Mol. Cells, Vol. 25, No. 2, pp.

178-183

Molecules
and
Cells
KSMCB 2008

Molecular Changes in Remote Tissues Induced by

Electro-Acupuncture Stimulation at Acupoint ST36
Sam-Woong Rho1, Gi-Soon Choi2, Eun-Jung Ko1, Sun-Kwang Kim1, Young-Seop Lee1, Hye-Jung Lee1,
Moo-Chang Hong1, Min-Kyu Shin1, Byung-Il Min2, Hyun-Jung Kee3, Cheol-Koo Lee3,*, and Hyun-Su Bae1,*
1

College of Oriental Medicine, Kyunghee University, Seoul 130-701, Korea;

Department of East-West Medicine Graduate School, Kyunghee University, Seoul 130-701, Korea;
3
Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea.
2

(Received June 4, 2007, Accepted November 29, 2007)

To investigate the effects of electro-acupuncture (EA)

treatment on regions remote from the application, we
measured cellular, enzymatic, and transcriptional activities in various internal tissues of healthy rats. The
EA was applied to the well-identified acupoint ST36 of
the leg. After application, we measured the activity of
natural killer cells in the spleen, gene expression in the
hypothalamus, and the activities of antioxidative enzymes in the hypothalamus, liver and red blood cells.
The EA treatment increased natural killer cell activity
in the spleen by approximately 44%. It also induced
genes related to pain, including 5-Hydroxytryptamine
(serotonin) receptor 3a (Htr3a) and Endothelin receptor
type B (Ednrb) in the hypothalamus, and increased the
activity of superoxide dismutase in the hypothalamus,
liver, and red blood cells. These findings indicate that
EA mediates its effects through changes in cellular
activity, gene expression, and enzymatic activity in
multiple remote tissues. The sum of these alterations
may explain the beneficial effects of EA.
Keywords: Acupoint ST36; Electro-Acupuncture; Gene
Expression; Oxidative Stress; Reactive Oxygen Species;
Remote Tissues.

Introduction
Acupuncture has a long history, but its scientific evaluation has only begun rather recently. Successive EA stimu* To whom correspondences should be addressed.
Tel: 82-2-961-0323; Fax: 82-2-962-9316
E-mail: hbae@khu.ac.kr (HSB)
Tel: 82-2-3290-3008; Fax: 82-2-953-0737
E-mail: cklee2005@korea.ac.kr (CKL)

lation increases the activity of splenic natural killer (NK)

cells in rats (Sato et al., 1996). An increase of endogenous
cytokines, such as interleukin-2 and interferon-gamma,
was suggested as the possible mechanism of this splenic
NK cell activation (Yu et al., 1997). At the transcriptional
level, Kim et al. proposed up-regulation of CD94, Protein
tyrosine kinase (PTK) and Vascular cell adhesion molecule-1 (VCAM-1) genes, and down-regulation of Protein
tyrosine phosphatase (PTP) and Tyrosine phosphatase-1
(SHP-1) genes as transcriptional changes responsible for
the NK cell activation (Kim et al., 2005a). According to
clinical studies with human subjects, acupuncture alleviates pain and improves knee joint function in osteoarthritis patients (Witt et al., 2005), though the molecular basis
of these changes is not clear. A functional magnetic resonance imaging (fMRI) study showed that acupuncture at
ST36 (Zusanli), a major acupoint in the leg, modulated
the neural activity of the human central nervous system
(Hui et al., 2005). Other fMRI studies in animals have
also demonstrated that acupuncture directly increases
brain activity, especially in the region of the hypothalamus (HT) (Chiu et al., 2001; 2003). These findings suggest that acupuncture modulates various brain functions in
the area of the HT, which is itself a center for descending
pain-control and endocrine-immune modulation. As shown
by these examples, acupuncture treatment has been applied
for a wide range of symptoms and has produced some
benefits.
Previous studies showed that acupuncture or EA treatment can affect several major molecular events such as
transcription and signaling. Therefore, it is important to
study the associated molecular events and search for robust molecular markers to evaluate its effectiveness for
each symptom. To better understand how EA affects variAbbreviation: EA, electro-acupuncture.

