ipsi- and contralateral moxibustion generate similar

Research ArticleIpsi- and Contralateral Moxibustion Generate Similar AnalgesicEffect on Inflammatory Pain

Chuan-Yi Zuo Peng Lv Cheng-Shun Zhang Ru-Xue Lei Wei Zhou Qiao-FengWu Ling Luo Yong Tang Hai-Yan Yin and Shu-Guang Yu

Acupuncture and Tuina School Chengdu University of Traditional Chinese Medicine Chengdu 610075 Sichuan China

Correspondence should be addressed to Yong Tang tangyongcn126com Hai-Yan Yin yinhaiyancdutcmeducnand Shu-Guang Yu ysgcdutcmeducn

Received 30 August 2018 Revised 17 October 2018 Accepted 21 November 2018 Published 5 February 2019

Academic Editor Olumayokun A Olajide

Copyright copy 2019 Chuan-Yi Zuo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to investigate whether contralateral moxibustion would generate a similar analgesic effect with ipsilateralmoxibustion Contra- and ipsilateral moxibustion were separately applied to Zusanli (ST36) acupoints of inflammatory painmice The analgesic effect was evaluated respectively by lickingbiting time (LBT) of formalin-induced inflammatory pain andthermal withdrawal latency (TWL) of complete Freundrsquos adjuvant- (CFA-) induced inflammatory pain For formalin-induced paincompared with formalin group the total LBT of ipsi- and contralateral moxibustion reduced in both phase I and phase II but therewas no significant difference between ipsi- and contralateral moxibustion For CFA-induced inflammatory pain compared withCFA group TWL of ipsi- and contra-Moxi groups increased immediately after moxibustion intervention however there was noobvious difference between ipsi- and contralateral moxibustion at any timepoint It indicated that contralateral moxibustion had asimilar analgesic effect with ipsilateral moxibustion in both formalin- and CFA-induced pain These results suggest that both ipsi-and contralateral moxibustion could be applied for pain relief

1 Introduction

Moxibustion as an important member of traditional Chi-nese medicine (TCM) therapies shares equal values withacupuncture in treating diseases in the traditional medicalsystem of China [1] It has been applied for more than3000 years for a variety of diseases especially for pain [2]Different from the mechanical stimulation of acupuncturethe function ofmoxibustionmainly depends on heat stimula-tion produced by igniting moxa sticks that are mostly madefrom mugwort (Artemisia vulgaris from traditional Chinesemedicine) Moxibustion therapy ismanipulated with ignitingmoxa stick at acupoints or some specific parts of the body [3]Since most acupoints are symmetrically distributed on bothsides of the body here is a question which side of acupointscould generate a better pain-relief effect Previous studiesdemonstrated that contralateral acupuncture (contra-Acumanual acupuncture on contralateral side) and contralateralelectroacupuncture (contra-EA electronic-acupuncture oncontralateral side) could be applied in various diseases

including episodic cluster headache [2] chronic shoulderpain [4] hemiplegia following acute ischemic stroke [5]female lumbago and pain of the lower abdomen [6] spino-genic dizziness [7] and so on These studies illustrated asimilar effect of contra-Acu or contra-EA with ipsilateralacupuncture (ipsi-Acu manual acupuncture on ipsilateralside) or ipsilateral electroacupuncture (ipsi-EA electronic-acupuncture on ipsilateral side) while it still remains unclearthat whether contralateral moxibustion (contra-Moxi mox-ibustion on contralateral side) could also generate a similareffect in pain relief with ipsilateral moxibustion (ipsi-Moximoxibustion on ipsilateral side)

Inflammatory pain is a common clinical symptom ofinflammatory diseases characterized by hyperalgesia dueto the sensitization of primary nociceptive neurons result-ing from wounds surgical incisions burn injury arthritisinfection allergic reactions autoimmune diseases tumorgrowth and other forms of tissue injury or disease [8]Both complete Freundrsquos adjuvant- (CFA-) and formalin-induced inflammatory pain models are commonly used to

