j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0

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Electroacupuncture ST36 prevents postoperative

intra-abdominal adhesions formation

Ming-Hua Du, MD,a,b Hong-Min Luo, MD,a,c Yi-Jun Tian, MD,a

Li-Jian Zhang, MD,a Zeng-Kai Zhao, MD,a Yi Lv, MD,a Rui-Jiang Xu, MD,b
and Sen Hu, MDa,*
a
Research Laboratory of Shock and Multiple Organ Dysfunction, Burns Institute, First Hospital Affiliated to the PLA
General Hospital, Beijing, China
b
Department of Pediatric Orthopedic Surgery, Chinese PLA General Hospital, Beijing, China
c
Department of Burns, Guangdong General Hospital, Guangzhou, China

article info abstract

Article history: Background: We have recently proved electroacupuncture (EA) ST36 exerted an anti-
Received 24 August 2014 inflammatory effect in the early phase of intra-abdominal adhesion formation. Evidences
Received in revised form indicate that the anti-inflammatory effect of EA ST36 involves a cholinergic anti-
4 December 2014 inflammatory pathway-dependent mechanism via the vagus nerve. However, the exact
Accepted 23 December 2014 effects and accurate vagal modulation of acupuncture in prevention of postoperative intra-
Available online xxx abdominal adhesion formation has not been thoroughly evaluated.
Materials and methods: SpragueeDawley rats subjected to abdominal adhesion lesions
Keywords: operation at the cecum and abdominal wall were randomly divided into six groups as
Abdominal adhesions follows: (a) EAN: EA non-channel acupoints; (b) EA: EA ST36 after abdominal lesions; (c)
Electroacupuncture VGX/EA: vagotomy (VGX) after abdominal lesions, then EA ST36; (d) VGX/EAN: VGX after
ST36 abdominal lesions, then EAN; (e) a-BGT/EA: intraperitoneal injection of a-bungarotoxin (a-
Inflammation BGT, an antagonist of a7 subunit of cholinergic nicotinic receptor) before EA ST36, and (f) a-
Angiogenesis BGT/EAN group: a-BGT injection before EAN. Seven days after abdominal surgical lesions,
the levels of tumor necrosis factor-a (TNF-a) and vascular endothelial growth factor (VEGF)
in the adhesive tissue were evaluated, macroscopic observation and histopathologic
evaluation of adhesion formation and assessment of angiogenesis by immunohisto-
chemical staining of platelet endothelial cell adhesion molecule-1 (CD31) were performed.
Results: EA ST36 reduced TNF-a and VEGF levels in adhesive tissue homogenates 7 d after
surgery, whereas vagotomy or intraperitoneal injection of a-BGT before EA ST36 reversed
its suppressive effects. EA at non-channel acupoints with or without vagotomy or intra-
peritoneal injection of a-BGT before EA had no suppressive effects on TNF-a and VEGF
levels. EA ST36 alleviated the adhesion formation, with both of macroscopic and histo-
pathologic adhesion scores significantly lower than those of the EAN group (1.56
0.29
versus 3.00
0.82, 1.35
0.4 versus 3.91
0.8, respectively, both P < 0.05). Compared with
the EAN group, EA ST36 significantly decreased angiogenesis evidenced by reduced CD31
positive microvessel density in adhesive tissue.