Sam-Woong Rho et al.

ous organs remote from the application site and causes molecular changes within them, we applied EA on a popular
acupoint called Zusanli in rats. This point is found on the
front of the leg, just below the knee, and is also known as
stomach meridian point #36 or ST36. Acupuncture treatment on this spot is known to improve digestive disorders,
anemia, fatigue, and general weakness. We found evidence
that the EA stimulation affected cellular activity, global
gene expression and enzymatic activity.

Materials and Methods

Rats, EA treatment and tissue preparation We used 12 Sprague-Dawley rats weighing 200230 g and from 6 to 8 weeks old.
The rats were maintained and killed according to the ethical
guidelines of the Committee on Animal Investigation of Kyung
Hee University. For EA stimulation, six randomly selected rats
were treated with an acupuncture needle (length: 30 mm, diameter: 0.25 mm, Dong Bang Acupuncture Co., Korea) at the
Zusanli spot (ST36), and 35 volts of 2-Hz electricity was applied through the pin from a pulse generator (Model PG 306,
Korea). The EA stimulation was carried out for 30 min by an
oriental medical doctor at one time each day for three days. At
the same time, the control group of rats (n = 6) were restrained
in holders without EA stimulation or with EA stimulation at a
non-acupoint. Both the control and EA-treated rats were killed
by cervical dislocation to dissect or collect the HT, SP, LV, and
RBCs. All tissues were either used immediately or quick-frozen
in liquid nitrogen and stored at 80C until use.
Measuring NK cell activity The cytotoxicity of active NK cells
was measured by a standard 4-hour 51Cr release assay (Brunner
et al., 1968). Briefly, we crushed the entire SP through a syringe
needle, filtered it through a cell strainer (Falcon, BD Biosciences, USA), and collected isolated splenic NK cells. One hundred l of NK cell suspension and 51Cr-labeled YAC-1 cell suspension (KCBL, Korea) containing 2 105 cells were mixed in
96-well U-bottom microtiter plates at 10:1, 30:1, and 100:1
ratios of effector to target (E:T). We co-cultured the cells at
37C with 5% CO2 for 4 h. The plates were then centrifuged at
400 g for 5 min, and 51Cr release (ECr-51) was measured with a
gamma scintillation counter (Hewlett-Packard, USA) in 100 l
of supernatant. The target cells were incubated under the same
conditions without effector cells to measure spontaneous 51Cr
release (SCr-51), and incubated in 1 N HCl to measure maximum
51
Cr release (MCr-51). Using these measurements, we calculated
the percent specific lysis (PSL) from the formula:
PSL = {(ECr-51 SCr-51)/(MCr-51 SCr-51)} 100.
Because a single PSL value is often misleading, we used the
LU (lytic unit)10, which is defined as the quantity of effector
cells needed to kill 10% of target cells. The LU10 was calculated
by linear regression of log E:T vs. PSL, and is reported per 106

