• 617 •Chin J Integr Med 2017 Aug;23(8):617-624

Chinese tuina is the earliest treatment method found in Chinese medicine (CM), originally being called massage or anqiao in ancient times.(1) Chinese Tuina is a therapeutic approach guided by the theory of CM used to treat diseases through massage manipulations or by means of some massage tools applied to certain parts or points on the human body.(2) It belongs to the larger category of external treatment methods in CM. As its advantage is preventing and curing diseases without side effects, Chinese Tuina has become one of the most popular therapies in complementary and alternative medicine (CAM) in China.

Peripheral nerve injury (PNI) is one of the most common diseases in departments of tuina in hospitals. PNIs are becoming increasingly familiar due to the increasing frequency of trauma incidents resulting from motor vehicle accidents. Fractures, lacerations, crush injuries, and surgical complications that cause direct or indirect nerve compression from oedema and haematomas are just a few examples of the growing list.(3,4) PNI results in motor (amyosthenia and amyotrophy)(5,6) and sensory (hyperalgesia, allodynia, analgesia and/or numbness)(6-8) disability. Although it is not always life threatening, it usually results in limited mobility for an

extented period of time or even life-long disability.(9,10) Despite rigorous surgical techniques and different repair methods, full functional recovery is rarely achieved. Non-surgical approaches like electrical stimulation,(11-15) laser phototherapy,(16-19) acupuncture(20) and exercise(15) have been developed as alternatives or complements to surgery, which can enhance nerve recovery.

Tissue plasminogen activator (tPA) is widely expressed in the nervous system by neurons and

PAN Fan1, YU Tian-yuan1, Steven Wong1, XIAN Si-tong1, LU Meng-qian1, WU Jian-cong2, GAO Yu-feng3, LI Xiao-qin4, GENG Nan5, and YAO Bin-bin1

©The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2015Supported by the National Natural Science Foundation of China (No. 81373759), Research Fund for the Doctoral Program of Higher Education (No. 20130013110016), Natural Science Foundation of Beijing (No. 7142097)1. Collage of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing (100029), China; 2. The 2nd Massage Department, Beijing Massage Hospital, Beijing (100035), China; 3. Department of Encephalopathy of Mogol Medicine, Affi liated Hospital of Inner Mongolia University for the Nationalities, Tongliao, Inner Mongolia Autonomous Region (028000), China; 4. Department of Acupuncture and Moxibustion, Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang (330019), China; 5. Department of Massage Physiotherapy, Beijing University of Chinese Medicine Dongfang Hospital, Beijing (100078), ChinaCorrespondence to: Prof. YU Tian-yuan, Tel: 86-10-64286744, E-mail: yutianyuan@sina.comDOI: 10.1007/s11655-015-2142-1

ABSTRACTABSTRACT ObjectiveObjective: To elucidate the mechanism of Chinese tuina in treating sciatic nerve crush injury, and : To elucidate the mechanism of Chinese tuina in treating sciatic nerve crush injury, and to detect the levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), which to detect the levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), which is thought to play an important role in nerve regeneration. is thought to play an important role in nerve regeneration. MethodsMethods: Thirty-two adult male Sprague-Dawley rats : Thirty-two adult male Sprague-Dawley rats were subjected to sciatic nerve crush injury and 16 rats (sham-operated group) went through a sham operation. were subjected to sciatic nerve crush injury and 16 rats (sham-operated group) went through a sham operation. Control group was given no treatment while tuina group received tuina therapy since day 7 post-surgery. Tuina Control group was given no treatment while tuina group received tuina therapy since day 7 post-surgery. Tuina treatment was performed once a day and lasted for 20 days. The sciatic functional index was examined every 5 treatment was performed once a day and lasted for 20 days. The sciatic functional index was examined every 5 days during the treatment session. The rats' gastrocnemius muscles were evaluated for changes in mass and days during the treatment session. The rats' gastrocnemius muscles were evaluated for changes in mass and immunohistochemistry techniques were performed to detect the levels of tPA and PAI-1. immunohistochemistry techniques were performed to detect the levels of tPA and PAI-1. ResultsResults: Tuina therapy : Tuina therapy improved the motor function of sciatic nerve injured rats (improved the motor function of sciatic nerve injured rats (P<0.05), however, it did not increase muscle volume <0.05), however, it did not increase muscle volume (P<0.05). Tuina downregulated the levels of tPA and PAI-1 (<0.05). Tuina downregulated the levels of tPA and PAI-1 (P<0.05). <0.05). ConclusionsConclusions: The present study implies that : The present study implies that tuina treatment could accelerate rehabilitation of peripheral nerve injury.tuina treatment could accelerate rehabilitation of peripheral nerve injury.KEYWORDSKEYWORDS Chinese medicine, physical therapy, peripheral neuropathy, massage Chinese medicine, physical therapy, peripheral neuropathy, massage

