DOI:10.1142/S0219635210002433 3
ELECTROACUPUNCTURE AT BAIHUI ACUPOINT (GV20)
4
REVERSES BEHAVIOR DEFICIT AND LONG-TERM
5
POTENTIATION THROUGH N-METHYL-D-ASPARTATE
6
AND TRANSIENT RECEPTOR POTENTIAL VANILLOID
7
SUBTYPE 1 RECEPTORS IN MIDDLE CEREBRAL
8
ARTERY OCCLUSION RATS
9
YI-WEN LIN†,‡ and CHING-LIANG HSIEH†,‡,§,∗,¶
10
†Graduate Institute of Acupuncture Science 11
China Medical University, Taichung, Taiwan 12
‡Acupuncture Research Center 13
China Medical University, Taichung, Taiwan 14
§Department of Chinese Medicine 15
China Medical University Hospital, Taichung, Taiwan 16 ¶[email protected] 17 Received 18 Accepted 19
Vascular dementia is one of the most important causes that account for 20–40% of all
20
dementia cases. The aim of this study was to investigate whether electroacupuncture can
21
reduce behavior deficit and long-term potentiation (LTP) in vascular dementia. Here we
22
used a middle cerebral artery occlusion (MCAo) technique to induce a vascular dementia
23
model with additional electroacupuncture (EA) manipulation. Behaviors were impaired in
24
animals with MCAo, and similar results were observed with long-term potentiation
induc-25
tion. MCAo decreased the expression of LTP from 180.4±14.9% to 112.5±18.3%,
suggest-26
ing that cerebral ischemia could impair the hippocampal LTP. In addition, immunostaining
27
results showed that the expressions of N-methyl-d-aspartate receptor subtype 1 (NR1)
28
and transient receptor potential vanilloid subtype 1 (TRPV1) receptors were significantly
29
increased in the hippocampal CA1 areas. Noticeably, these phenomena can be reversed
30
by 2 Hz EA at Baihui acupoint (GV20) for six consecutive days. Our results support a
31
rescue role of 2 Hz EA for MCAo-induced behavior and LTP impairment. These results
32
also suggest that NMDAR1 and TRPV1 may be involved in this pathway.
33
Keywords: Stroke; acupuncture; hippocampus; Traditional Chinese Medicine; NMDA; 34
LTP.
35
1. Introduction
36
Neurodegenerative diseases resulting from stroke, ischemia and brain trauma
37
are characterized by the presence of extremely high glutamate concentration or
38
∗Corresponding author. Graduate Institute of Acupuncture Science, and Acupuncture Research Center, China Medical University. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, R. O. C.
N-methyl-d-aspartate (NMDA) subtype glutamate receptors in the brain [4, 14]. The
1
majority of excitotoxic cascade is due to the overactivation of N-methyl-d-aspartate
2
(NMDA) receptors, which in turn causes abnormal Ca2+ influx. Na+-dependent 3
glutamate transporters, which are located on nerve terminals and astrocytes, can
4
modulate the concentration of excess glutamate to reduce neuronal excitotoxicity [9,
5
34]. Glutamate transporters, which are located in the brain vascular, may play a key
6
role in controlling extracellular glutamate levels through a brain-to-blood glutamate
7
efflux [11, 29, 36].
8
The potency of neuronal transmission in the central nervous system (CNS) can
9
undergo noticeable usage-dependent changes. These phenomena are usually
repre-10
sented as long-term potentiation (LTP) [2, 3, 24] and long-term depression (LTD)
11
[1, 10, 17] of synaptic efficacy. LTP can be induced with high-frequency
stimu-12
lation (HFS) or low-frequency stimulation (LFS) paired with postsynaptic
mem-13
brane potentiation [12, 41]. In contrast, LTD can be induced with relatively low
14
frequency stimulation from 1 to 3 Hz, presynaptic LFS with small postsynaptic
15
potentiation, or two independent pathways paired within a narrow period of time
16
[20, 23]. Interestingly, it is known that induction of both LTP and LTD requires
17
activation of the NMDA receptors [10, 28].