179

effector cells.
Real-time reverse transcription-polymerase chain reaction
(RT-PCR) analysis To select possible candidate genes induced
by EA stimulation, we used the GeneChip Rat Neurobiology
U34 array (Affymetrix, USA) only once. This gene chip included probes of 1,263 genes encoding kinases, cell surface
receptors, cytokines, growth factors, and oncogenes selected by
academic and industrial neuroscientists as being relevant to the
study of neurobiology. We chose two HT tissues from the rats
restrained with and without EA. These rats showed the closest
NK cell activity to the average of each group. We followed the
protocols provided by the manufacturer for the gene chip assay
strictly. Based on the analysis of Affymetrix algorithms implemented with GeneChip Operating Software (GCOS), we identified 22 genes that increased more than 1.5-fold in expression in
the EA-treated HT. We randomly selected nine of these genes
and performed real-time RT-PCR on all 12 HT samples. DNase
treatment, first strand cDNA synthesis, and SYBR green I RTPCR were performed as described (Vandesompele et al., 2002).
Total RNA was extracted from the HT using TRIZOL reagent,
and DNase I (Invitrogen Life Technologies, USA) was used to
eliminate genomic DNA contamination. The first cDNA strand
was synthesized from 5 g of total RNA. Total RNA was mixed
with 0.5 g of oligo(dT)12-18 primer (Invitrogen Life Technologies, USA), and the mixture was heated at 70C for 10 min.
Then 0.5 mM dNTP mix, 10 mM DTT, and 200 U Superscript II
RNase H- reverse transcriptase were added on ice, and the reaction was carried out for 50 min at 42C. The mixture was diluted
four fold, and 2 l of the diluted solution was used for the realtime PCR performed with a Gene Amp 5700 Sequence Detection System (PE Biosystems, UK). Quantitative PCR reaction
was carried out in a final volume of 25 l. Each reaction contained 2 l cDNA, 12.5 l 2 SYBR Green PCR master mix
(Applied Biosystems, UK), 0.3 M forward primer, and 0.3 M
reverse primer. The real-time PCR was performed for 40 cycles
at 95C for 15 s and at 62C for 45 s after initial denaturation at
95C for 10 min. We performed three technical replicates for
each sample and six biological replicates for each gene. The list
of genes and primer sequences are listed in Table 1.
Measuring superoxide dismutase (SOD) and glutathione
peroxidase (GPx) activities, and production of reactive oxygen species (ROS) We measured total SOD and GPx activities
from HT, LV and RBCs. Fractionated RBCs were treated with
chloroform-ethanol (5:3) in 50 mM phosphate buffer (PBS). The
LV and HT were homogenized in 50 mM PBS. Each tissue extract was prepared by collecting the supernatant after centrifugation at 12,000 g for 40 min. SOD activity was measured by the
xanthine-xanthine oxidase-cytochrome c reduction method (Flohe
et al., 1988). This method measures cytochrome c reduced by
superoxide anions by the activity of xanthine oxidase. SOD
slows down the reduction of cytochrome c. Each reaction contained 50 l tissue extract, 1 ml cytochrome c solution containing 5 M xanthine (Sigma, USA), and 50 l 0.2 U xanthine

180

Effects of Electro-Acupuncture

Table 1. List of genes and primers for the real-time PCR analysis.
Gene*
Gapdh
Gsta5
Ednrb
Slc6a15
Htr3a
Mif
Sod1
Unknown (AF023087)
Avp
Sst

Primer sequences
(forward and reverse)
5-GGC ATG GAC TGT GGT CAT GA-3
5-TTC ACC ACC ATG GAG AAG GC-3
5-TGC CAA AAT CAA GGA CAA AG-3
5-GGC TGC CAG GCT GAA GAA ACT-3
5-TGA GTC TAT GTG CTC TGA GTA TTG ACA-3
5-ACC TAT GGC TTC AGG GAC AGC-3
5-ATA GCC TGT ACT TAT CTT TGG-3
5-TAA CCC TCG ATC ACA TCT GG-3
5-GAT GTG AGT CCT GCA TCC TG-3
5-GCC ATA ATC ACA GAA GTA CTG-3
5-CGC CCA GAA CCG CAA CTA CA-3
5-CCG GAA GGT GGC CAT CAT TAC-3
5-TTG GGC AAA GGT GGA AAT GA-3
5-CGG GAG GCT ATG GGT CTG-3
5-CCT CTC CGG GCT CCT CTA CCT A-3
5-GCT CCC CTC CCT TTG CTC TC-3
5-CTG CCG CGG GCA TCT GCT GCA G-3
5-GCT CAG TAG ACC CGG GGC TTG-3
5-GCT GGC CGC GCT CTG CAT CG-3
5-CTG GTT GGG CTC GGA CAG CAG-3

Size of PCR
product (bp)
236
243
161
162
159
160
160
160
202
160

* Abbreviations: Avp, Arginine vasopressin; Ednrb, Endothelin receptor type B; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Gsta5,
Glutathione S-transferase A5; Htr3a, 5-Hydroxytryptamine (serotonin) receptor 3a; Mif, Macrophage migration inhibitory factor; Slc6a15,
Solute carrier family 6 (neurotransmitter transporter), member 15; Sod1, Superoxide dismutase 1; Sst, Somatostatin.