Acupuncture Research

Chinese Tuina Downregulates the Elevated Levels of Tissue Plasminogen Activator in Sciatic Nerve Injured

Sprague-Dawley Rats

Available online at link.springer.com/journal/11655Journal homepage: www.cjim.cn/zxyjhen/zxyjhen/ch/index.aspxE-mail: cjim_en@cjim.cn

hinese Journal of Integrative MedicineC

• 618 • Chin J Integr Med 2017 Aug;23(8):617-624

some glial cells, and its activity is upregulated by neurons during both normal and pathological events that lead to neural plasticity.(21) Mice lacking the tPA or plasminogen gene showed increased axonal degeneration and myelin sheath breakdown causing delayed functional recovery after sciatic nerve injury.(22,23) The tissue plasminogen activator system degrades fi brin and facilitates the degradation of other extracellular matrix (ECM) proteins.(24) It is thought to assist cell migration, chemotaxis, and tissue remodeling during many physiological and pathological processes.(25,26) As one of the inhibitors of tPA, plasminogen activator inhibitor-1 (PAI-1) can inhibit and modulate these interactions. PAI-1, which is synthesized by macrophages and monocytes is a potent chemoattractant molecule that induces cell migration through changes in cell morphology and cytoskeleton organization in response to tissue injury or infl ammatory processes.(27) Via this mechanism, PAI-1 can protect against the neuronal cell injury caused by tPA. Apart from this, PAI-1 prevents the disintegration of already formed neuronal networks by maintaining or promoting neuroprotective signaling through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway.(28)

Laboratory research has not yet been conducted or reported on how tuina affects the tissue plasminogen activator system, since most current studies on the effects of tuina therapy on PNI are in the form of clinical reports. Therefore, the primary aim of this study was to determine whether the neuronal tPA system is infl uenced by tuina treatment after nerve crush injury.

METHODS

AnimalsThe protocols were conducted in compliance

with the Guidance Suggestions for the Care and Use of Laboratory Animals formulated by the National Institute of Health, as well as the 3R principle: reduction, replacement, and refinement. All experiment procedures were approved by the Animal Care and Use Committee at Beijing University of Chinese Medicine. Forty-eight young adult male Sprague-Dawley rats (Charles River, Beijing, China, body weight 170–195 g) were kept in a temperature-controlled room (23±2 ℃) , 45% humidity, with a 12-h light/dark cycle (lights on at 8:00 a.m.) and free access to water and food. Experiments were performed between 8:00 a.m. and 12:00 a.m. All

efforts were made to minimize the number of animals used and their discomfort.

Surgical ProcedureThe animals were anaesthetised with an

intraperitoneal injection of a premixed solution containing 10% chloral hydrate (350 mg/kg body weight). After shaving and preparing the skin with 10% povidone iodine, the right sciatic nerve was exposed using the gluteal-splitting approach.

Thirty-two rats were subjected to nerve crush injury and the remaining 16 rats were subjected to gluteal-splitting without damaging the sciatic nerve to serve as the sham-operated group (n=16). The sciatic nerves of 32 rats were exposed at midthigh level and crushed for 30 s with a pair of non-serrated forceps and then the skin was sutured with 4–0 stitches.

Following surgery, the rats given crush injury were randomly divided into two groups: the rats that received no treatment were used as a control (n=16), whereas the rats in the tuina group (n=16) were given tuina therapy once a day since the 7th day after surgery.

TreatmentA tuina manipulation emulator (patent No.