* Corresponding author. Research Laboratory of Shock and Multiple Organ Dysfunction, Burns Institute, First Hospital Affiliated to the
PLA General Hospital, No.51 Fu Cheng Road, Beijing 100048, China. Tel.: þ86 10 66867397; fax: þ86 10 68989139.
E-mail address: bs0425@163.com (S. Hu).
0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jss.2014.12.043
2 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
Conclusions: EA ST36 might reduce the postoperative local inflammatory response, atten-
uate the angiogenesis, and alleviate the adhesion formation partly via activating the
cholinergic anti-inflammatory mechanism.
1. Introduction
Postoperative intra-abdominal adhesion, a common compli-
cation secondary to surgical procedures of the abdomen, has
constantly been a matter of difficult task for surgeons all over
the world. Published researches document that intra-
abdominal adhesions appear to be at a modestly high preva-
lence after laparotomy [1,2] and one of the most elusive causes
of morbidity [3] or severe-related complications such as small-
bowel obstruction, infertility, chronic abdominal and pelvic
pain, difficult reoperative surgery, and other discomforts
[4e6]. The current treatment for intra-abdominal adhesion
focuses on reduction of tissue damage and inflammation,
lowering vascular permeability or inhibition of angiogenesis,
prevention of fibrin exudation, and deposition or enhancing
fibrinolytic activity. Unfortunately, up to date, no ideal remedy
for protection and treatment of postoperative intra-
abdominal adhesion has been established in clinical prac-
tice. The high risk of adhesion-related complications needs to
be carefully investigated and prevented, and seeking in-
terventions to prevent formation of intra-abdominal adhesion
after surgery is of great importance.
Acupuncture, as one of therapeutic maneuvers in tradi-
tional Chinese medicine, has been widely accepted and suc-
cessfully applied in the clinic for functional gastrointestinal
disorders for years. It has also been found to be an appropriate
adjunctive treatment for the management of postoperative
nausea and vomiting, prolonged postoperative ileus, and
various functional gastrointestinal disorders [7e9]. We have
demonstrated that electroacupuncture (EA) ST36 may activate
the cholinergic anti-inflammatory pathway (CAP), with
marked effects on promoting gastrointestinal function, pre-
serving intestinal barrier function, alleviating tissue inflam-
mation, and decreasing gut permeability [10,11]. Research has
put forward that inflammatory reactions seem to be the key-
driving mechanism in the pathogenesis of postoperative
adhesion formation [12e14]. And we have recently verified the
inhibitive effect of EA ST36 on inflammatory mediators in the
early phase of postoperative intra-abdominal adhesions [15],
suggesting that EA ST36 may have a negative impact on intra-
abdominal adhesion formation. The exact effects and
accurate vagal modulation by acupuncture in postoperative
intra-abdominal adhesion formation, however, has not yet
been thoroughly evaluated.
The CAP is a neural mechanism that inhibits the expres-
sion of proinflammatory cytokines through the interaction of
the principal vagus nerve neurotransmitter, acetylcholine,
and the cholinergic a7 nicotinic acetylcholine receptor
(a7nAChR) subunit located on cytokine-expressing cells,
which can be activated through stimulating the vagus nerve
either via electrical or pharmacologic methods [16,17]. The
anatomic physiology have proved that the nerves supplying
the viscera of the abdomen involve branches and fibers of the
ª 2015 Elsevier Inc. All rights reserved.
vagus nerve, which plays a crucial role in the peritoneal cavity
and innervates abdominal viscera, such as the entire gastro-
intestinal tract, liver and biliary system, and pancreas [18e20].
Besides, evidence demonstrates that acetylcholine, abundant
in peritoneum, and the serous membrane of the abdominal
viscera can directly modulate immune function in peripheral
tissues including the gastrointestinal tract [21].
Given the vagal modulative effects by EA [22] and the
supplying of the vagus nerve in the abdomen [18e20], we
hypothesized that acupuncture at bilateral ST36 might exert a
preventive effect on postoperative intra-abdominal adhesion
formation through activation of the vagus nerve pathway and
its cholinergic receptor. The objectives of our present study
are to investigate whether EA at ST36 could attenuate the in-
flammatory reactions evidenced by the release of tumor ne-
crosis factor-a (TNF-a), reduce the histopathologic scores of
adhesion formation, attenuate the angiogenesis and micro-
vessel density (MVD) by decreased expression of modulating
factors of vascular endothelial growth factor (VEGF) and
platelet endothelial cell adhesion molecule-1 (CD31) in adhe-
sive tissue, thus prevent adhesion formation after surgery
through activating the cholinergic anti-inflammatory-
dependent mechanism.
2. Materials and methods
2.1. Animals
Male SpragueeDawley rats (8e10 wk, 240e260 g) were pur-
chased from the Experimental Animal Center of Military
Medical Sciences of the Chinese PLA. Rats were acclimatized
for at least 1 wk before surgical procedures in mesh cages in a
temperature-controlled room with a 12-h lightedark cycle in
the animal quarter of our laboratory, provided with standard
rodent chow and water ad libitum. Animals were fasted over-
night but allowed free access to water until 4 h before surgery.
The research protocols were approved by the Committee of
Scientific Research of the First Hospital Affiliated to General
Hospital of PLA, China. The experiment was conducted in
compliance with the National Institute of Health Guide for the
Care and Use of Laboratory Animals.
2.2. Surgical procedures
All the surgical procedures were performed by two surgeons
who were supervised and standardized by the senior author
S.H. and were not aware of which group the animals were to
be assigned to at the time of the adhesion lesions. Adhesion
lesions at cecum and abdominal wall were performed on
anesthetized rats according to the established protocols by
previous studies [23e26]. Rats were anesthetized and instru-
mented with 3% isoflurane inhalation (Yeeran Technology
 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
Limited, Beijing, China). Ketamine, 10 mg/kg, was hypoder-
mically injected for local anesthesia. Isoflurane (0.7%) was
used to maintain anesthesia during the experiments. While
anesthetized, each rat was laid flat on the back in a supine
position on a surgical table. The abdomen was then
completely shaved with a razor and disinfected with alcohol
and an iodine solution. A 3-cm laparotomy was performed to
gain access to the abdominal cavity and expose the wall of the
cecum. The cecum was abrased with sterile wet gauze
repeatedly until subserosal hemorrhage and punctate
bleeding developed over an area of 2  1 cm2. Defects
(1  1 cm2) were taken from the peritoneal surface on the left
side of the abdominal wall with surgical scissors. Then, the
cecum was returned to its anatomic position. After the
adhesion lesions, the experimental rats were randomly
assigned to six groups as follows: EAN, EA, VGX/EA, VGX/EAN,
a-BGT/EA, and a-BGT/EAN groups, with corresponding treat-
ments as described in the following. Then, for rats in all
groups, the gastroesophageal junction was identified, and the
dorsal and ventral vagus nerve was exposed on the distal
esophagus with a Phenix XLT165-LB stereomicroscope (Phe-
nix Optical Instrument Group Company, Jiangxi Province,
China), and for those in VGX/EA and VGX/EAN groups, va-
gotomy of the dorsal and ventral vagus nerve on the distal
esophagus was performed. To prevent stimulation of perito-
neal adhesions formation due to the presence of surgical su-
ture material, four sutures at 1-cm intervals were placed using
absorbable catgut (Pudong Jinhuan Medical Products Co Lit,
Shanghai, China). Fascia and skin were closed with four su-
tures at 1-cm intervals using a nonabsorbable silk (Pudong
Jinhuan Medical Products Co Lit). Finally, the given area of the
skin was again disinfected, and the rats were left at a suitable
temperature to become conscious. External sutures were
removed on the seventh day of the treatment under general
anesthesia.
2.3. Animal grouping and treatments
The experimental rats were randomly assigned to six groups
with 10 rats each:
(a) EAN group: Animals underwent EA at non-channel acu-
points located 5 mm lateral and distal from ST36 points
[27]; EA parameters and treatment protocols were the
same as the EA group.
(b) EA group: Animals underwent EA at ST36 points, which
located at the posterior and lateral side of the knee joint,
5 mm below the capitulum fibulae [27], 30 min after sur-
gery in conscious conditions and fixed in a self-made cloth
bag in accordance with previous study [28]. EA at ST36,
with an EA apparatus (HANS, LH202 H), was performed as
described previously [10,11]. Briefly, both hind limbs were
shaved and the skin was disinfected. ST36 acupuncture
point was punctured with a depth of 7 mm, and then the
needle was connected with an EA apparatus. The electric
current with the intensity of 2e3 mA and 2e100 Hz was
continued for 1 h for 7 d, with stimulus intensity up to the
point that it can be tolerant and induce slight limb shaking
but cause no struggling to the rats.
3
(c) VGX/EA group: Animals underwent vagotomy of the dorsal
and ventral vagus nerve on the distal esophagus after
abdominal adhesion lesions and before EA at ST36 the
same as the EA group.
(d) VGX/EAN group: Animals underwent vagotomy similar to
the VGX/EA group before EA at non-channel acupoints the
same as the EAN group.
(e) a-BGT/EA group: a-bungarotoxin (a-BGT, 1 mg/kg, an
antagonist of a7 subunit of cholinergic nicotinic receptor
(a7nAChR), which inhibits the a7 subunit of acetylcholine
receptors by blocking a pivotal communication pathway
between the efferent vagus and intestinal immune cells
[29,30]), was injected intraperitoneally 30 min before EA at
ST36 the same as the EA group.
(f) a-BGT/EAN group: a-BGT was injected intraperitoneally
similar to the a-BGT/EA group 30 min before EA at non-
channel acupoints the same as the EAN group.
2.4. Samples of adhesive tissues
Rats were sacrificed with an overdose of ether for adhesive
tissue harvest 7 d after operation. The samples of adhesive
cecal tissues in the corresponding injured site were removed
and snap frozen in liquid nitrogen before storage at 80 C
for enzyme-linked immunosorbent assay (ELISA) or fixed
in 4% paraformaldehyde for histologic evaluation and
immunohistochemistry.
2.5. Macroscopic observation
Blinded gross observation was evaluated through a reversed
U-shaped incision across the abdominal cavity 7 d after
operation. For this, the abdomen of each rat was opened and
the adhesions severity, extent, and appearance were evalu-
ated by three independent observers, who were blinded to the
animals’ group assignment or treatment and did not take part
in the surgical procedures, using a well-documented adhesion
severity scoring system on a scale from grade 0egrade IV
(Table 1) published by RKS Phillips and HAF Dudley [31]. Each
rat was assessed by the three observers separately, and the
average grade of the three was accepted as the macroscopic
adhesive grade of the rat.
2.6. Histopathologic evaluation
Specimens of adhesion tissue from each animal were har-
vested at the second laparotomy and dissected, rinsed in PBS,
Table 1 e Macroscopic criteria for adhesions’ scaling.
Grade Criteria Score
0 No adhesions 0
I The ratio of adhesive area/the total 1
treated area in the vermiform
processes <20%
II The ratio is about 40% 2
III The ratio is about 60% 3
IV The ratio is 60% 4
4 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0