18
Transient receptor potential vanilloid subtype 1 (TRPV1) was first identified as
19
capsaicin receptor, the biting element in peppers. It is a non-selective cation channel
20
expressed mainly in sensory neurons. TRPV1 is involved in both pronociceptive and
21
protective roles in many inflammatory and neuropathic pain syndromes [15]. Recent
22
reports suggest that TRPV1 may be sensitized and upregulated during
inflamma-23
tion and neuropathic pain. It is present not only in primary sensory neurons but
24
also in brain and non-neuronal tissues, such as the urothelium, alveolar cells, mast
25
cells, fibroblasts, and smooth muscle. TRPV1 is usually recommended for sensing
26
heat, pain, learning and mechanical stimuli [18, 38]. It has also been observed in
27
the hippocampus and is considered an important factor in maintaining the
expres-28
sion of LTP [19, 27]. Recent studies have shown that TRPV1 may be involved in
29
neurotoxicity due to the function of calcium permeable characterization [15].
30
Acupuncture is an ancient Chinese method used to treat diseases, and has been
31
documented in Traditional Chinese Medicine (TCM) literature for more than 3000
32
years. It is widely used to treat several diseases including stroke [21]. Low- and
33
high-frequency electroacupuncture (EA) stimulation selectively induces the release
34
of enkephalins and dynorphins to reduce pain sensation [37]. EA on Baihui acupoint
35
(GV20) can improve syndromes in stroke patients [21], reduce post-stroke anxiety
36
[40], reduce cerebral infarct accompanied dopamine increase [7], as well as restore
37
learning and memory impairment in diabetes mellitus (DM) and cerebral ischemia
38
in rats [13].
39
It is well known that stroke-induced cerebral ischemia can induce deficits in
40
long-term potentiation expression in the hippocampus CA1 areas. Our hypothesis is
41
that stroke-induced cerebral ischemia can increase the excitotoxicity by increasing
42
NMDA and TRPV1 receptors, and EA can restore the deficit of behavior and LTP
induction. To verify our hypothesis, we induced a stroke model with middle cerebral
1
artery occlusion (MCAo) and found that the stroke-induced impairment of behavior
2
and LTP induction can be repaired by EA at Baihui acupoint. These processes
3
were accompanied by changes in the expression levels of NMDA receptor 1 and
4
TRPV1 receptors. This implies that NMDAR1 and TRPV1 may be involved in
5
stroke-induced behavior and LTP induction.
6
2. Materials and Methods
7
2.1. Animals
8
A total of 48 male Sprague-Dawley rats weighing between 300 g and 350 g were used
9
in this study. Rats were fasted overnight with free access to water. Usage of these
10
animals was approved by the Institute Animal Care and Use Committee of China
11
Medical University and guidelines in The Guide for the Use of Laboratory Animals
12
(National Academy Press) were followed.
13
2.2. Establishment of MCAo model
14
The MCAo model was established in the SD rats using an intraluminal suture
15
method as previously described [22]. The right common carotid artery (CCA) and
16
the internal carotid artery (ICA) were exposed through a neck midline incision,
fol-17
lowed by ligation of the pterygopalatine artery close to its branch under
anestheti-18
zation with chloral hydrate (400 mg/kg, i.p.). A 3-0 nylon filament suture, blunted
19
at the tip by a flame and coated with poly-L-lysine (Sigma, USA), was inserted into
20
the right external carotid artery (ECA) through the CCA and up to the ICA, a
21
distance of 20–25 mm, to block the origin of right middle cerebral artery (MCA).
22
The suture was removed slowly to re-establish blood flow 10 minutes after MCAo.
23
2.3. Preparation of electrodes
24
The head of the rat was fixed in a stereotactic apparatus in a prone position 10
min-25
utes after MCAo. The scalp was incised from midline and the skull and neck muscle
26
were exposed. The electrodes were implanted and fixed on the Baihui acupoint which
27
is located at the midmost point of parietal bone. The needles were inserted into the
28
muscle at a depth of 0.5 mm of Baihui acupoint to serve as a cathode, while another
29
electrode was placed on the neck muscle, serving as an anode. Electrical square
30
pulses were delivered for 20 minutes with a 100µs in duration and 2 Hz in frequency
31
generated from the (Trio 300, MDSS GmbH, Burckhardtstr. 1D-30163, Hannover,
32
Germany) stimulator, at an amplitude of 2 mA.
