Research Article
Acupotomy Alleviates Energy Crisis at Rat Myofascial
Trigger Points

Yi Zhang ,1 Ning-Yu Du,2 Chen Chen,1 Tong Wang,1 Li-Juan Wang,1 Xiao-Lu Shi ,3

Shu-Ming Li,4 and Chang-Qing Guo1

1School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing 100029, China
2Center for Early Childhood Development, Shijiazhuang Maternal and Child Health Care Hospital, Shijiazhuang 050051, China
3Beijing Key Laboratory of TCM Basic Research on Prevention and Treatment of Major Disease, Experimental Research Center,
China Academy of Chinese Medical Sciences, Beijing 100700, China
4Department of Pain Medicine, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University,
Beijing 100010, China

Correspondence should be addressed to Yi Zhang; zhangyi.acupotomology@gmail.com

Received 20 November 2019; Accepted 18 January 2020; Published 28 February 2020

Academic Editor: Manel Santafe

Copyright © 2020 Yi Zhang et al. *is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*e aim of this study was to determine the effects of acupotomy on energy crises in rat trigger points (TrPs) by measuring
mechanical pain thresholds (MPTs) and levels of acetylcholinesterase (AChE), free sarcoplasmic calcium (Ca2+), adenosine 5′-
triphosphate (ATP), adenosine 5′-monophosphate (AMP), substance P (SP), and calcitonin gene-related peptide (CGRP) in rat
muscle TrP tissue. Male Sprague Dawley rats (n � 32) were randomly divided into four groups: control, TrP, acupotomy, and
lidocaine injection. Enzyme-linked immunosorbent assays were used to measure AChE, and free sarcoplasmic Ca2+ concen-
trations were determined by fluorescent staining with Fura-2 AM; high-performance liquid chromatography was used to measure
ATP and AMP, and SP and CGRP were evaluated by immunohistochemistry. Compared with the control group, free sarcoplasmic
Ca2+, AMP, SP, and CGRP were higher in the model group, while MPT, AChE, and ATP were lower. Treatment with acupotomy
or lidocaine injection reduced free sarcoplasmic Ca2+, SP, and CGRP and increased MPTs and AChE levels compared with the
model group. However, only acupotomy also led to decreased AMP and increased ATP levels relative to the model group. We
conclude that acupotomy can alleviate energy crises at TrPs.

1. Introduction

Myofascial pain syndrome (MPS) is a group of clinical
disorders characterized by chronic pain arising from soft
tissue, associated with one or multiple trigger points (TrPs)
[1]. Myofascial TrPs are irritable points characterized by taut
skeletal muscle bands. *ey can cause soft tissue pain that is
activated by pressing, pulling, or overuse. TrPs can con-
tribute to local pain, referred pain, and local twitch re-
sponses. *ey can also decrease muscle strength, work
endurance, and coordination as well as cause other types of
muscle dysfunction. TrPs are also associated with autonomic
nerve manifestations, such as hyperhidrosis and arrector pili
muscle response. Among patients with soft tissue pain, 20%–
95% suffer from MPS [2].

Although the exact nature of TrPs is unknown, Simons
proposed the energy crisis hypothesis, which was subse-
quently developed to generate the integrated hypothesis
[3, 4]. It contends that contributing factors lead to the
malfunction of motor endplates and excessive acetylcholine
(ACh) leaking from the endplates, causing continuous de-
polarization of muscle cell membranes and calcium (Ca2+)
release from the sarcoplasmic reticulum, which cannot be
reabsorbed. *erefore, the sarcoplasm continually has high
Ca2+ concentrations, causing persistent muscle fiber con-
tractions and the formation of taut intramuscular bands that
can be palpated [5]. Continuous muscle contraction leads to
local hypoxia and hypermetabolism, resulting in local energy
crises and the release of sensitizing substances such as 5-
hydroxytryptamine, histamine, bradykinin, and substance P

Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2020, Article ID 5129562, 11 pages
https://doi.org/10.1155/2020/5129562

mailto:zhangyi.acupotomology@gmail.com
https://orcid.org/0000-0002-8933-0051
https://orcid.org/0000-0002-5351-0894
https://creativecommons.org/licenses/by/4.0/
https://doi.org/10.1155/2020/5129562


(SP). *is stimulates nerve endings, causing pain and
sympathetic neuronal symptoms [6].

Acupotomy, also referred to as miniscalpel-needle
acupuncture, is a fairly recent development in the ancient
practice of acupuncture. Acupotomy involves a combination
of acupuncture needle insertion and surgical incision and is
effective for relieving TrPs [7]. While TrPs are common sites
for acupotomy, the mechanism underlying the effects of
acupotomy on TrPs remains unclear. In this study, me-
chanical pain thresholds (MPTs) and levels of acetylcho-
linesterase (AChE), free sarcoplasmic Ca2+, adenosine 5′-
triphosphate (ATP), adenosine 5′-monophosphate (AMP),
calcitonin gene-related peptide (CGRP), and SP were
evaluated in rat TrPs, and the effects of acupotomy on these
indicators were compared with the effects of local lidocaine
injection. *e results provide a theoretical basis for different
interventions to treat TrPs, including needle insertion at ashi
points as described in TCM.