ZL200710187403.1) was designed in 2007. It is designed to stimulate tuina techniques while maintaining qualitative and quantitative controls. The emulator's key structural elements are a stepper motor, disc and contact point. The pressure sensor and the contact point are connected by a metallic strip. This structure guarantees the sensitivity of the sensor and prevents abrasion due to long-term use. The amount of pressure is adjusted using a lead screw. The amount of pressure is displayed on the screen. The contact point of the stimulator is a cylinder with a diameter of 10 mm. The emulator was used to perform pressing method, strumming method, and circular rubbing method on Huantiao (GB30), Yanglingquan (GB34), and Chengshan (BL57) in turn on the affected side. The frequency was set to 30 times/min and the force was set to 0.98 N. Each method was used for 1 min on each acupoint. Tuina treatment was performed once a day and lasted for 20 days. According to the principle of comparative anatomy, GB30 is situated at the junction of the lateral 1/3 and medial 2/3 of the line connecting the prominence of the greater trochanter of the femur with the sacral hiatus on the rat. GB34

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is located in the depression anterio-inferior to the capitulum of the fi bula. BL57 is located in the center of the posterior of the leg, between the two heads of the gastrocnemius.

the rats were anesthetized with chloral hydrate and perfused intracardially with normal saline, followed by 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS, pH 7.4). The gastrocnemius muscle and a 1-mm-long sciatic nerve segment were carefully dissected from a point 2 mm distal to the crush site; half of each group was dissected at 7 days post-injury, and the other half at 28 days post-injury. As an initial indication of trophic variation, wet muscles were immediately weighed with an analytical balance (AUX120, Shimadzu, Japan). The gastrocnemius muscles and sciatic nerves were immersed in the same fixative for 6 h and then placed into 30% sucrose in 0.1 mol/L PBS solution overnight.

Quantifi cation of Muscle AtrophySciatic nerve injury (SNI) can lead to gastrocnemius

atrophy as a result of muscle denervation; the severity of atrophy depends on nerve damage and nerve regeneration.(30) A direct measurement of sciatic nerve functional recovery after crush can be obtained by measuring muscle atrophy index (MAI). MAI is defi ned by muscle weight (mg) divided by body weight (g). The empty stomach weights of the rats pre-dissection and their gastrocnemius muscles (post-dissection) were measured.

ImmunohistochemistryTransverse sections (4 μm) were cut using a

rotary microtome (Leica RM2235) and mounted on glass slides. Sections were deparaffi nized with alcohol and xylene, incubated in 1% hydrogen peroxide, and blocked with 2% bovine serum albumin. Primary antibodies against tPA (No. ab157469, Abcam, USA) and PAI-1 (No. ab66705, Abcam, USA) were added at a dilution of 1:5000 and 1:600. Secondary antibodies (Haopoly-HRP, Shanghai, China) were added at a dilution of 1:200. To visualize the resulting stains, DAB kits (ZSGB-BIO, Beijing, China) were used. Images were acquired using a light microscope (BX53, Olympus, Tokyo, Japan). The protein expressions were quantifi ed by integral optical density (IOD) using Image Pro Plus 6.0 software. For each rat, 3 to 4 nerve sections were analyzed and a mean IOD value was obtained, the microscope technician was blinded to the identity of the slides. Standard optical density was corrected for by selecting the brightest areas of the images as background, and hue-intensity-saturation (HIS) color mode was used to select and count tPA- or PAI-stained areas, respectively.

Figure 1. Tuina Manipulation Emulator Applied as Intervention

Walking Track AnalysisRats were tested in a conf ined walkway

measuring 80-cm-long and 10-cm-wide, with a dark shelter at the end. A white paper was placed on the fl oor of the rat walking corridor. The hind paws of the rats were pressed down onto a finger paint-soaked sponge, and the rats were then allowed to walk down the corridor leaving their hind footprints on the paper. Several measurements were taken from the footprints: (1) distance from the base of the heel to the tip of the third toe, the print length (PL); (2) distance from the first to the fifth toe, the toe spread (TS); and (3) the distance from the second to the fourth toe, the intermediary toe spread (ITS). All three measurements were taken on the experimental (E) and normal (N) sides. The SFI was calculated as described by Bain, et al(29) according to the following equation:

SFI=–38.3 (EPL–NPL

NPL )+109.5 (ETS–NTSNTS )+13.3 (EIT–NIT

NIT ) –8.8

The SFI oscillates around 0 for normal nerve function, and –100 or less represents total dysfunction. Records of the footprints were obtained before surgery and on the following days post-treatment: day 0, 5, 10, 15 and 20.