33
2.4. Grouping
34
Rats were randomly divided into three groups of 3–4 rats each as follows: (1) sham
35
operation group: electrodes were implanted into the Baihui acupoint and neck muscle
without electrical stimulation or MCAo; (2) model group: MCAo was induced for
1
10 minutes followed by reperfusion; (3) EA group: the method was identical to
2
that in the model group, but 2 Hz electrical stimulation was applied at the Baihui
3
acupoint for 20 minutes, beginning 24 hours after reperfusion and continuing for six
4
days in awake and free movement states. The intensity of stimulus was based on the
5
slightly visible muscle twitch.
6
2.5. Measurement of neurological severity score
7
The behavior status of each rat was measured at 24 hours (24 h) and at seven days
8
(D7) after reperfusion by an investigator blinded to the treatment group. Motor,
9
sensory, balance and reflex functions were assessed based on a behavior deficit score
10
(18-point scale) as previously described [5]. Briefly, motor tests: with the rat on the
11
floor, an inability to walk straight was scored as 1, circling toward the paretic side
12
was scored as 2 and falling down to the paretic side was scored as 3; raising each
13
rat by its tail, flexion of the forelimb was scored as 1, flexion of the hindlimb was
14
scored as 1 and head moving> 10◦ was scored as 1.
15
Sensory tests: tactile deficiency was scored as 1 while pushing paw against table
16
edge subtest deficiency was scored as 1. Ability to balance on the beam: rats grasping
17
the side of the beam was scored as 1, hugging the beam and one limb slipping off the
18
beam was scored as 2; hugging the beam and two limbs slipping off the beam was
19
scored 3, attempting to balance but falling off (> 40 sec) was scored as 4, attempting
20
to balance but falling off (< 20 sec) was scored as 5, falling off the beam without
21
attempting to balance was scored as 6. Reflex tests: pinna reflex deficiency was
22
scored as 1, corneal reflex deficiency was scored as score 1, and startle reflex subtest
23
deficiency was scored as 1; rat experiencing a seizure was also scored as 1.
24
2.6. Immunostaining
25
The animals were deeply anaesthetized with chloral hydrate (400 mg/kg, i.p.), and
26
perfused with normal saline via the cardiac vascular system followed by a fixative
27
containing 4% paraformaldehyde (Merck, Frankfurt, Germany) and in 0.1 M
phos-28
phate buffer saline (PBS, pH = 7.4). The brains were removed and put in the same
29
fixative overnight at 4◦C. After a brief wash with PBS, the brains were transferred
30
to 30% sucrose in 0.01 M PB for cryoprotection and the coronal sections containing
31
the hippocampal area were cut into 20µm thickness using a frozen sectioning
tech-32
nique. The sections were then preincubated (2 hours, 25◦C) with 10% horse serum
33
and 0.3% Triton X-100 in phosphate-buffered saline (PBS) to avoid non-specific
34
binding. Sections were then incubated overnight at 4◦C with a mixture of rat
mono-35
clonal antibody against NR1 (1:200; Chemicon, Temecula, USA and TRPV1 (1:500;
36
Chemicon, Temecula, USA), 0.1% horse serum, and 0.1% Triton X-100 in PBS.
37
Sections were subsequently incubated (2 hours, 25◦C) with biotinylated-conjugated
38
secondary antibody (1:200 diluted; Vector, Burlingame, USA), followed by
incuba-39
tion with avidin-horseradish peroxidase complex (ABC-Elite, Vector), and finally
were visualized with 3,3-diaminobenzidine as a chromogen. Sections were washed
1
with PBS between incubation steps three times for 10 minutes each time.