oxidase (Sigma). Absorbance was read using a spectrophotometer at 550 nm. GPx activity was measured by a minor modification of the method of Flohe et al. (1973). Reaction mixtures
consisted of tissue extract, 0.1 M PBS, glutathione reductase
(Roche, Germany), and 10 mM glutathione (Calbiochem, USA).
After incubation at 37C for 10 min, NADPH was added and
incubation continued at 37C for 3 min. Then, t-butyl hydroperoxide (Sigma) was added and GPx activity was measured spectrophotometrically at 340 nm. The levels of ROS production in the
liver were determined by the oxidative conversion of nonfluorescent 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) to the
highly fluorescent 2,7-dichlorofluorescein (DCF) (Ali et al.,
1992). The tissue extracts (40 l) were incubated at 37C for 60
min with 50 l of 2 mM H2DCFDA (Sigma) in methanol. Fluorescence was then recorded at 485 and 527 nm for excitation
and emission, respectively, using a Fluoroscan Ascent FL (Type
374, Labsystems, Finland). In the experiment assaying ROS, the
NA-EA group (n = 6, EA stimulation at a non-acupoint located
on the proximal part of the tail) was used as a control for the
ST36-EA group to examine specifically the effect of an acupoint.

Results and Discussion

EA stimulation increases overall NK cell activity Consistent with previous observations (Kim et al., 2005a; Sato et

al., 1996), we observed that EA treatment activated NK

cells in the SP. We assessed NK cell activities by measuring
LU10 in six EA-treated rats and six control rats. The LU10
averages were 13.2 0.5 and 9.2 0.6, respectively. The
difference was statistically significant, and the EA stimulation increased NK cell activity by 43.5% (Fig. 1). Therefore, splenic NK cell activity may be a useful marker for
the effects of EA.
EA stimulation alters transcriptional activity A series of
fMRI studies showed that needling on acupoints elicited
hypermetabolic signals in the HT, and these effects were
exclusively associated with acupoint stimulation (Chiu et
al., 2003). To test whether the effects were associated with
any changes in transcription, we examined the gene expression profiles of HTs from a control rat and an EAstimulated rat using the Rat Neurobiology U34 array. We
detected 332 genes (26.3% of total) in the arrays, 66 of
which (19.9% of the detected genes) were either increased
or decreased in the EA-stimulated HT. We identified 22
candidate genes that were up-regulated more than 1.5-fold,
most of which could be categorized into synaptic function
and pain (data not shown). For quantitative RT-PCR
analysis, nine of the 22 genes were randomly selected:
Glutathione S-transferase A5 (Gsta5), Endothelin receptor type B (Ednrb), Solute carrier family 6 (neurotrans-

Sam-Woong Rho et al.

Fig. 1. EA stimulation increases NK cell activity. NK cell activity was measured in the SP from six rats in each group. Each bar
is the average with SEM (standard error of the mean). The LU10
difference between groups was statistically significant by t-test
(*** p value = 0.0004).

mitter transporter), member 15 (Slc6a15), Serotonin receptor 3a (Htr3a), Macrophage migration inhibitory factor (Mif), Superoxide dismutase 1 (Sod1), Arginine vasopressin (Avp), Somatostatin (Sst), and an unknown gene
AF023087. The real-time RT-PCR analysis was done using total RNA prepared from the HTs of six EAstimulated mice and six non-stimulated mice. Seven of
the nine genes were significantly up-regulated (p-value <
0.05) (Fig. 2). They were Gsta5 (4.4-fold), Ednrb (2.3fold), Slc6a15 (2.0-fold), Htr3a (2.4-fold), Mif (1.9-fold),
Sod1 (2.9-fold) and AF023087 (1.9-fold). The other two