Perfusion and Tissue ProcessingAt the end of the postoperative treatment period,

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Statistical AnalysisData were presented as means ± standard

error of mean (SEM). SPSS 20.0 (SPSS Inc, Chicago, USA) was utilized for one-way ANOVA data analysis after the test of normal distribution and homogeneity of variance were performed, followed by post hoc multiple comparison. A P-value less than 0.05 was considered to be statistically signifi cant.

RESULTS

Effects of Tuina Treatment on SFIThe baseline SFI values were similar for all three

groups of rats prior to surgery (Figure 2). However, a remarkable reduction (P<0.01) in SFI was observed in animals subjected to sciatic nerve crush injury in the control and tuina groups compared with the sham-operated group (–110.05±14.56 and –107.33±10.77 vs. –15.20±6.72, respectively) 1 week post-operation (on treatment day 0). On the 5th day post-treatment, the control and tuina groups presented a recovering tendency, but remained signif icantly different (F=183.03, P<0.01) as compared with the sham-operated group (–88.97±12.19 and –73.57±6.28 vs. –9.90±6.51, respectively). On the 10th day, the tuina group (–44.68±8.55) showed a more obvious positive tendency than the control group (–80.46±4.88). C o m p a r e d w i t h t h e s h a m - o p e r a t e d g r o u p (–4.38±6.67), the differences were still statistically signifi cant (F=245.64, P<0.01). On the 15th day post-treatment, there were signifi cant differences (F=245.64, P<0.01) among the sham-operated (–9.16±4.08), control (–49.21±9.23) and tuina groups (–28.36±7.40). The tuina group showed significant improvement compared with the control group (P<0.05). After 20 days of treatment, the SFI of the control and tuina groups were still signifi cantly lower (F=19.28, P<0.01) compared with that of the sham-operated group (–35.29±6.50 and –13.56±6.42 vs. –5.97±14.31, respectively), and significant differences were found between the control and tuina groups as well (P<0.05).

Effects of Tuina Treatment on Muscle RecoveryThe consecutive changes of the gastrocnemius

we igh t and MAI be fo re and a f te r tu ina a re demonstrated in Figure 3.

On day 0 post-treatment (1 week post-operation), gastrocnemius muscles from the control and tuina groups weighed less than those from the sham-operated group (703.0±122.7 and 708.0±140.0 vs. 1324.0±193.1 mg,

Figure 2. Assessment of SFI in Rats Subjected to Sciatic Nerve Crush Injury ( ±SEM, n=8)

Notes: P<0.05, compared with the sham-operated group; △P<0.05, compared with the control group

SF

I

Baseline

20.00

0

–20.00

–40.00

–60.00

–80.00

–100.00

–120.00

–140.00

Day 0 Day 5 Day 10 Day 15 Day 20Sham-operated

Control

Tuina

△

△

△

△

Figure 3. Gastrocnemius Wet Weight (A) and MAI (B) for Each Group ( ±SEM, n=8)

Notes: P<0.05, compared with the sham-operated group; △P<0.05, compared with the control group

Gas

troc

nem

ius

wet

w

eigh

t (m

g)

2500

2000

1500

1000

500

0Day 0 Day 20

Sham-operated

Control

Tuina

A

Gas

troc

nem

ius/

body

w

eigh

t (m

g/g)

8.00

6.00

4.00

2.00

0Day 0 Day 20

Sham-operated

Control

Tuina

B

△

△

respectively), indicating that sciatic nerve crush injury elicited significant atrophy (F=53.26, P<0.01) in the gastrocnemius muscles. On 20th day post-treatment, gastrocnemius weights in the control and tuina group were significantly lighter (F=79.41, P<0.01) compared with those in the sham-operated group (703.0±182.6 and 708.0±145.3 vs. 2027.0±183.5 mg, respectively), whereas no signifi cant differences were found between the control and tuina groups.