2
2.7. Electrophysiology
3
Adult male SD rats were anesthetized with halothane and decapitated. The brains
4
were quickly removed and placed in ice-cold artificial CSF (ACSF) containing the
5
following (in mM): 119 NaCl, 2.5 KCl, 26.2 NaHCO3, 1 NaH2PO4, 1.3 MgSO4, 2.5 6
CaCl2, and 11 glucose (the pH was adjusted to 7.4 by gassing with 5% CO2 and 95% 7
O2). Transverse hippocampal slices (450µm thick) were cut with a vibrating tissue 8
slicer (Campden Instruments, Loughborough, UK) and transferred to an
interface-9
type holding chamber at room temperature (25◦C). The slices were recovered for
10
at least 90 minutes and then transferred to an immersion-type recording chamber,
11
perfused at 2 ml/min with ACSF containing 100µM picrotoxin at room
tempera-12
ture. The border between the CA1 and CA3 areas was cut to prevent epileptiform
13
discharge of pyramidal neurons. For extracellular field potential recording, a glass
14
pipette filled with 3 M NaCl was positioned in the stratum radiatum of the CA1
15
area and the field excitatory post-synaptic potential (fEPSP) was recorded. Bipolar
16
stainless steel stimulating electrodes (Frederick Haer Company, Bowdoinham, ME)
17
were placed in the striatum radiatum to stimulate Schaffer collateral branches. The
18
fEPSP was elicited by adjusting the intensity of stimulation to about 40–50% of
19
the maximum response, with population spikes after the fEPSP began to appear.
20
Stable baseline fEPSP activity was recorded by applying a short-duration voltage
21
pulse (∼1 msec) at the determined intensity every 30 seconds for at least 10 minutes.
22
High-frequency stimulations were used to induce LTP expression with three trails
23
of 100 Hz at 30-second intervals. All signals were filtered at 2 kHz using the low-pass
24
Bessel filter provided with the amplifier and digitized at 5 kHz using a CED micro
25
1401 interface running Signal software (Cambridge Electronic Design, Cambridge,
26
UK). All drugs were purchased from Sigma (St. Louis, MO, USA). The initial slopes
27
of the fEPSP were measured for data analysis. Synaptic responses were normalized
28
to the average of the baseline. The average size of the slope of the fEPSPs recorded
29
40–50 minutes after 100 Hz stimulation was used for statistical comparisons. All
30
data are presented as the mean ± standard error. Statistical significance among
31
sham, stroke, and EA-treated slices was tested using the Mann–Whitney U test,
32
with p < 0.05 considered statistically significant.
33
3. Results
34
3.1. Effect of 2 Hz EA at Baihui acupoint on behavior
35
deficit in MCAo rats
36
In the sham operation group, animals did not show any deficit with respect to
behav-37
ior scores. In contrast, animals that received MCAo showed deficits with respect
38
to behavior, including inability to walk straight, circling toward the paretic side,
Fig. 1. Relationship of different manipulation and neurological scores in subgroups. ∗∗denotes
p < 0.01, by comparing MCAo with EA at D7.##denotes p < 0.01, by comparing 24 h and D7 with EA manipulation.
falling down to the paretic side and flexion of the forelimb; the scores were 7 and
1
6.25 ± 0.25 for 24 hours and D7, respectively. Interestingly, the abnormal behavior
2
can be improved by 2 Hz EA at the Baihui acupoint for six continuous days, from
3
scores of 6.25 ± 0.25 to 3.75 ± 0.25. This implies that the 2 Hz EA treatment was
4
effective to suppress the deficit of behaviors caused by MCAo in rats. All the data
5
are presented as a bar chart in Fig. 1.