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 1807287 7 pageshttpsdoiorg10115520191807287

2 Evidence-Based Complementary and Alternative Medicine

investigate inflammatory pain CFA-induced inflammatorypain is a well-characterized model of chronic inflammatorypain [9] The formalin-induced acute inflammatory paincould be divided into two phases the first phase (phase I0ndash5 min) is caused by the initial tissue injury and a directactivation of peripheral small afferent fibers by formalinthe second phase (phase II 15ndash60 min) is considered tobe mediated by a low level of peripheral nerve activitywhose effect is then enhanced in the spinal level by cen-tral sensitization [10] In this study both formalin- andCFA-induced inflammatory pain models were establishedto compare the analgesic effect between contra-Moxi andipsi-Moxi

2 Materials and Methods

21 Animals Adult male C57BL6J mice weighing 20-25gwere purchased from Beijing Hua Fu Kang Bioscience CoLtd All mice were maintained on 12-h lightdark cycle24∘C and 40-50 humidity conditions with free access tofood and water After being adaptively fed for one weekmice for formalin-induced inflammatory pain research wererandomly assigned into control group formalin group ipsi-Moxi group and contra-Moxi group and mice for CFA-induced inflammatory pain research were randomly dividedinto control CFA ipsi-Moxi contra-Moxi saline + ipsi-Moxi saline + contra-Moxi ipsi-smoke-free Moxi and moxasmoke groups After all the experiments mice were inhaledwith isoflurane for anesthesia and then applied with cervicaldislocation All efforts were made to minimize sufferingAll animal experiments were conducted in accordance withthe National Institutes of Health (NIH) Guide for the Careand Use of Laboratory Animals [11] and the ethical guide-lines of the International Association for the Study of Pain[12] All experimental procedures were approved by theAnimal Care and Use Committee of Chengdu Universityof Traditional Chinese Medicine (No AECCDUTCM-2015-04)

22 Inflammatory PainModel Theformalin-induced inflam-matory pain models were prepared by injecting 10 120583l of 1 (vv) formalin solution (Sigma-Aldrich St Louis MOthe concentration was adjusted using sterile saline) into theplantar surface of the left hind paw of mice [10] Mice inthe control group were injected 10120583l sterile saline (KelunIndustry Group Sichuan China) instead of formalin TheCFA-induced inflammatory pain models were establishedby injecting 20 120583l of 05 mgml complete Freundrsquos adjuvant(CFA Sigma-Aldrich St Louis MO) into the plantar surfaceof left hind paws [9] In the control group mice were injected20 120583l normal saline

23 Moxibustion Intervention Moxibustion was carried outrespectively for 15 min on formalin-induced pain modelbefore formalin injection (Figure 1(a)) and for 30 min onthe fifth day on CFA-induced pain model after CFA injec-tion (Figure 1(b)) Before moxibustion intervention the furon moxibustion site was shaved to expose ST36 (Zusanliacupoint located 2 mm lateral to the anterior tubercle

of the tibia in the anterior tibial muscle and 4mm distalto the knee joint lower point Figure 1(c)) Ipsi-Moxi wasdone over the left ST36 while contra-Moxi was appliedat the right ST36 (Figure 1(c)) For ipsi-smoke-free Moxigroup moxa smoke was eliminated by C200 smoke purifier(Shenzhen Conyson Company Guangzhou China) Duringmoxibustion operation the distance between the skin of ST36and the lighted end of animal-used moxa sticks (length120 mm diameter 5 mm Nanyang Hanyi MoxibustionTechnology Development Co Ltd China Figure 1(e)) wascontrolled within 1-15 cm All procedures were performed atroom temperature (24∘C) The control and the model groupswithout moxibustion operation were under the same condi-tion