On day 0 post-treatment, the MAI values in the control and tuina groups decreased significantly (F=62.40, P<0.01) in comparison with that in the sham-operated group (2.81±0.47 and 2.80±0.50 vs. 5.35±0.75, respectively). On the 20th day post-treatment, the MAI value of the sham-operated group (4.99±0.32) was significantly higher (F=87.22, P<0.01) compared with that of the other two groups. However, the tuina group showed a significant increase compared with the control group (3.70±0.44 vs. 2.98±0.26, P<0.05).

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Effects of Tuina Treatment on tPAAs shown in Table 1 and Figure 4A, a signifi cant

increase (F=853.15, P<0.01) in tPA was found in the sciatic nerves of rats subjected to sciatic nerve crush injury. Immunostaining-positive cells in the endoneurium and myelin sheath in the control and tuina groups became visibly darker in color. On the 20th day post-treatment, the expression of tPA in the control and tuina groups were significantly higher compared with that of the sham-operated group (F=119.71, P<0.01; Table 1, Figures 4B and 4C). Treatment with tuina exhibited significant decreases (LSD post hoc test, P<0.05) in tPA in the sciatic nerve as compared with that of the control group.

Effects of Tuina Treatment on PAI-1A significant increase in PAI-1 was found in

the control and tuina groups due to the surgery (F=41.74, P<0.01; Table 1). As shown in Table 2 and Figures 4E and 4F), on the 20th day post-treatment, IOD values in the control and tuina groups of PAI-1 were significantly increased compared with that in the sham-operated group (F=81.12, P<0.01). The expression of PAI-1 in tuina group presented a decline tendency, and this decrease was signifi cant in comparison with control group (P<0.05). However,

the IOD value of PAI-1 in the control group showed the opposite tendency.

DISCUSSION

As shown by research on the mechanism of Chinese tuina for several diseases, it can: (1) facilitate the circulation of blood and lymph fluid by exerting pressure on the skin and muscles, therefore increasing the body surface temperature;(31) (2) decrease blood viscosity and erythrocyte aggregation(32) by adjusting the endocrine function of vascular endothelial cells;(33) (3) relieve pain both instantly and between treatments sessions,(34) by increasing the β-endorphin levels and decreasing substance p levels in the blood.(35)

Our previous study demonstrated that tuina treats SNI by increasing the expression of both nerve growth factor (NGF) and its high receptor TrkA,(36) however, the expression of the low affi nity NGF receptor p75NTR was decreased.(37) We also found that tuina promoted the expression of two axoplasmic transporting motor proteins kinesin and dynein.(38) These fi ndings provide evidence that tuina accelerates the transportation of rat sciatic nerve axoplasm.

To further determine whether Chinese tuina

Table 1. IOD Value of tPA ( ±SEM)

GrouptPA PAI-1

Day 0 Day 20 Day 0 Day 20

Sham-operated 18.00±1.15 21.70±1.44△ 3.17±0.63 3.10±0.77△

Control 52.50±2.23 33.63±1.94 5.57±0.87 8.72±0.96

Tuina 53.63±2.85 23.88±2.07 △ 5.91±0.67 5.02±1.22 △

Notes: P<0.05, compared with the sham-operated group; △P<0.05, compared with the control group

Figure 4. Representative Photomicrographs of tPA and PAI-1 in Each Group (Immunohistochemical staining, ×400) Notes: A, D: control group on day 0; B, E: control group on day 20; C, F: tuina group on day 20. Scale bar=50μm

Control group (Day 0)

tPA

PAI-1

Control group (Day 20) Tuina group (Day 20)

A B

D E F

C

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has an impact on synaptic plasticity, in this study we evaluated the effects of Chinese tuina therapy on functional recovery and tissue plasminogen activator levels after sciatic nerve crush injury. The major fi nding of the present study is that Chinese tuina improved functional recovery and modulated the expression of tPA and PAI-1 in sciatic nerve injured rats.