2. Materials and Methods

2.1. Animals. Male specific pathogen-free Sprague Dawley
rats (n � 32, 9 weeks old, weight 300±15 g) were provided
by Beijing Vital River Laboratory Animal Technology Co.,
Ltd. (animal batch number: SCXK(Jing)2016-0006). Rats
were raised in the animal house at the Beijing University of
Chinese Medicine with five rats per cage, free access to feed
and water, natural lighting, indoor temperature of
22°C±2°C, humidity of 40%–60%, and regular ultraviolet
disinfection. *e 32 rats were randomly divided into 4
groups (n � 8 each; control, TrP model, acupotomy, and
lidocaine injection) using the random number table
method. During the experiment, the rats were treated and
handled with strict adherence to the standards set by the
Ethical and Animal Research Committees, with approval
from the National Natural Science Foundation (no.
81503653).

2.2. Experimental Reagents and Equipment. Experimental
materials included acupotomes (0.4 × 40 mm; ZYHY, Bei-
jing, China), lidocaine hydrochloride injections (2%; Yoo-
kon, Beijing, China), normal saline (Yookon), iodophor
(Tiangen, Kunming, China), and disposable sterile syringes
(Yuekang, Changzhou, China). Other reagents used were rat
AChE enzyme-linked immunosorbent assay (ELISA) kit
(CSB-E11304r; Huamei, Wuhan), phosphate-buffered saline
(PBS) solution (Hyclone, Logan, UT, USA), Dulbecco’s
modified Eagle’s medium (DMEM; Hyclone), Krebs-Hen-
seleit solution (in mmol/L: NaCl, 118.50; NaHCO3, 25.00;
KCl, 4.70; MgSO4, 1.20; KH2PO4, 1.20; glucose, 11.00; CaCl2,
1.80; NaOH to pH 7.4), Fura-2 AM powder (F1221; Invi-
trogen), Fura-2 Calcium Imaging Calibration Kit (F6774;
Invitrogen), 5′-adenosine triphosphate disodium salt hy-
drate (Sigma), sodium adenosine 5′- monophosphate
(Sigma), rat SP immunohistochemical kits (ab14184,
GR206575-1; Abcam), and rat CGRP immunohistochemical
kits (ab81887, GR291095-3; Abcam).

2.3. Model Preparation. *e rats, except those in the control
group, were prepared as TrP models by applying “blunt
striking injury and eccentric exercise” as previously de-
scribed [5]. Briefly, the rats were anesthetized with 20%
urethane solution injected into the abdominal cavity
(0.5 mL/100 g). Rats were then fixed onto a plank under a
striker, and the middle of the left vastus medialis was marked
as the target. *e striker was allowed to fall freely from a
height of 20 cm to hit the marked position. On the second
day, the rats were placed on a − 16° treadmill set, so that they
ran downhill continuously for 90 min, with the speed
gradually rising to 16 m/min, once per week, for 8 weeks.
After the 8th week, rats in the control and model groups
were examined by electromyography to confirm TrP model
establishment.

2.4. Interventions. Rats in the four experimental groups
described above were treated as follows: (1) Control, no
intervention; (2) TrP model, no intervention after model
preparation; (3) Acupotomy, acupotomy was applied 1 week
after TrP model preparation; and (4) Injection group, li-
docaine injections given 1 week after model preparation
(20% urethane solution injected into the abdominal cavity at
a dosage of 0.5 mL/100 g, iodophor application to the TrP in
the vastus medialis, and injection of 0.5 mL of 1% lidocaine
into the taut band once a week for 3 weeks). Acupotomy
intervention was conducted once per week for 3 weeks. *e
specific operation conducted at each point was as follows: (1)
insertion of an acupotome into a TrP in the vastus medialis
to pierce the taut band; (2) insertion of an acupotome
perpendicular to the belly muscle, with the edge of the
acupotome parallel to the taut band; and (3) the acupotome
was then withdrawn and the point pressed for 30 s.

2.5. Index Assessments

2.5.1. Electromyography. Electromyography assessments
were performed in the control and model groups. Rats were
anesthetized by intraperitoneal injection of 20% urethane
(0.5 mL/100 g) and then fixed on a board. *e skin was cut to
expose the left vastus medialis, and an electrode (0.3 mm)
was inserted into the tail as a reference electrode. For the
control group, an electrode was slowly inserted into the
middle of the vastus medialis, and another electrode was
inserted longitudinally 3 mm away from the last electrode
into the vastus medialis. Finally, the wound was sutured. *e
same procedure was performed for the TrP group, but local
twitching was considered confirmation of insertion into the
TrP. Electromyography recordings were performed when
the muscle was at rest (Z2J-MB-NCC08; NCC, Shanghai,
China).

2.5.2. TrP MPTs. After the last intervention, 32 rats were
anesthetized by 20% urethane. Anesthesia induction was
confirmed by squeezing their tails using a power of 600 g [8]
to see if it caused stable abdominal muscle contraction. *e
mechanical sensitivity of rat TrPs was assessed using the Von

2 Evidence-Based Complementary and Alternative Medicine



Frey mechanical pain stimulator (Ugo Basile, Genomio,
Italy) to stimulate the TrP skin area until ipsilateral hind
limb movement occurred [9]. *e MPT was defined as the
minimum stimulus intensity that caused the movement of
the hind limbs. TrP sensitivity was assessed on days 1, 2, 3, 5,
and 7, next week after the last intervention. For each
measurement, MPTs were determined three times, with an
interval of 1 min between each test, and the mean of the
results was calculated.

