought to be responsible for
aging and key pathogenesis of relevant senile diseases. The merid-
ian theory is one of important parts of the traditional Chinese Med-
icine ttheories, which deals with physiologic regulation and
pathologic changes in the human body, and guides the diagnosis
and treatment of oriental medicine in many aspects, especially in
relation to acupuncture. According to the theory, meridians are
invisible channels through which qi circulates throughout the
body. There are 12 main meridians, which form a network of en-
ergy channels throughout the body. The acupuncture points are
linked together in the network of meridians and the location of
them are mostly at where the qi of the channels rises close to
the surface of the body. Under pathologic conditions, qi becomes
 X. Zhang et al. / Neurochemistry International 65 (2014) 23–29 27

Fig. 4. Mitochondrial oxidative stress in the cortex of MID rats. Mitochondria were isolated from the cortical brain tissue of MID rats by differential centrifugation and the
level of oxidative stress markers in mitochondria was assessed using commercial kits. (A) Activities of mitochondrial total SOD, CuZnSOD and MnSOD. (B) Content of
mitochondrial reduced glutathione (GSH) and oxidized glutathione (GSSG). (C) Level of mitochondrial malondialdehyde (MDA). (D) Level of mitochondrial superoxide anion
(O2 ). Data are expressed as means ± S.D. ⁄P < 0.05 and ⁄⁄P < 0.01, compared as indicated.

Fig. 5. Level of respiratory control index (RCI) and P/O ratio in the cortex of MID rats. Mitochondria extracted from the cortical brain tissue in MID rats were used for the
measurement of oxygen consumption, state 3 respiration and state 4 respiration using a Clark type oxygen electrode. RCI was calculated as the ratio state 3/state 4. The P/O
ratio was calculated from the added amount of ADP and total amount of oxygen consumed during state 3. Data are expressed as means ± S.D. ⁄P < 0.05 and ⁄⁄P < 0.01,
compared as indicated.