and fixed overnight in 4% paraformaldehyde. After dehydra-
tion with increasing concentrations of ethanol, the preserved
tissues were embedded in paraffin. Then, 5-mmethick, trans-
verse incisions were sectioned and stained by hematoxylin
and eosin to observe the morphology of the tissues. Histo-
pathologic evaluations of fibrosis and inflammation were
viewed via light microscopy and reviewed by three patholo-
gists who were blinded to the experimental groups from the
Pathology Department of the First Hospital Affiliated to the
PLA General Hospital. At least three randomly selected high-
power fields were reviewed for each pathologic section, and
at least two sections for each rat were confirmed and graded
using a scoring system as previously described, and the
average score of all fields was accepted as the histopathologic
adhesive grade of the rat (Table 2) [32].
to block nonspecific binding sites. The sections were incubated
at 4 C overnight with Mouse monoclonal anti-CD31 antibody
(diluted 1:200; Zhongshan Jinqiao Biotechnology Co, Ltd) diluted
in PBS containing 0.1% BSA and 2% normal mouse serum. The
following day, after washing with PBS three times, sections were
incubated for 1 h with biotinylated goat anti-mouse IgG in 1%
BSA for 1 h at room temperature. Sections were subsequently
stained with freshly prepared 3,3N-Diaminobenzidine-
hydrogen-peroxide complex and counterstained with hema-
toxylin. Images were viewed using the Olympus microscope
(BX51-DP71, Olympus Corporation, Japan) with exposure-
matched settings.
2.9. Identification of immunohistochemical staining
results