2.5.3. Sample Collection. Samples were collected after be-
havioral assessment. Rats were deeply anesthetized using
20% urethane and then fixed on an operating table with the
left vastus medialis fully exposed. An experienced physician
confirmed the position of the TrP in the vastus medialis by
lightly pressing the taut band using the thumb or forefinger.
To collect the samples, the vastus medialis was divided at the
marked TrP, the fascia was carefully removed, and muscle
tissue samples (1 × 0.2 cm) were excised. After sample col-
lection, 32 rats were sacrificed by anesthesia overdose.

2.5.4. AChE Elisa. Tissue samples were placed in 900 μL PBS
(pH 7.4), rapidly minced with ophthalmic scissors, and
homogenized using an ultrasonic homogenizer (FS-100T;
sxsonic, Shanghai, China) and vibration table (KJ-201C;
Kangjian, Jiangsu, China). Tissue homogenates were
centrifuged with 6,000 g at 4°C for 15 min, and the super-
natants were separated. AChE levels in the samples were
tested by an AChE ELISA kit according to the manufac-
turer’s protocol. Optical density (OD) was read at 450 nm on
a microplate reader (Multiskan MK3; *ermo Fisher Sci-
entific, Waltham, MA, USA). Concentrations were calcu-
lated according to a standard curve, using the CurveExpert
1.3 software (https://www.curveexpert.net/).

2.5.5. Fluorescent Staining to Detect Free Sarcoplasmic Ca2+.
*e basement membrane of isolated muscle tissue was
carefully removed to expose the muscle fibers. *en, a 1 cm
length of muscle tissue was cut along the direction of the
muscle fibers and rinsed with PBS buffer to load with
fluorescent dye. Fura-2 AM stock solution was prepared by
dissolving Fura-2 AM powder (F1221, Invitrogen) in 20%
Pluronic F127 DMSO solution. *e muscle tissue was in-
cubated with fluorescent staining solution (final Fura-2
concentration was 2.5 μmol/L in DMEM solution) in the
dark at the 37°C temperature. After 30 min loading, replace
the fluorescent loading solution by KH solution and incu-
bated another 5 min for twice at the 37°C temperature to
remove unloaded Fura-2 AM. *e fluorescence-stained
samples in KH solution were then placed in 35 mm glass
plates, and only the resting free sarcoplasmic Ca2+ in intact
skeletal muscle fibers was measured using an Andor iXon3
EMCCD (Andor, Belfast, UK) with a 10x fluorescence ob-
jective lens. A Sutter DG4 Xenon lamp system (Sutter,
Sacramento, CA, USA) was used as the excitation light
source at 340±10 nm and 380±10 nm, separately, with
emitted light measured at 510±25 nm. Ca2+ images were

collected from three different positions for each sample, and
three muscle fibers were analyzed in each image. *e mean
value for the muscle fibers was considered the Ca2+ level in
the tissue sample. Background fluorescence without staining
was also measured for each sample. Fluorescence intensities
at 340 and 380 nm were corrected by background intensity.
*e fluorescence intensity ratio was determined using the
340 and 380 nm excitation wavelengths, and tissue Ca2+
concentration was subsequently calculated using the fol-
lowing formula [10]: [Ca2+] = Kd × (R–Rmin)/(Rmax–R) × Sf2/
Sb2 (in the formula, Kd is the effective dissociation constant,
R is the fluorescence ratio, Sf2 is the proportionality coef-
ficient for free dye measured at wavelength λ2, and Sb2 is the
proportionality coefficient for Ca2+-bound dye at λ2). *e
values of Rmax and Rmin were the ratio values measured by
Fura-2 Calcium Imaging Calibration Kit (F6774,
Invitrogen).

2.5.6. ATP and AMP Measurement. Tissue samples were
weighed and added to precooled 0.4 mol/L perchloric acid
(5 μL/mg) and then homogenized in an ultrasonic cell
grinder (SCIENTZ-1200E; Scientz, Zhejiang, China). Ho-
mogenates were centrifuged at 10000 g (4°C, 10 min). *en,
100 μL supernatant and 50 μL perchlorate were added into a
1 mol/L potash-methanol mixture (volume 4 :1), and the
mixture was centrifuged at 10,000 g (4°C, 10 min). Next, a
100 μL aliquot of the supernatant was removed, diluted with
100 μL water, and assessed by high-performance liquid
chromatography. *e conditions were as follows: mobile
phase, 30 mM NaH2PO4 (pH 6.25), 3% methyl alcohol;
detection wavelength, 254 nm; sample size, 20 μL; column
temperature, room temperature; sample temperature, 4°C.