6
3.2. MCAo-induced deficit of LTP was improved by 2 Hz EA
7
at Baihui acupoint
8
To investigate the effect of MCAo on the expression of LTP, we first tried to confirm
9
whether LTP can be induced in slices taken from MCAo animals. Figure 2(a) shows
10
LTPs induced in slices taken from sham control (solid circles), MCAo (open circles)
11
and 2 Hz EA-treated animals (open triangle). In the sham control group, LTP can be
12
induced successfully in hippocampal CA1 areas with a brief 100 Hz high-frequency
13
stimulation (180.4 ± 14.9%, n = 8, paired t-test). In contrast, LTP could not be
14
induced in hippocampal CA1 areas taken from MCAo-treatment slices (112.5 ± 17.3,
15
n = 9, paired t-test). Interestingly, LTP can be restored and successfully induced in
16
CA1 areas from slices taken from MCAo animals treated by 2 Hz EA (167.1 ± 10.5,
17
n = 9, paired t-test). These results suggest that MCAo treatment per se can depress
18
the induction of LTP in hippocampal slices. Furthermore, EA treatment can prevent
19
a deficit in LTP expression from MCAo treated hippocampal slices.
20
3.3. MCAo-induced ischemia did not influence basal synaptic
21
transmission and presynaptic glutamate release
22
The basal synaptic properties were tested to evaluate the effect of MCAo on
Schaf-23
fer collateral-CA1 synaptic transmission. Input–output curves were confirmed by
(a) (b)
(c) (d)
Fig. 2. LTPs induced in sham (solid circles), MCAo (open circles) and EA (open triangle) pre-treated slices (a). In sham group, LTP can be successfully induced with 100 Hz HFS. In contrast, LTPs were failed to be induced in slices taken from MCAo. In EA group, LTPs can be reversely induced in slices from MCAo treated rats. Inserts are representative basal and LTP-induced fEPSP tracings. Traces 1 and 2 are recordings from a sham slice, traces 3 and 4 are from a MCAo pretreated slice, while traces 5 and 6 are recordings from an EA slice. Each tracing represents the compos-ite of 10 sweeps. (b). Quantitative analysis of LTP responses in slices from sham, MCAo and EA subgroups.∗∗denotes p < 0.01, by comparing sham with MCAo groups.##denotesp < 0.01, by comparing MCAo with EA groups. (c). Input/output (I/O) ratio curves of EPSP amplitudes in the sham group (solid circle), MCAo (open circle) and EA (open triangle) groups. (d). Relationships of paired-pulse facilitation ratio and inter-pulse intervals. Paired-pulse stimulation from 25 to 100 ms intervals was applied to the Schaffer collateral branches.
plotting a graph with the fEPSP amplitude against increasing intensities of
stimula-1
tion ranging from 3 to 24 V, as shown in Fig. 2(c). There is no significant difference
2
between the input–output curves of the sham, MCAo and 2 Hz EA groups,
sug-3
gesting that the basal synaptic transmission of CA1 neurons and synapses was not
4
affected by the MCAo and 2 Hz EA treatment. Moreover, the paired-pulse ratio was
5
measured with interpulse intervals of 25, 50, 75 and 100 ms in each group. The results
6
showed that the MCAo and 2 Hz EA treatment did not destroy the probability of
presynaptic glutamate release because there was no significant difference between
1
the groups (p > 0.05, Fig. 2(d)).
2
3.4. 2 Hz EA at the Baihui acupoint can reverse the extreme
3
overexpression of NR1 and TRPV1 in MCAo rats
4
To investigate the effect of MCAo on hippocampal cell death, we first examined the
5
neuron density using hematoxylin and eosin (HE) staining through the hippocampal
6
areas. The hippocampal neurons showed homogenous immunostaining with HE in
7
the sham (Fig. 3(a)), MCAo (Fig. 3(b)), and EA groups (Fig. 3(c)). These results
8
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Fig. 3. Immunohistochemistry staining of HE, NR1, and TRPV1 in hippocampal slices from sham, MCAo and EA pretreated groups. (a–c). HE staining (blue) of Sham, MCAo, and EA groups. (d–f). NR1 (brown) imaging of hippocampus at D7 after pretreatments was pictured with 400× magnifi-cation (g–i). TRPV1 (brown) imaging of hippocampus at D7 after pretreatments was pictured with 400× magnification.