2.7. Enzyme-linked immunosorbent assay
TNF-a and VEGF levels were measured from adhesive tissue
extracts of cecum harvested at 7 d after surgery. Two com-
mercial ELISA kits for TNF-a (Nanjing Jiancheng Corp, Nanjing,
China) assay and VEGF (R&D Systems, Minneapolis, MN) assay
were used according to the manufacturer’s protocol. Briefly,
after adding 50 mL of assay diluent, 50 mL of samples or stan-
dard concentration for TNF-a or VEGF were incubated for 2 h
at room temperature. After five washes, the plates were
incubated with 100 mL conjugate for 1 h at room temperature,
followed by five washes. Then the plates were incubated with
100 mL substrate solution for 30 min at room temperature, and
the reaction was stopped with stop solution. The absorbance
rate was read at 450 nm. The concentrations of the samples
were calculated according to the standard curve. The tissue
TNF-a and VEGF levels were expressed as picograms per
milligram of protein.
2.8. Immunohistochemical evaluation
Immunohistochemical investigations were performed on
paraffin-embedded 5-mm sections using peroxidase-
conjugated, affinity-isolated immunoglobulins. Briefly, after
deparaffinization, the adhesive tissue sections were rehydrated
and incubated with 3% hydrogen peroxidase in PBS to block the
endogenous peroxidase, followed by washing in PBS (three
washes, 5 min each), and then the slides were immersed in
citrate buffer (Zhongshan Jinqiao Biotechnology Co, Ltd, Beijing,
China) for heat-induced antigen retrieval. After three washes
with PBS, sections were incubated with 3% bovine serum albu-
min (BSA) (Zhongshan Jinqiao Biotechnology Co, Ltd) for 30 min
Counting of MVD in adhesion tissues was in accordance with
Paola Bossi’s standards with a minor modification [33]. Briefly,
slides were examined at low-power magnification (100) to
identify the areas with the highest density of microvessels. In
each case, five areas of most vascularized areas were selected
for counting under 200 magnification (field diameter,
0.9 mm). The highest and the average counts of the five 200
fields were recorded for analysis. The average of the five areas
was recorded as the MVD level of this case. Single endothelial
cell or small clusters of endothelial cells, with or without a
lumen, were considered as individual vessels. Vessels of a
caliber larger than approximately eight red blood cells and
vessels with a thick muscular wall, ranging from none to less
than 2% of the overall vessel count in the microscopic fields
investigated, were excluded from the final count. The micro-
vessel count was independently evaluated in all cases by three
blinded pathologists and the average grades of the three were
recorded as the microvessel counts. Microvessel counts were
expressed as the absolute number of vessels per 200 field.
2.10. The statistical analysis
SPSS 13.0 statistical software was used, and all results were
expressed as mean
the standard error of the mean. One-way
analysis of variance was used for comparison among all
groups, followed by the Student-Newman-Keuls test for
comparison between two groups. Differences were considered
to be statistically significant when P  0.05.
3. Results
3.1. Gross observation