2.5.7. Immunohistochemical Analysis of SP and CGRP.
Muscle biopsies were fixed in 10% formalin and embedded
in paraffin. After sectioned at 4 μm thickness, sections were
dehydrated in xylene, rehydrated in graded alcohol, and then
rinsed with PBS. *en, the antigen was retrieved by mi-
crowave (800 W) in 0.01 M citric acid buffer (pH 6.0) for
10 min, washed with PBS three times, and then placed in the
3%H2O2 at room temperature for 60 min. *e antibodies
were diluted with PBS buffer. Slides were incubated (4°C)
overnight with rabbit anti-SP (1 : 500; Abcam) or rabbit anti-
CGRP (1 : 200; Abcam) and then with the appropriate sec-
ondary biotinylated antibody (Boster) for 60 min at room
temperature. *e slides were then incubated with the avidin-
biotinylated enzyme complex and then put into a peroxidase
reaction solution containing diaminoaniline. Afterward,
fresh DAB was added. *en, the slides were washed with
PBS, counterstained in hematoxylin, blued in running water,
dehydrated with gradient alcohol, cleared with xylene, and
mounted with neutral gum. Areas positive for SP or CGRP
in randomly selected fields of vision (original magnification
×200) were counted using Image-Pro Plus 6.0 (Media Cy-
bernetics, Rockville, MD, USA). *e resulting data were
collected as immunohistochemical staining-positive signal
cumulative optical density (IOD) values, which represent the

Evidence-Based Complementary and Alternative Medicine 3

https://www.curveexpert.net/


average positive area. *ese IOD values were used as
quantitative measures of tissue expression levels.

2.6. Statistical Analysis. Data were analyzed using the SPSS
22.0 statistical software (v22.0; IBM Corp., Armonk, NY,
USA). For normally distributed data with homogeneous
variance, analysis of variance (ANOVA) was used to
compare four groups. For non-normally distributed data,
the Kruskal–Wallis test was used. All data are expressed as
mean±standard deviation. P<0.05 and P<0.01 were
considered to indicate significant and very significant dif-
ferences, respectively (Figure 1).

3. Results

3.1. Electromyography of Model Group. Electromyography
was recorded at 10 ms/D and 20 μV/D. Figure 2(a) shows a
representative typical endplate potential, which turns neg-
ative from baseline. Figure 2(b) shows the fibrillation po-
tential, which turns positive from baseline and then turns
negative. *is suggests the presence of TrPs in the vastus
medialis of the model group rats [6].

3.2. Acupotomy Effect on TrP MPTs. Compared with the
control group, the MPTof the model group decreased on the
1st, 2nd, 3rd, 5th, and 7th days after intervention (P<0.05,
Figure 3). Moreover, compared with the model group, the
MPT increased in the acupotomy group on the 1st, 2nd, 3rd,
5th, and 7th days after intervention (P<0.05, Figure 3).
Further, relative to the model group, the MPT was higher in
the lidocaine injection group on the 1st, 2nd, 3rd, 5th, and
7th days after intervention (P<0.05, Figure 3). Comparisons
between the acupotomy and injection groups showed no
significant difference in MPTs at any time point tested
(P>0.05, Figure 3).

3.3. Acupotomy Effect on AChE. AChE levels were signifi-
cantly lower (P<0.05) in the model group than the control
group (P<0.05). However, AChE levels in the acupotomy
(P<0.05) and injection (P<0.05) groups were significantly
increased compared with the model group (Figure 4, Ta-
ble 1). *ere was no significant difference between the
acupotomy and injection groups (P>0.05, Figure 4, Ta-
ble 1). *ese data show that acupotomy can increase AChE
levels in TrPs.

3.4. Acupotomy Effect on Free Sarcoplasmic Ca2+. Free sar-
coplasmic Ca2+ in the model group was significantly higher
than in the control group (P<0.05). Relative to the model
group, free sarcoplasmic Ca2+ in the acupotomy and in-
jection groups was decreased significantly (both P<0.05,
Figure 5, Table 2). *ere was no significant difference be-
tween the acupotomy and injection groups (P>0.05, Fig-
ure 5, Table 2). *ese findings demonstrate that acupotomy
can reduce free sarcoplasmic Ca2+ levels in TrPs.

3.5. Acupotomy Effect on ATP and AMP. ATP levels in the
model group were significantly lower than those in the
control group (P<0.05). Relative to the model group, ATP
increased significantly in the acupotomy group (P<0.05,
Figure 6, Table 3); however, there was no significant dif-
ference between the model and injection groups (P>0.05,
Figure 6, Table 3). *ese findings indicate that acupotomy
can enhance ATP levels in TrPs.

AMP levels in the model group were significantly higher
than those in the control group (P<0.05), but those in the
acupotomy group were significantly lower than those in the
model group (P<0.05, Figure 6, Table 3). Again, there was
no significant difference between the model and injection
groups (P>0.05, Figure 6, Table 3). *ese results show that
acupotomy can decrease AMP levels in TrPs.

3.6. Acupotomy Effect on SP and CGRP. In Figure 7, SP
expression was yellowish brown in muscle fibers. SP levels
were significantly higher in the model group than the control
group (P<0.05), but they were significantly lower in the
acupotomy group than the model group (P<0.05, Figure 6,
Table 4). Furthermore, compared with the model group, SP
levels were significantly lower in the injection group
(P<0.05, Table 4). *ere was no significant difference be-
tween the acupotomy and injection groups (P>0.05, Fig-
ure 7, Table 4). Hence, our findings indicate that acupotomy
can reduce SP levels in TrP muscle tissue.