blocked, stagnated or weakened, so stimulation on acupuncture
points will correct the imbalance of qi and promote qi flow
smoothly through the meridians. In this way, acupuncture can
effectively rebalance the energy system and restore health or pre-
vent the development of disease. The ‘‘Sanjiao’’ acupuncture meth-
od created by Han mainly regulates qi within Sanjiao by
stimulating specific acupoints. These acupoints are all located
along the meridians, so acupoint stimulation can trigger qi flow
along the meridians, which leads to a restoration toward balance
of qi activity within Sanjiao and production of special therapeutic
effects. On the contrary, the sham-acupoints are not located at
any meridians, even though a minor response was found after nee-
dle stimulation, which is not specific to the physical conditions or
the properties of the disorder. As a result, the therapy effects of
acupoint stimulation are much better than sham-acupoint
stimulation.
Hypoperfusion is an important risk factor in the development of
cognitive decline and vascular dementia in older patients. The data
28 X. Zhang et al. / Neurochemistry International 65 (2014) 23–29
Fig. 6. Activities of mitochondrial respiratory chain enzyme complexes in the
cortex of MID rats. Mitochondria extracted from the cortical brain tissue in MID rats
were used for the measurement of enzyme complexes, including complex I, II and
IV. Data are expressed as means ± S.D. ⁄P < 0.05 and ⁄⁄P < 0.01, compared as
indicated.
from a large population-based study indicated that higher CBF
velocity was related to a lower prevalence of cognitive decline,
and low CBF may contribute directly to the progression of demen-
tia (Ruitenberg et al., 2005). In MID patients, regional CBF reduc-
tions are found particularly in the distribution of both middle
cerebral arteries, and each regional CBF reduction has been shown
to correspond to a zone of cerebral ischemia or infarction. These
ischemic zones in MID frequently involve the thalamus bilaterally
as well as the frontal and temporal cortex surrounding or adjacent
to both sylvian fissures (Meyer et al., 1988). Reductions of CBF in
these regions correlate directly with cognitive impairments. In this
study, the Gi rats exhibited declined regional CBF and impaired
learning and memory abilities; most of them had trouble finding
the platform in the Morris water maze tasks. Acupuncture treat-
ment could significantly improve the cognitive abilities of the Ga
rats, making their searching process more spatially precise and
efficient. However, sham-acupuncture had little or no effect on
any of the neurobehavioral parameters analyzed, indicating that
the acupuncture points were of therapeutic specificity.
Reactive oxygen species (ROS), such as superoxide anion, are
generated from mitochondrial electron transport chain and have
been implicated in the pathogenesis of stroke. Increased levels of
ROS are a major cause of tissue injury after cerebral ischemia, in
which there is an overproduction of ROS, inactivation of antioxi-
dant enzymes, and consumption of antioxidants, so natural de-
fense mechanisms fail to protect neurons from oxidative damage
(Loh et al., 2006). Endogenous antioxidants such as superoxide dis-
mutases (SOD), glutathione peroxidase and glutathione could con-
trol ROS levels (Sugawara and Chan, 2003). Some studies have been
demonstrated the impact of ROS on cognitive function by studies
of SOD isoenzyme transgenic mice. Overexpression of mitochon-
drial SOD has a neuroprotective role against drug-induced neuro-
toxicity, overexpression of cytoplasmic SOD improves age-related
impairments in LTP, and overexpression of extracellular SOD is cor-
related with better spatial memory in laboratory rats (Hu et al.,
2006). In this study, we found significantly increased levels of
superoxide anion and MDA in mitochondria of the Gi rats, indicting
that overproduction of ROS had caused mitochondria damage after
ischemia by participating in destruction of mitochondrial mem-
brane lipids. Moreover, the declined activities of total SOD, CuZn-
SOD and MnSOD, and altered GSH metabolism were unable to
prevent the elevated oxidative stress, which further accentuated
mitochondrial abnormalities of the Gi rats. Acupuncture treatment
could reduce mitochondrial oxidative stress damage by increasing
SOD activities and regulating GSH pool, indicating acupuncture
might be a useful tool to maintain mitochondrial homeostasis
and improve endogenous antioxidant defense.
Altered activities of respiratory enzymes and aerobic O2 utiliza-
tion are critical processes for ROS production under physiological
conditions (Raza et al., 2011). Production of ROS is markedly en-
hanced in many pathological conditions in which the respiratory
chain is impaired. In this study, we found that the activities of
mitochondrial respiratory complex enzymes (complex I, II and
IV) were increased in the Ga rats, which indicated an excessive
electron flow in the mitochondria. This increase might presumably
due to improved cerebral blood flow, which could offer adequate
substrates (oxygen and glucose). There are several studies sug-
gested that altered respiratory complex activities led to increased
production of ROS. Complex I has been reported to be directly in-
volved in ROS production, which is dependent on a number of fac-
tors, including the number of carriers, electron supply and release
of electrons from each carrier. These factors, in turn, are affected by
the biological state of tissues, respiratory rate, reverse electron
flow, inner membrane potential, oxidation of mitochondrial gluta-
thione and post-translational modification of the complexes (Meh-
ta et al., 2012). Under hypoxic conditions or ischemia, a reduced
complex IV activity augments the production of ROS. Studies have
also shown that alteration in mitochondrial GSH and ROS plays an
important role in regulating the activities of respiratory complexes
under oxidative stress conditions (Atif et al., 2009).
Neurons are particularly dependent on mitochondrial energy
production (Graham and Chen, 2001). Mitochondria are closely
associated with synapses and tethered to vesicle release sites
(Rowland et al., 2000). Synaptic transmission requires mitochon-
drial ATP generation and control of local [Ca2+]i for neurotransmit-
ter exocytosis, vesicle recruitment, activation of ion conductances,
signaling at metabotropic receptors, potentiation of neurotrans-
mitter release, and synaptic plasticity (Chang et al., 2006). Mito-
chondria dysfunction following cerebral ischemia can not meet
the energy demands for neuronal activity, resulting to synaptic
impairment and degeneration. In this study, a decrease in RCI
and P/O ratio suggested altered bioenergetics in the mitochondria
of the Gi rats, which presumably due to altered oxygen metabolism
by respiratory complexes and increased production of ROS. Acu-
puncture treatment could improve mitochondrial efficiency of oxi-
dative phosphorylation, ameliorate hypoxia-induced suppression
to enzyme activities of respiratory complexes and then compen-
sate energy deficit of neurons.
To maintain normal brain function, cells depend on a steady
supply of energy substrates, so inadequate supply of glucose and
inefficient glucose utilization may cause neuronal damage, even
lead to cognitive function decline. GLUT1 is the main glucose trans-
porter in the brain. GLUT1 (55 kD) primarily expresses in the cap-
illary endothelial cells of the BBB and is responsible for glucose
transport across the BBB (Tanaka et al., 2010). The expression of
GLUT1 is regulated not only by blood glucose level to maintain a
constant supply of energy substrates, but also by hypoxia–ische-
mia in the brain. Under normal conditions, oxidative phosphoryla-
tion is the rate-limiting step for converting the glucose into energy
due to sufficient supply of oxygen and glucose; whereas under
pathologic conditions, glucose transport is the rate limiting step
for glucose utilization. In this study, acupuncture treatment
slightly increased GLUT1 expression level in the cortex of the Ga
rats compared to the Gi rats, but this difference did not reach sta-
tistical significance, indicating acupuncture could not enhance
brain glucose transport.
In summary, this study demonstrated that acupuncture treat-
ment could improve cognitive performance of MID rats; and this
improvement might be due to an increased supply of glucose and
 X. Zhang et al. / Neurochemistry International 65 (2014) 23–29
oxygen to neural cells mediated by increased CBF, which amelio-
rated mitochondrial dysfunction induced by ischemia and endoge-
nous oxidative stress system of brain. The sham-acupuncture
stimulation did not show the same effect, indicating that the acu-
puncture points were of therapeutic specificity.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (No. 81202739 and No. 81072882) and the Special-
ized Research Fund of the Doctoral Program of Higher Education
(20111210120004).
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