Seven days after operation, the tissue adhesion extent was

Table 2 e Histopathologic criteria for adhesions’ scaling.
Score Degree of inflammation
0 No inflammation
1 Giant cells, lymphocytes, and
plasma cells
2 Giant cells, plasma cells,
eosinophils, and neutrophils
3 Inflammatory cell infiltration and
microabscess formation
graded and averaged after the surgical exploration of the
Degree of fibrosis injured sites. Adhesion formation was prevalent in the EAN
No fibrosis group (Fig. 1), and the organs adhered to rubbed cecum were
Mild the small bowel, colon, and abdominal wall, respectively. EA
at ST36 reduced tissue adhesion, and adhesions formations
Moderate were rarely revealed between the cecum and the abdominal
wall, while vagotomy or the intraperitoneal injection of a-BGT
Severe
before EA at ST36 reversed its anti-adhesion effects. All the
animals in the a-BGT/EA and a-BGT/EAN groups showed large
 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0 5

There were defects in the EAN group filled with numerous

lymphocytes, plasma cells, macrophages, and abundant
deposition of obvious fibroplasia (Fig. 2A). EA group showed
the least fibroplasia with a reduced accumulation of the in-
flammatory cells (Fig. 2B). In contrast, when abdominal va-
gotomy or administration of a-BGT was performed and as
such the cholinergic anti-inflammatory neurenteric axis was
interrupted, EA ST36 failed to prevent the histologic changes,
evidenced by massive diffused lymphocytes infiltration and
aggravated fibroplasia reaction (Fig. 2C and D). Full-thickness
fibroplasias and lymphocyte infiltration appeared in a-BGT/
EA and a-BGT/EAN groups similar to the EAN group. The
observed results could not reveal any statistical significance in
the EAN, VGX/EA, VGX/EAN, a-BGT/EA, and a-BGT/EAN
groups. Taken together, these data demonstrated that an
intact vagus nerve was necessary for the biological effect of EA
ST36 and a7nAChR was involved in this effect. The histo-
pathologic adhesion score is shown in Table 3.

3.3. EA ST36 decreased rising levels of TNF-a in cecal

adhesive tissues

Table 4 illustrates the effect of EA ST36 on TNF-a level of ad-

hesive tissues 7 d after abdominal adhesion lesions operation.
Adhesion tissues in the EAN group exhibited significantly
higher levels of TNF-a than those in the EA group. EA at ST36
reduced TNF-a level in adhesive tissues, whereas vagotomy or
the intraperitoneal injection of a-BGT before EA at ST36
Fig. 1 e Typical adhesion 7 d after operation in the EAN
reversed its anti-inflammatory effects. TNF-a level in VGX/EA,
group. Gross observation in the EAN group showed
VGX/EAN, a-BGT/EA, and a-BGT/EAN groups had no difference
prevalent adhesion formation, with small bowel, colon,
than that in the EAN group. These evidences suggested that
and/or abdominal wall adhered to rubbed cecum. (Color
EA at ST36 attenuated the release of TNF-a in the presence of
version of the figure is available online.)
an intact vagus nerve.