In Figure 8, CGRP expression was yellowish brown in
muscle fibers. CGRP levels were significantly higher in the
model group than the control group (P<0.05), but those in
the acupotomy (P<0.05) and injection (P<0.05) groups
were significantly lower than those in the model group.
*ere was no significant difference in CGRP content be-
tween the acupotomy and injection groups (P>0.05, Fig-
ure 8, Table 4). *ese results suggest that acupotomy can
reduce CGRP content in TrP muscle tissue.

4. Discussion

*e results of this study demonstrate that the MPT, AChE,
free sarcoplasmic Ca2+, ATP, AMP, SP, and CGRP levels
were altered in rats after TrP model generation, and acu-
potomy improved these effects. Other than the changes in
ATP and AMP levels, which were improved by acupotomy
but not lidocaine injection, there were no significant dif-
ferences between the effects of acupotomy and those of li-
docaine injection.

4.1. Energy Crisis at Trigger Points. Despite several theories,
the exact nature of TrPs remains unknown [11, 12]; however,
electrophysiological and histological evidence support the
integrated hypothesis of the cause of TrPs [13], which
contends that an abnormal increase in ACh causes increased
energy consumption in TrPs. Specifically, motor endplates in
active TrPs release excessive ACh in the resting state,
resulting in continuous depolarization of muscle fibers. *is
causes the sarcoplasmic reticulum to release excessive Ca2+
into myoplasm, followed by continuous muscle fiber

4 Evidence-Based Complementary and Alternative Medicine



contraction that consumes excessive energy. Inadequate
blood flow due to continuous contraction of muscle fibers
cannot effectively replenish energy and oxygen. In energy
crises, Ca2+ pumps in the sarcoplasmic reticulum fail to
reduce the free Ca2+ in myoplasm, aggravating muscle fiber
contraction. Energy crises increase algogenic substances at
local levels, which further perpetuates Ach release at motor
endplates, creating a vicious circle [14].

4.2. AChE Indicates Endplate Function. *is neuromuscular
junction is responsible for passing nerve impulses to the
skeletal muscles, causing muscle contractions. When an action
potential reaches the presynaptic terminal of a motor neuron,
ACh is released into the synaptic space where it binds to
nicotinic ACh receptors (AChRs) on the muscle membrane.
Binding of ACh to an AChR depolarizes the muscle fibers.
ACh can be hydrolyzed by AChE, which is the main factor
controlling the duration of ACh effects. *erefore, AChE
measurement is often used to indirectly assess ACh levels.

Excessive ACh at the endplate, induced by diisopro-
pylfluorophosphate, leads to the formation of the contrac-
ture TrP nodules [15], while injection of botulinum toxin
into TrPs in New Zealand rabbits can prevent ACh release
into the synaptic space, thus reducing endplate noise levels
in myofascial trigger spot regions [16]. In addition, TrP
pathogenesis is related to the sympathetic nervous system
[17] through regulation of motor nerve synaptic vesicle
release and levels of postsynaptic membrane AChR via its
effects on the Gαi2-Hdac4-Myogenin-MuRF1 pathway [18].

In this study, AChE levels were decreased in the TrP
model compared with control and acupotomy or lidocaine
increased AChE levels. Similarly, a previous investigation
reported that ACh and AChR levels were decreased after dry
needling at precise TrPs, but AChE was increased [19].
Overall, the evidence suggests that acupotomy or dry needle
treatment may affect TrPs through regulation of AChE levels
in neuromuscular junctions.

In addition to inactivating ACh, AChE also promotes
neuromuscular junction survival [20]. In addition to

Control
 group 8 

Model
 group 8 

Acupotomy
 group 8

Injection 
group 8 

Model 
preparation 

Acupotomy

Injection

Electromyo
 graphy

TrP MPTs Samplecollection 

AChE

Free sarcoplasmic
Ca2+

ATP and AMP

SP and CGRP

1st–8th week

Statistical analysis

9th week 10–12th week 14th week and beyond13th week

Experimental flow chart

Figure 1: *e flow chart shows procedures applied to the animals in timeline. Model preparation was in the 1st–8th week. Electro-
myography detection was in the 9th week. Acupotomy and injection were conducted in the 10–12th week. MPT was detected in the 13th
week. Sample collection and other tests were in the 14th week and beyond.

20μV/D 10 ms/D

(a)

20μV/D 10 ms/D

(b)

Figure 2: (a) *e typical endplate potential, which turns negative from baseline. (b) *e fibrillation potential, which is positive from baseline
and then turns negative. *e scanning speed and sensitivity are 10 ms/D and 20 μV/D, respectively.

Evidence-Based Complementary and Alternative Medicine 5



reflecting the functional state of motor neurons [21], AChE
activity can also indicate endplate degeneration and re-
generation [22, 23]. AChE activity is markedly decreased in
denervated skeletal muscle, and the presence of AChE can be
considered a marker of nerve fiber regeneration. Hence, the
decrease in AChE in TrPs may indicate that TrPs are as-
sociated with changes in nerve or endplate function. Indeed,
neuroaxonal degeneration and neuromuscular transmission
are disordered in TrP-containing muscles [24]. In addition,
the type of muscle excitation can also regulate AChE activity
in the motor endplate, suggesting that the type of synaptic
transmission can also regulate AChE [25]. How continuous
contraction of muscle fibers at TrPs is related to decreased
AChE levels warrants further study.