Nerve crush injury is a well-established PNI model in experimental neural regeneration studies used to investigate the impact of various treatments. (39) It is relatively inexpensive, easy to handle, and the capacity for regeneration is equivalent in rats and subhuman primates.(40)

Since the introduction of the SFI in 1982,(41) it has become a mainstay in the assessment of global functional recovery after SNI.(42) In the present study, the SFI was analyzed in each group consecutively up to the 20th day post-treatment in order to observe differences between the groups over time. On the 20th day, SFI values of subjects from the tuina group were close to those of the sham-operated group. On day 20, gastrocnemius muscle wet weight from the control group was less than that from tuina group. Although this difference did not quite reach statistical signifi cance, it refl ects a potential for tuina to protect against muscle loss in sciatic nerve injured rats. However, as there were losses of both body weight and muscle weight, when muscle weights were normalized to body weight, the MAI of the tuina group was signifi cantly higher than that of the control group. These data demonstrate the ability of tuina to stimulate increases in local muscle mass. Both SFI and MAI suggest tuina therapy can accelerate functional recovery of damaged peripheral nerves.

It has been shown that exogenous tPA or tPA/plg promotes nerve regeneration after injury via two pathways: one is proteolytic lysis of fibrin and the other is a nonproteolytic pathway associated with macrophage activation and matrix metallo proteinase (MMP)-9 expression.(43)

According to the results, the expression of tPA and PAI-1 were unregulated in the injured sciatic nerve in response to the crush injury on day 0 post-treatment. The PA system converts plasminogen to the proteolytically active plasmin, which includes PAI-1 and tPA. In regenerating nerves, it has been

suggested that the degradation of the ECM by the PA system helps pave a road for regenerating axons at the site of injury.(44) However, after 20 days of treatment, tPA and PAI-1 levels declined in the tuina group with statistical signifi cance in comparison with those of the control group. We propose that tuina treatment could accelerate the removal of fibrin deposition and myelin debris and prevent collagen scar formation, thereby supplying a similar function as that of the PA system while both decreasing the secretion of tPA and PAI-1 and increasing peripheral nerve regeneration.

According to CM, SNI refers to Bi syndrome (arthromyodynia) or Wei syndrome (fl accidity), usually caused by meridian obstruction. Prof. YU Tian-yuan proposed the "acupoint-nerve-muscle" theory based on years of clinical practice and scientific research and it was used in selecting acupoints for this study. Huantiao (GB30), Chengshan (BL57), Yanglingquan (GB34) are distributed along the sciatic nerve, common peroneal nerve and tibial nerve respectively. The three acupoints are respectively located on the gluteus maximus muscle, gastrocnemius muscle, and anterior tibial muscle. Based on CM channel theory, Huantiao (GB30) and Yanglingquan (GB34) belong to Shaoyang Gallbladder Channel of the Foot and Chengshan (BL57) belongs to Taiyang Bladder Channel of the Foot. Therefore, the tuina manipulations were designed to stimulate both anatomic structures and meridians.

One limitation of the present study is the use of an animal model. The treatment procedure may be misconstrued by the rats as a threat and cause them to panic, which may influence treatment efficacy. Second, considering the individual differences in size and sensitivity between animal subjects, the force and duration of each manipulation applied should be changed accordingly. Third, in order to minimize the number of animals sacrificed for this study, the expression of tPA and PAI-1 were only observed on day 0 and day 20 post-treatment. Therefore, we were unable to measure tPA and PAI-1 secretion levels at intermediary times to clarify the intermediate rate of change.

An increase in gastrocnemius muscle wet weight suggests that tuina can increase muscle volume, although this increase was not statistically signifi cant.

• 623 •Chin J Integr Med 2017 Aug;23(8):617-624

However, the MAI value did increase signifi cantly. More importantly, the expression of tPA and PAI-1 suggests tuina intervention can facilitate the regeneration of injured peripheral nerves. In summary, our study suggests that Chinese tuina can bring a relevant contribution to the rehabilitation of peripheral nerve injury.

Confl ict of InterestAll authors have no confl ict of interest to disclose.

Author ContributionsPan F and Xian ST were responsible for the design and

performance of the main experiments. Yu TY was responsible

for the design and guidance in all works. Steven W modified

language expressions throughout the article. Lu MQ took charge of

immunohistochemistry analysis. Wu JC and Gao YF took charge

of functional evaluation. Li XQ and Geng N were responsible for

perfusion and tissue processing. Yao BB helped with data analysis.

Acknowledgment The authors wish to thank YU Yue (The People's Hospital

of Ji County, Tianjin) for her technical assistance.

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(Received August 27, 2014; First Online May 13, 2015)Edited by YUAN Lin

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