and severe adhesions. Macroscopic adhesion scaling results 3.4. EA ST36 reduced angiogenesis by inhibition of VEGF
are summarized in Table 3. The average score of the EA group and CD31 in cecal adhesive tissues
was lower than that of the EAN group (1.56
0.29 versus
3.00
0.82, P < 0.05). The adhesion score did not reveal sig- VEGF and CD31 are two major proteins involved in angio-
nificant differences among the EAN, VGX/EA, VGX/EAN, a- genesis during adhesion formation after operation [34]. We
BGT/EA, and a-BGT/EAN groups. performed the ELISA assay for VEGF levels (Table 4) and
immunohistochemical methods for CD31 staining (Fig. 3) to
3.2. EA ST36 lowered histopathologic intra-abdominal evaluate angiogenesis in the cecal adhesive tissues. Tissue
adhesion degree lesion and stimulation during surgery resulted in a pro-
nounced increase in the levels of VEGF of cecal adhesive tis-
Hematoxylin and eosin staining revealed an intense forma- sues at 7 d after surgery. EA ST36 significantly reduced the
tion of adhesions in the scathed area of the cecum (Fig. 2). levels of VEGF in adhesive tissue compared with the EAN
group. There was no significant difference in the levels of
VEGF among EAN, VGX/EA, VGX/EAN, a-BGT/EA, and a-BGT/
EAN groups.
Table 3 e Macroscopic and histopathologic adhesion
score at 7 d after operation. The mature vessels were evaluated by CD31 immunohis-
tochemical observations with respect to an active agent. In
Groups Macroscopic Histopathologic
adhesion score adhesion score evaluation of CD31 as a marker known to mediate angiogen-
esis and wound healing, we saw more CD31þ cells in the EAN
EAN 3.00
0.82y 3.91
0.8y group than in the EA group (Fig. 3A and B). The expression
EA 1.56
0.29* 1.35
0.4*
pattern of CD31 in VGX/EA, VGX/EAN, a-BGT/EA, and a-BGT/
VGX/EA 3.12
0.64y 3.70
1.4y
VGX/EAN 3.33
0.87y 4.26
0.5y
EAN groups showed a fairly large number of small, newly
a-BGT/EA 2.98
0.92y 4.00
0.7y formed vessels evidenced by CD31 present on endothelial
a-BGT/EAMN 3.10
1.06y 3.86
1.0y cells, just the same as the EAN group. Microvessels count in
adhesive tissues was measured by immunohistochemical
* versus EAN group, P  0.05; y versus EA group, P  0.05.
staining with CD31 (Table 4).
6 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0

Fig. 2 e Hematoxylin and eosin staining of intra-abdominal adhesion. EA ST36 protected against intra-abdominal adhesion
formation after surgical lesions, whereas EA at non-channel acupoints and EA ST36 after abdominal vagotomy or
administration of a-BGT attenuated such protection. All images are taken at 3200 magnification with black bar [ 5 mm. (A)
Animals in the EAN group, (B) animals in the EA group, (C) animals in the VGX/EA group, (D) animals in the VGX/EAN group,
(E) animals in the a-BGT/EA group, and (F) animals in the a-BGT/EAN group. (Color version of the figure is available online.)

activity, angiogenesis and delayed gastrointestinal transit,

4. Discussion
As a common and serious surgical relevant problem, intra-
abdominal adhesion is a notorious phenomenon of the
repairing processes leading to morbidity after surgical
abdominal injury. Also of problem is an incalculable social,
economic, physical, and emotional impact on the patients
after adhesion formation, which may lead to hospital read-
missions and consecutive interventions [35]. To understand
the mechanism of abdominal adhesion is very important for
the prevention of its formation. At present, it is regarded as
consequences and complex interplay of fibrin exudation and
deposition resulting from the interaction among initial sur-
gical injury and/or ischemia of the peritoneal cavity, the local
excessive inflammatory reaction, decreased fibrinolytic
tissue lesion, and stimulation during surgery. Attributed to a
variety of chemical messengers release, these interactions at
the injury site in turn might dedicate to promoting their
release and leading to a series of events [36e40]. During the
healing process after peritoneal injury, the key components of
the sequence of events occurred are angiogenesis, inflam-
mation, and fibrosis. And interventions to inhibit one or more
components of the succedent events, exhibiting anti-
inflammatory, antiangiogenic, and/or antifibrogenic proper-
ties, after surgery are believed to represent key targets in
strategies to prevent abdominal adhesion formation [41].
Despite the extensive efforts undertaken to prevent peri-
toneal adhesion formation, no single method has so far been
successfully applied in the clinic. A clinically desirable alter-
native therapy on the prevention of adhesion formation after
 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0 7