4.3. Two Possible Reasons for Regulating Free Sarcoplasmic
Ca2+. Ca2+ is a chemical signal that can communicate
messages within cells [26]. Originating from motor end-
plates at myofascial TrPs, spontaneous electrical activity
(SEA) can reflect muscle fiber excitability, depending on TrP
sensitivity [27]. Decreased AChE activity increases the
amplitude of miniature excitatory postsynaptic currents,
prolonging their attenuation time and causing sustained
AChR activation that induces a Ca2+ influx [28]. Increases and
decreases in free sarcoplasmic Ca2+ directly influence skeletal
muscle fiber contraction and relaxation, and one important
characteristic of TrPs is their continuous contraction that
causes taut band formation. *e Ca2+ channel blocker ve-
rapamil can effectively inhibit SEA in myofascial trigger spots
in rabbit biceps femoris muscles [29]. Verapamil prevents
Ca2+ influx through voltage-dependent Ca2+ channels on the
plasma membrane, and it removes the inhibition of sodium
(Na+) pumps caused by increased Ca2+ loads, thereby re-
ducing intracellular Ca2+ and ensuring normal physiological
cell function. *is fully shows that intracellular Ca2+ content
can have a significant impact on the trigger point.

*is investigation provides direct proof that free sar-
coplasmic Ca2+ concentrations in the model group were
significantly higher than those in the control group. Fur-
thermore, acupotomy effectively reduced free sarcoplasmic
Ca2+ in TrP muscle cells. *e reason why acupotomy re-
duces free sarcoplasmic Ca2+ requires further study, but it
may be associated with changes in AChE and ACh and/or it
may be related to transient muscle cell Ca2+ levels.

Firstly, ACh release is related with muscle spindle.
Stecco et al. [30] believe that if the fascia outside the muscle
fiber is denser, it will hinder the shortening of the
muscle spindle and even produce a chronic stretch on the
muscle spindle, thus causing the continuous excitement of
the muscle spindle. *is explains the increased ACh from
endplates at the trigger point and the continued muscle
fibers contraction. Acupotomy or dry needling may reduce
the excitability of muscle spindles by reducing fascia
tension and ultimately reducing the ACh release and in-
tracellular Ca2+.

Secondly, Na+-Ca2+ exchanger deserves attention. *e
Na+-Ca2+ exchanger NCX is an ATP-independent bidi-
rectional transporter with two working modes. Hypoxia
leads to decreased Na+/potassium (K+)-ATPase activity and
increased intracellular Na+ concentrations, causing NCX-
mediated exchange of Ca2+ into cells and consequent Ca2+
overload and damage. In a study of the effects of acu-
puncture on Ca2+ in skeletal muscle cells, Zhuqing et al.
inferred that local acupuncture for delayed onset muscle
soreness may affect NCX function, thereby inhibiting cy-
toplasmic Ca2+ overload [31].

∗

#

Control Model Acupotomy Injection
30

40

50

60

70

Figure 4: AChE concentrations (pg/mg) in each group (control,
model, acupotomy, and injection; n � 8 per group). ∗P<0.05
compared with the control group, #P<0.05 compared with the
model group, ΔP<0.05 compared with the model group, and
▲P>0.05 compared with the acupotomy group. *e error bars
represent the standard deviations.

Table 1: Changes in AChE levels after acupotomy (x ± s).

Groups N AChE (pg/mg)
Control 8 50.91±14.88
Model 8 32.18±7.27∗
Acupotomy 8 46.70±7.88#
Injection 8 46.35±9.99▲Δ

Versus control group: P∗<0.05; versus model group: P#<0.05; versus
model group: PΔ<0.05; versus acupotomy group: P▲>0.05.

50

100

150

200

250

300

350

1 2 3 5 7

MTrPs VF threshold (g) of four groups

Control
Model

Acupotomy
Injection

V
F 

th
re

sh
ol

d 
(g

)

Time (day)

Δ
Δ

Δ
Δ

Δ

Figure 3: MPT changes (g) in each group (control, model, acu-
potomy, and injection; n � 8 per group) determined by the Von
Frey mechanical pain stimulator test. ■P<0.05 versus the model
group. ΔP<0.05 versus the model group. ◆P<0.05 versus the
model group. *e error bars represent standard deviations.

6 Evidence-Based Complementary and Alternative Medicine



4.4. ATP and ADP Indicate Energy Supply. *e persistent
contraction of muscle fiber TrPs increases local energy
consumption and restricts blood circulation. ATP produced
by oxidative phosphorylation is the direct source of energy
for muscle contraction and sarcoplasmic reticulum Ca2+
reabsorption. A lack of ATP in the muscle reduces its ability
to function, including both contraction and relaxation.
Adenosine 5′-diphosphate (ADP) and AMP are degradation
products of ATP and have important functions in fatigue
[32, 33]. In this study, ATP levels were lower in the model
group than control group, which is consistent with the
integrated hypothesis. Furthermore, this decrease was at-
tenuated following acupotomy. *is effect may be caused by
improved blood circulation in TrPs in response to decreased
free sarcoplasmic Ca2+.