indicated that MCAo did not induce cell death. Furthermore, we used a specific
1
NR1 antibody to mark hippocampal slices in the three groups. In the sham group,
2
hippocampal neurons exhibited low density with NR1 staining in the hippocampal
3
CA1 areas (Fig. 3(d), indicated with arrows). In slices taken from MCAo animals,
4
the NR1 was highly expressed in the hippocampal CA1 areas (Fig. 3(e), indicated
5
with arrows). Importantly, in slices taken from 2 Hz EA-treated animals, the extreme
6
increase was rescued to sham control conditions, as shown in Fig. 3(f) (indicated with
7
arrows). We also wanted to investigate the role of TRPV1 in MCAo-induced cerebral
8
ischemia. In the sham control group, neurons in the hippocampus show relatively
9
low density with TRPV1 immunostaining in the hippocampal CA1 areas (Fig. 3(g),
10
indicated with arrows). In contrast, in slices from MCAo animals, TRPV1 was
11
relatively high in the hippocampus especially in the CA1 areas (Fig. 3(h), indicated
12
with arrows). Significantly, in the EA group, the extreme increase in TRPV1 was also
13
rescued to basal conditions as shown in Fig. 3(i). These results indicated that the
14
MCAo treatment increases both NR1 and TRPV1 expressions and the phenomenon
15
can be reduced with 2 Hz EA treatment.
16
4. Discussion
17
In this study, we tested the hypothesis that MCAo treatment can induce
behav-18
ior impairment. It induced mild to moderate behavior dysfunction with a score of
19
approximately 7. Interestingly, 2 Hz EA treatment can reduce these dysfunctions
20
seven days after reperfusion. Consistent with previous reports, we perceived that
21
LTP induction can be impaired with MCAo treatment. Notably, 2 Hz EA at the
22
Baihui acupoint for six continuous days can reduce the deficit of LTP. The IO
23
curves and paired-pulse ratio did not change with MCAo or 2 Hz EA treatment,
24
which means that these manipulations did not alter the basal synaptic transmission
25
or presynaptic glutamate release. Finally, we examined NR1 and TRPV1 expression
26
under MCAo treatment. The results showed that both NR1 and TRPV1 increased
27
significantly with MCAo treatment, supporting its use for this condition.
Further-28
more, 2 Hz EA at the Baihui acupoint can reverse this phenomenon at seven days
29
after reperfusion. To our knowledge, this study represents the first demonstration
30
that 2 Hz EA at the Baihui acupoint can reduce the dysfunction evoked by MCAo
31
treatment, including behavior and LTP impairment, suggesting that NMDAR1 and
32
TRPV1 may be involved in this pathway.
33
A previous study showed that two-vessel occlusion, a stroke-induced model, can
34
successfully induce global cerebral ischemia and an impairment of long-term
potenti-35
ation (LTP) expression with no alteration of basal synaptic transmission and paired
36
pulse facilitation in the hippocampal CA1 areas. This transient brain hypoperfusion
37
was light and did not induce histological changes in cell death with cresyl violet
38
immunostaining [26]. In contrast, four-vessel occlusion, the density of CA1
pyrami-39
dal neurons decreased significantly when observed with histological staining. Basal
40
synaptic transmission also decreased and showed a deficit of LTP induction [8].
The MCAo model in this study was similar to the two-vessel occlusion model as
1
they both have normal basal synaptic transmission and paired pulse facilitation
2
but not cell death. This demonstrated that 2 Hz EA manipulation can reverse the
3
MCAo-induced of mild to moderate stroke.
4
Recent study suggested that EA can improve learning and memory in
experimen-5
tally impaired DM rats with cerebral ischemia. The study utilized passive avoidance
6
test, active avoidance test and Morris water maze to ensure the effective role of
7
EA on dementia rats. In addition, it was also demonstrated that EA treatment can
8
rescue the LTP expression impaired by both DM and cerebral ischemia. The EA
9
treatment was given to each conscious rat once per day for 30 consecutive days at
10
Baihui and bilateral Zusanli acupoint [13]. Here, we demonstrated that EA at Baihui
11
acupoint alone can restore MCAo-induced behavior and LTP impairment at day 7
12
after treatment. We suggested that this maybe due to the different dementia models
13
and EA manipulation methods.