postoperative adhesions [15] and activation of CAP by EA ST36

Table 4 e The effects of EA at ST36 on the concentrations
of TNF-a and VEGF and quantitative study of [10,11,42,43], the following two reasons have further made us
microvessels by CD31 immunohistochemical staining in to assume that CAP might involve in the prevention of post-
cecal adhesive tissue (mean ± SD, n [ 10). operative intra-abdominal adhesion formation. First, activa-
Groups TNF-a VEGF Microvessels count tion of the CAP by vagus nerve stimulation have been
demonstrated to affect the activation of coagulation and
y y
EAN 3.00
0.82 3.91
0.8 42.43
6.27y
fibrinolysis during endotoxemia in rats, thus illustrating a far
EA 1.56
0.29* 1.35
0.4* 11.93
3.93*
unrecognized effect of vagus nerve stimulation and suggest-
VGX/EA 3.12
0.64y 3.70
1.4y 40.33
4.63y
VGX/EAN 3.33
0.87y 4.26
0.5y 41.32
5.30y ing that the CAP not only impacts on inflammation but also on
a-BGT/EA 2.98
0.92y 4.00
0.7y 39.17
6.22y the coagulanteanticoagulant balance [44]. The vagus nerve
a-BGT/EAN 3.10
1.06y 3.86
1.0y 39.92
7.62y supply of the viscera of the abdomen [18e20] is the other
reason that assures us to assume EA ST36 might produce a
The content of TNF-a and VEGF in cecal adhesive tissue was
expressed as picogram per milligram protein in rats. * versus EAN marked effect on postoperative intra-abdominal adhesion
group, P  0.05; y versus EA group, P  0.05. formation through activation of the vagus nerve, CAP, and its
Microvessels count: * versus EAN group, P  0.05; y versus EA group, cholinergic receptor.
P  0.05. This study demonstrates that EA ST36 has preventive ef-
fects on postoperative adhesion formation. Our data support
the hypothesis that surgical damage and/or ischemia caused
surgery needs to be established. Clinically, acupuncture and the local inflammation reaction and activated the expression
EA have been successfully applied for functional gastrointes- of VEGF and CD31 to develop angiogenesis and fibrovascular
tinal disorders. In our previous experimental studies, we did a adhesion. Our results further demonstrate that EA at ST36
lot of work on the effects of EA at ST36, which can alleviate treatment inhibits postoperative local inflammation action,
intestinal inflammation [42], promote gastric emptying [43], resulting in reduced VEGF and CD31 expression, which
and protect intestinal barrier [10,11] through activation of CAP therefore might lower the expression and evolution of fibrosis
in rats with different model preparation. We have also and inflammation action in the injured area, and thus protects
recently verified the inhibitive effect of EA ST36 on inflam- against surgical trauma-induced postoperative adhesion. We
matory mediators of postoperative intra-abdominal adhe- also proved that this biological effect is dependent on an
sions [15]. However, the accurate effects of EA ST36 and vagal intact vagus nerve. Disrupting the neurenteric axis via the
modulation of CAP by acupuncture on postoperative intra- surgical abdominal vagotomy weakened the protective effect
abdominal adhesion formation need to be evaluated. In light of EA at ST36 points. Combined with our previous studies
of the confirmed inhibitive effect on inflammation in demonstrating the promotion effects on gastrointestinal