4.5. CGRP Enhances ACh Effect at Motor Endplate.
Continuous contraction leads to local ischemia and
hypoxia in TrPs, which can cause the tissue to release
vasoactive substances that act on nociceptors, leading to
nerve sensitization, pain, and Ach release from nerve
endings [34]. Using microanalytical techniques, Shah
et al. found that concentrations of protons, bradykinin,
CGRP, SP, tumor necrosis factor-alpha, interleukin-1
beta, serotonin, and norepinephrine were significantly
higher in active TrPs [6, 35, 36]. Muscle pain is associated
with muscle nociceptor activation by a variety of

endogenous substances including neuropeptides and
inflammatory mediators. SP and CGRP can cause plasma
extravasation, inflammation, and nociceptive effects [6]. CGRP
exists in the ends of ɑ motor neurons and promotes ACh
release into the synaptic cleft [15]. Importantly, in relation to
TrPs, CGRP increases the number of AChRs and inhibits
AChE activity at motor endplate [37, 38], thereby enhancing
the effect of ACh at the motor endplate. Gerwin and Shah
hypothesized that CGRP enhances the motor endplate re-
sponse to ACh by increasing the activity and synthesis of
AChRs [39]. In this investigation, we found significantly ele-
vated levels of SP and CGRP in tissue from TrPs. Furthermore,
both SP and CGRP were significantly lower in the acupotomy
group than the model group, which is consistent with the
common observation of decreased pain after TrP treatment.
Acupuncture increases local nitric oxide and microcirculation
[40, 41], so local microcirculation in TrPs should be evaluated
before and after needling to determine whether it dilutes
algogenic substances.

In Figures 7 and 8, images of acupotomy and injection
groups show lymphocyte infiltration and destruction of the
fiber order, which may indicate the healing process of in-
juries. Because both acupotomy and injection are invasive, is
the healing process of injuries related to treatment effect?

Table 3: Changes in ATP/AMP levels after acupotomy (x ± s).

Groups N ATP (ng/mg) AMP (ng/mg)
Control 8 1322.15±516.51 85.60±114.29
Model 8 298.34±181.82∗ 239.18±71.05∗∗
Acupotomy 8 734.62±344.53# 108.76±89.89##
Injection 8 490.73±280.36Δ 208.49±101.65ΔΔ

Versus control group: P∗<0.05; versus model group: P#<0.05; versus
model group: PΔ>0.05; versus control group: P∗∗<0.05; versus model
group: P##<0.05; versus model group: PΔΔ>0.05.

∗

∗∗

#

##

Δ

ΔΔ

0
200
400
600
800

1000
1200
1400
1600
1800
2000

ATP ng/mg AMP ng/mg

A
P/

A
M

P 
(n

g/
m

g)

Control
Model

Acupotomy
Injection

Figure 6: ATP and AMP levels (ng/mg) in each group (control,
model, acupotomy, and injection; n � 8 per group). ∗P<0.05 for
ATP levels compared with the control group, #P<0.05 for ATP
levels compared with the model group, ΔP>0.05 for ATP levels
compared with the model group, ∗∗P<0.05 for AMP levels
compared with the control group, ##P<0.05 for AMP levels
compared with the model group, and ΔΔP>0.05 for AMP levels
compared with the model group. *e error bars represent the
standard deviations.

∗

#

70

90

110

130

150

170

190

210

Control Model Acupotomy Injection

Figure 5: Free sarcoplasmic Ca2+ concentrations (nmol/L) in each
group (control, model, acupotomy, and injection; n � 8 per group).
∗P<0.01 compared with the control group, #P<0.05 compared
with the model group, ΔP<0.05 compared with the model group,
and▲P>0.05 compared with the acupotomy group. *e error bars
represent the standard deviations.

Table 2: Changes in free sarcoplasmic Ca2+ levels after acupotomy
(x± s).

Groups N Free sarcoplasmic Ca2+ (nmol/L)
Control 8 74.02±32.71
Model 8 161.81±46.14∗
Acupotomy 8 105.52±26.52#
Injection 8 109.1±24.41▲Δ

Versus control group: P∗<0.05; versus model group: P#<0.05; versus
model group: PΔ<0.05; versus acupotomy group: P▲>0.05.

Evidence-Based Complementary and Alternative Medicine 7



Stecco et al. [30] believe that the increased fascia density
outside the muscle fiber may excite muscle spindle, causing
increased ACh from endplates and continued contraction.
Acupotomy or dry/wet needling may reduce the excitability
of muscle spindles by changing the inner environment
around the fibers.

4.6. Relationship between TrP and Ashi Points. Herman et al.
proposed the concept of TrPs in 1942 [42]. Since accepting
the concept of TrPs, some Chinese scholars believe that
TrPs and TCM ashi points exhibit considerable overlap in
their positions, needling sensation, and indications [43].
When investigations into TrPs began, TCM practitioners
believed that TrPs can be considered typical representative
ashi points. But a contrary opinion is that the overlap of a
number of pain points does not change that basic dif-
ference between acupoints and TrPs. Acupoints and TrPs
are derived from vastly different concepts [44]. No matter
the two concepts have similarities or differences in es-
sence, it will be an hint to promote the reseach of ashi
points in TCM. Although ashi points have been under-
stood by practitioners of TCM for several centuries and
various acupuncture methods have been applied in TCM
to treat ashi points, deep research into ashi points has
been lacking.