181

genes, Avp and Sst, were up-regulated 1.3- and 1.4-fold,

respectively, although the changes were not statistically
significant. This evidence confirms that EA can affect
global gene expression in a remote tissue.
The up-regulated genes included pain modulators, such as
Htr3a and Ednrb. The protein encoded by Htr3 gene is
known to mediate transcutaneous electrical nerve stimulation
(TENS)-induced antihyperalgesia in rats (Radhakrishnan et
al., 2003). Therefore, induction of Htr3a might help to
reduce pain, and regulation of the receptors channel activity might be promising for screening useful chemicals
for pain modulation (Lee et al., 2005). The protein encoded by Ednrb is known to trigger an endogenous analgesic cascade at sites of peripheral injury mediated by
endothelin-1 (Edn1) (Khodorova et al., 2003). Edn1, a
pain mediator, could either trigger pain through endothelin-A (ETA) receptors (Pomonis et al., 2001) or produce analgesia through endothelin-B (ETB) receptors
(Khodorova et al., 2003). There are no probes for the ETA
receptor transcript on the array, but we saw no change in
Edn1 expression (data not shown). The induction of
Ednrb might also contribute to reducing pain. In addition,
there was induction of Chemokine (C-X3-C motif) ligand
1 Cx3cl1 (data not shown), and Cx3cl1 (also called fractalkine), which is known to enhance nociception in rats
(Milligan et al., 2004). It was interesting to observe this
kind of transcriptional event in the HT, because it may
also happen in organs such as the skin where the EA was
applied, because of the puncture damage.

Fig. 2. Nine of the genes up-regulated in the microarray analysis showed consistent trends in the real-time RT-PCR analysis in the HT.
The y-axis indicates the relative expression level of each gene normalized by GAPDH. A cross line indicates the mean value of six
observations. The mean value for the control group was scaled to 100. * p < 0.05, ** p <0.01, and *** p < 0.001.

182

Effects of Electro-Acupuncture

Fig. 4. Levels of ROS in the LV. The fluorescent intensities were

scaled to 100 for the control in each tissue. Control (n = 6): restraint without EA stimulation. ST36-EA (n = 6): EA stimulation
at the ST36 acupoint. NA-EA (n = 6): EA stimulation at the nonacupoint. * p < 0.05, between the indicated two groups by the
Newman-Keuls multiple comparison test following one-way
ANOVA.

Fig. 3. Enzymatic activities of SOD and GPx in the HT, LV and

RBCs. The activities were scaled to 100 for the control in each
tissue. Five replicates were measured, except in the HT control
for SOD (n = 4), RBCs control for SOD (n = 4), and LV EA
group for SOD (n = 3). * p < 0.05 and ** p < 0.01.

EA stimulation increases SOD activity and reduces

ROS production Both the mRNA and protein levels of
SOD and GPx in the hippocampus are increased by acupuncture treatment of damaged rat brains (Liu et al.,
2006). This is indirect evidence that acupuncture reduces
oxidative stress. To test whether the EA treatment also
reduces overall oxidative stress in normal rats by increasing the activities of the enzymes involved, we measured
SOD and GPx activities in the HT, LV, and RBCs. Interestingly, SOD activity was significantly increased by the
EA stimulation in these tissues, but GPx activity was unchanged (Fig. 3). SOD activity increased 1.9-fold in the
HT, 5.7-fold in the LV and 3.6-fold in the RBCs. This
suggests that the EA treatment enhances oxidative stress
resistance by activating the major antioxidative enzyme in
multiple tissues. The increase of SOD activity in the HT
is consistent with the transcriptional up-regulation of
SOD1. To determine whether EA stimulation indeed reduces ROS production, we measured ROS in the LV. The
ROS level was significantly reduced by the EA stimulation at the ST36 acupoint (28.2% decrease compared to
control), but not by EA stimulation at a non-acupoint (Fig.
4). Since these results are consistent with the results of
the measurements of SOD activity, they strongly suggest
that the EA treatment activates the antioxidative enzyme,

resulting in a decrease of oxidative stress. It also appears

that EA stimulation performed elsewhere on the body
does not produce the same effect as stimulation at a specific acupoint such as ST36 (Kim et al., 2005b; 2006).
Overall, our data suggest that EA stimulation can affect
major biological processes, including cellular activity,
transcription, and enzymatic activity. The sum of these
molecular changes could result in beneficial, or at least
neutral, effects on subjects. Therefore, it is important to
measure these different biological events to clarify the
mechanisms underlying acupuncture therapy. Also, the
altered genes could be robust biomarkers for evaluating
the effectiveness of EA treatment.

Acknowledgments This work was supported by grant from the

SRC/ERC program of Ministry of Science and Technology
(MOST)/Korea Science and Engineering Foundation (KOSEF)
R11-2005-014-01005-0 and from the Basic Research Program
of the Korea Science and Engineering Foundation No.2000-221300-009-3.

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