Fig. 3 e CD31-immunostained sections of adhesion tissue. Animals in the EAN group showed a high immunohistochemical
intensity in the adhesive tissue 7 d after surgery (A), whereas EA ST36 showed inhibition of CD31 staining (B), and
abdominal vagotomy or a-BGT administration performed before EA ST36 or EA non-channel acupoints had no preventive
effects against adhesion formation (CeF). All images are taken at 3200 magnification with black bar [ 5 mm. (Color version
of the figure is available online.)
8 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
motility by EA ST36 [43,45], our results have provided further
theoretic evidence for an extended role of acupuncture in
anti-adhesion treatment after surgery.
Inflammatory response [33] and the production of extra-
cellular matrix by fibroblasts are two processes linked in the
formation of fibrous adhesion after abdominal surgery [46].
We have already proved that EA ST36 can exert its anti-
inflammatory effects to provide protective effects against
gut injury and intestinal barrier dysfunction and improve
outcomes through activating the cholinergic anti-
inflammatory-dependent mechanism [10,11]. In this study,
we demonstrated EA ST 36 decreased surgical trauma-
induced increase of TNF-a in cecal adhesive tissue, whereas
vagotomy or the intraperitoneal injection of a-BGT before EA
at ST36 reversed its anti-inflammatory effects, further sug-
gesting that EA at ST36 attenuated the release of TNF-a level in
the presence of an intact vagus nerve. This protective effect is
consistent with decreased severity, extent, and appearance of
tissue adhesion.
In addition to alterations in the local tissue cytokines
concentration, studies strongly suggest that another pivotal
event determining the postoperative adhesion formation is
angiogenesis. Angiogenesis is a fundamental process that can
affect both inflammation and wound repair [47]. It is reported
that angiogenesis is regulated mainly through the modulation
of angiogenic factors expression, such as VEGF and CD31,
which have been proved to be evident on newly formed blood
vessels present in the adhesion tissue [33,48,49]. The upre-
gulation of VEGF during the progression of adhesion forma-
tion has been documented, and inhibition of VEGF or CD31
limited postoperative adhesion formation [47,48]. The results
of our present study are in agreement with these previous
articles. We observed the least level of VEGF or CD31 immu-
nohistochemical staining in tissues with the lowest adhesion
scores in animals treated with EA at ST36 acupoints. Futher-
more, we obtained supportive results with an abdominal va-
gotomy, or administration of CAP antagonist reversed this
protective effect of EA ST36.
Our study just demonstrates a preliminary success of the
EA in a postoperative model in vivo study. To fully understand
the exact importance of EA ST36 on prevention of adhesion
formation, further studies are required to fully elucidate the
molecular and cellular mechanism of acupuncture at ST36 on
activation of vagus. But the neuroanatomic distribution of
the vagus nerve and/or its branches has been proved to be in
correspondence with the efficacy of acupuncture [50], as we
assumed in this study the interaction of effects of EA ST36
and the vagus nerves supplying of the viscera of the
abdomen. Furthermore, by understanding the mechanisms
of peritoneal healing and its repair processes after surgery,
we may be able to define the components involved in adhe-
sion formation and to more precisely determine the role of
fibrin, the regulation of fibrinolysis and coagulation, and the
angiogenic factors in adhesion formation, which in turn will
dedicate to strategies to completely prevent intra-abdominal
adhesions.
The protective effects on adhesion formation of EA ST36
may affect macrophages, which can express a7nAChR
involved in the CAP. During the initiation period of peritoneal
adhesion, postsurgical macrophages increase and secrete
variable substances and factors, many of which can affect the
inflammation reaction, regulate fibrinolysis [1], and increase
vascular permeability. All those actions contribute to the
collection of a fibrin-rich exudate that covers the injured area
and elicitation of angiogenesis factors that initiates the
development of vascular structure and induces newly
occurred vessels in the healing area [35]. Besides, vascular
endothelial cells also express a lot of a7nAChR. EA ST36 might
exert the anti-adhesive effects by interaction with some of
these phages in adhesion formation.
Admittedly, there are several limitations in this study. One
shortcoming is that we only focused on the anti-inflammatory
and antiangiogenic effects of EA ST36 on prevention of
adhesion formation, its influence on other components after
surgery lesions remains unknown. Moreover, the unconscious
bias is inevitable as intensity and pressure of abrasion of cecal
abrasion cannot be completely the same in a different rat
during surgery. Besides, only one time point was examined,
which made it possible that the rats treated by EA ST36 may
have delayed adhesion formation rather than preventing it.
Thus, the intermediate and long-term efficiency of EA ST36 on
adhesion formation needs to be further identified.
5. Conclusions
In summary, this study showed that EA at ST36 might be
performed to prevent intra-abdominal adhesion formation
after abdominal surgery, which is consistent with attenuated
postoperative local inflammation action and decreased
angiogenesis by inhibition of VEGF and CD31. The protective
role of EA at ST36 is possibly related to an intact vagus nerve
and it might exert its effects via or at least in part, through the
regulation of cholinergic a7nAChR. It might be considered as
an alternative intervention to prevent intra-abdominal
adhesion formation in this difficult-to-treat postoperative
complication.
Acknowledgment
The authors thank Drs Jiang-Yang Lu, Yi-Duo Jin, Hui-Zhen
Ding, Xiu-Li Ma, and Na Feng from the Department of Pa-
thology, First Hospital Affiliated to the PLA General Hospital,
for their technical assistance concerning pathology in this
project.
Authors’ contributions: S.H. participated in the design of
the study. M.-H.D., Y.-J.T., L.-J.Z., and H.-M.L. carried out the
experimental studies and data analysis. Y.L., H.-M.L., and Z.-
K.Z. helped experimental measurements and data acquisi-
tion. M.-H.D. and S.H. participated in the article preparation.
S.H. and R.-J.X. helped with the article review and editing. All
authors have read and approved the final article.
Supported by the Special Foundation of the 11th Five-Year
Plan for Military Medical Project (No. 06Z055), and the National
Natural Science Foundation of China (No. 81471872).
Conflicting interest: none.
 j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 5 ) 1 e1 0
Disclosure
The authors reported no proprietary or commercial interest in
any product mentioned or concept discussed in the article.
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