(a) (b)

(c) (d)

∗

#

Control Model Acupotomy Injection

SP IOD

1400
1500
1600
1700
1800
1900
2000
2100

(e)

Figure 7: Immunohistochemistry for SP (light microscopy, ×200). SP expression was yellowish brown in muscle fibers. SP and CGRP levels
by group (control, model, acupotomy, and injection; n � 8 per group). ∗P<0.05 for SP compared with the control group; #P<0.05 for SP
compared with the model group; ΔP<0.05 for SP compared with the model group;▲P>0.05 for SP compared with the acupotomy group.
*e error bars represent the standard deviations. (a) Control group. (b) Model group. (c) Acupotomy group. (d) Injection group. (e) SP IOD.

Table 4: Changes in SP/CGRP IOD after acupotomy (x ± s).

Groups N SP CGRP
Control 8 1468±195.24 1412.63±178.38
Model 8 1866.38±165.38∗ 1770.3±186.90∗∗
Acupotomy 8 1591.25±102.97# 1502.38±156.35##
Injection 8 1640.75±197.48Δ 1578.13±148.58ΔΔ

Versus control group: P∗<0.05; versus model group: P#<0.05; versus
model group: PΔ<0.05; versus acupotomy group: P▲>0.05; versus control
group: P∗∗<0.05; versus model group: P##<0.05; versus model group:
PΔΔ<0.05; versus acupotomy group: P▲▲>0.05.

8 Evidence-Based Complementary and Alternative Medicine



5. Conclusion

Developed from ancient acupuncture techniques, acupot-
omy is similar to dry needling; both require accurate in-
sertion of needles into local TrPs, and they may also work via
similar mechanisms. *e observations from our study
support the hypothesis that energy crises affect TrP muscle
fibers. Furthermore, our data demonstrate that acupotomy
can influence TrP MPTs and levels of AChE, free sarco-
plasmic Ca2+, ATP, AMP, SP, and CGRP. Its effects were
similar to local lidocaine injection, except only acupotomy
affected ATP and AMP levels. It is indicated that acupotomy
has effect on trigger point pathophysiology, which is dif-
ferent from acupuncture analgesia. Future research will
focus on the mechanism by which acupotomy resolves
energy crises in TrPs, peripheric or central?

*e general understanding of acupuncture mechanism is
that the mechanical signals generated by needle insertion are
converted into nerve signals through the acupoint and
transmitted to the central nervous system and on to the
efferent system and effector organs via the neuro-endocrine-
immune system [45]. Further, acupuncture may have pe-
ripheral effects, independent of overall neurohumoral reg-
ulation [46, 47]. Zhuqing et al. [48] found that local
acupuncture is effective in delaying muscle soreness onset
because it adjusts the catabolism and anabolism of muscle
contractile proteins. *is differs from the mechanism in-
volved in acupuncture analgesia. *e same effect was ob-
served in isolated semitendinosus muscle experiments,
suggesting that acupuncture may have a peripheral mech-
anism independent of overall neurohumoral regulation. For
visceral disorders, it is necessary for acupuncture to work via

(a) (b)

(c) (d)

∗

#

1400

1500

1600

1700

1800

1900

2000

Control Model Acupotomy Injection

CGRP IOD

(e)

Figure 8: Immunohistochemistry for CGRP (light microscopy, ×200). CGRP expression was yellowish brown in muscle fibers. CGRP levels
were significantly higher in the model group than the control (P<0.05), acupotomy (P<0.05), and injection (P<0.05) groups. *ere was no
significant difference in CGRP content between the acupotomy and injection groups (P>0.05). SP and CGRP levels by group (control,
model, acupotomy, and injection; n � 8 per group). ∗P<0.05 for CGRP compared with the control group; #P<0.05 for CGRP compared
with the model group;▲P>0.05 for CGRP compared with the acupotomy group;ΔP<0.05 for CGRP compared with the model group. *e
error bars represent the standard deviations. (a) Control group. (b) Model group. (c) Acupotomy group. (d) Injection group. (e) CGRP IOD.

Evidence-Based Complementary and Alternative Medicine 9



the neuro-endocrine-immune system because the viscera
cannot be needled directly. But for myofascial lesions, it is
different because muscular fasciae can be needled directly.
For myofascial disorder, what will happen when it is needled
directly? Acupuncture was shown to trigger a local increase
in adenosine in human subjects [49, 50] and to increase local
microcirculation [40, 41]. It is still controversial whether a
trigger point is a central or peripheral phenomenon [11].
Accurate positioning at TrPs is an important requirement
for both acupotomy and dry needling, suggesting that the
effects of these procedures may have similar underlying
peripheral mechanisms; however, a review reported that due
to the scarcity of reliable studies, the current evidence on the
local effects of acupuncture is insufficient to draw reliable
conclusions [51]. Additional investigations are needed to
verify the local effects of acupuncture.

*e limitation of this study is that only one method was
used to test each variable; therefore, our data lack verifi-
cation with multiple experimental methods. And several
anesthesia procedures during the experiment may influence
the results.

Data Availability

*e data used to support the findings of this study are
available from the corresponding author upon request.

Conflicts of Interest

*e authors declare that there are no conflicts of interest
regarding the publication of this paper.

Acknowledgments

*e research program was funded by the National Natural
Science Foundation of China (no. 81503653).

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