14
Selective blockages of NR2B inhibited the induction of post-ischemic LTP in
15
ischemia model but did not affect physiological LTP. They suggested that
ischemia-16
induced excitotoxicity is a critical mechanism for neuronal death and could be
17
restored by blockage of NR2B [30]. They also demonstrated that the NR2B was
18
reduced at 48 hours after MCAo manipulation by correcting its assembly to the
19
NMDA receptors at synapses [30]. In this study, we suggest that 2 Hz EA at the
20
Baihui acupoint can reduce the deficit effect of behavior and LTP. This process did
21
not influence the basal synaptic transmission and presynaptic glutamate release.
22
We also found that the NR1 subunit increased significantly during MCAo-induced
23
ischemia and can be reversed with EA. This is in agreement with several
stud-24
ies which show that chronic blockade of NMDA receptors can induce a significant
25
reversible increase in NMDA receptor clusters [32].
26
The increase of glutamate release or glutamate receptors is critical for LTP
induc-27
tion in mammalian hippocampal CA3 and CA1 areas, respectively [31, 33, 35]. The
28
glutamate release from presynaptic vesicles would bind to postsynaptic receptors
29
to cause Ca2+ influx and switch on Ca2+-dependent signal transduction, including 30
calcium-calmodulin kinase II and protein kinases. However, an extremely elevated
31
glutamate may be induced in many pathological conditions such as trauma, stroke
32
and ischemia. It is well understood that a decrease in brain glutamate can remove
33
excitotoxicity and neuronal damage. A new approach to solving these questions is to
34
apply the glutamate scavenger oxaloacetate to decrease the content of glutamate in
35
the CSF. Previous studies support the assertion that the impaired LTP induced by
36
two-vessel occlusion ischemia can be reduced by glutamate scavenger oxaloacetate
37
[26]. In addition, EA is also known to be effective in ischemia treatment by reducing
38
glutamate neuroexcitotoxicity to decrease neuronal cell death. EA can decrease in
39
ischemic and reperfusion of cerebral blood flow than control rats in the ischemic
40
model of diabetic mellitus [6].
41
TRPV1, also known as vanilloid receptor 1 (VR1), is a noxious heat-sensitive
42
non-selective cation channel that is permeable to calcium. TRPV1 can be activated
by various ligands, including capsaicin (CAP), anandamide (AEA) and thermal
lig-1
ands [25, 38, 39]. The TRPV1 receptor is widely expressed in the brain, including
2
the hippocampus region, and plays important roles in pain, learning memory, LTP
3
induction and neurotoxicity [15, 19, 25]. However, whether or not TRPV1 receptor
4
mediates neuroprotection or neurotoxicity remains unknown. The TRPV1
recep-5
tor has a neuroprotective effect on global cerebral ischemia [42]. However, injection
6
of CAP into the rat brain resulted in cell death of dopaminergic (DA) neurons,
7
visualized with immunostaining. This phenomenon was also observed in vivo by
8
capsazepine (CZP), the antagonist of TRPV1, suggesting the role of TRPV1 in
9
neurotoxicity [16]. The degeneration of DA neurons was due to an increase in
intra-10
cellular Ca2+, mitochondrial damage and neuronal death. Our results suggest that
11
TRPV1 is highly increased in MCAo-induced ischemia. The extreme increase of
12
TRPV1 was observed in CA1 pyramidal neurons and can be reversed with EA
13
manipulation.
14
In this study, we report that MCAo-induced cerebral ischemia can induce
behav-15
ior and LTP impairment. This phenomenon is accompanied by overexpression of
16
NR1 and TRPV1 receptors. EA of 2 Hz at the Baihui acupoint can reverse the
17
deficit of behavior and LTP via reversal of NR1- and TRPV1- mediated
neurotoxi-18
city. This implies that 2 Hz EA can successfully rescue vascular dementia and could
19
be applied in clinical medicine.
20
Acknowledgment
21
This study was supported in part by Taiwan Department of Health Clinical Trial
22
and Research Center of Excellence (DOH99-TD-B-111-004).
23
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