24 Painreshold Measurements

241 LickingBiting Times Calculation As previouslydescribed [13] spontaneous behaviors following intraplantarformalin injection were measured Mice were firstlyacclimatized for 60 min in an individual Plexiglas cageplaced atop a glass surface and then were videotapedfollowing intradermal formalin injection and analyzedfor nociceptive behaviors (defined as licking lifting andflinching of the injected paw sum of lickingbiting timeswere counted) in five min bins for 60 min following formalininjection The first phase of spontaneous behavior wasdefined as 0ndash10 min after injection and the second phaseof testing was defined as 10ndash45 min after injection Theprocedure was done at room temperature (24∘C)

242 ermal reshold Measurement As previouslydescribed [14] the thermal threshold was assessed bymeasuring the thermal withdrawal latency (TWL) with aPlantar Test Apparatus (Hargreaves method PL-200 TaiMeng China) Mice were placed in a transparent squareand bottomless acrylic chamber (9 cmtimes8 cmtimes6 cm) After15 min of habituation period the infrared source placedunder a glass plate was positioned near the operator directlybeneath the plantar surface of the left hind paw Withdrawalof the paw indicating the sensation of pain in the micecaused the infrared source to switch to the off position andthe reaction time counter to stop To avoid tissue damage aresting interval of at least 5 min was set between tests andthe maximum time of heat focus was 20 sec Each mouse wastested for three times and the average value was set as theTWL All experiments were performed at room temperature(approximately 24∘C) and the stimuli were applied onlywhen the animals were calm but not sleeping or grooming

25 Statistical Analysis Results are expressed as mean plusmnstandard error of mean (SEM) Statistical evaluation wascarried out by GraphPad Prism 6 (GraphPad Software SanDiego CA USA) Lickingbiting time calculation resultswere evaluated by One-way Analysis of Variance (ANOVA)followed by Tukey-Kramer test Thermal threshold measure-ment results were evaluated by repeated measures two-wayANOVA followed by Tukey-Kramer test P values lt 005 wereconsidered statistically significant

Evidence-Based Complementary and Alternative Medicine 3

Moxi Lickingbiting time calculation

600 15

Formalin injection

Day 1 Day 2 Day 5Day 3

Moxi TWL threshold test0 30 150

Time (min)

(a)

(b)

(c)

(d)

(e)

(f)

Time (min)

CFA injection

Day 4

Baseline of TWL threshold test

Ipsi-moxi

LocalInflammation

Zusanli(ST36)

Zusanli(ST36)

Contra-moxi

CFA injection Paw edema

Moxa Stick

Zusanli(ST36)

05

10152025303540

0 5 110 115 220 225 330Time (min)

Tem

pera

ture

(C)∘

Figure 1 Schematic diagrams of experimental procedures and moxibustion intervention (a) Experimental procedure on formalin-inducedpain model moxibustion intervention for 15min was given before formalin injection mice receivedMoxi or restriction alone before formalinintraplantar injection (b) Experimental procedure on CFA-induced pain model On day 1 mice received CFA intraplantar injection afterbaseline of TWL threshold test On day 5 mice were intervened by moxibustion for 30 min and then got TWL threshold test for 2h (c) Micereceived formalinCFA injection in the left hind paw ipsicontra-Moxi was applied to ST36 in the leftright legs of mice respectively (d) CFAinjection and paw edema on day 5 (e)Moxibustion intervention (f) Temperature curves on ST36 the lowest temperature 287∘C the highesttemperature 375∘C temperature difference 88∘C

3 Results

31 Painreshold of Inflammatory PainMice Pain thresholdof formalin-induced inflammatory pain mice was detectedfrom 0 min to 60 min with a 5 min interval followingmoxibustion for 15 min and then formalin injection Theprocesses of formalin test can be divided into two phases anearly phase lasting the first 5 min is due to direct effect onnociceptors and a late phase lasting from 20 to 30 min afterthe injection of formalin is considered as an inflammatoryresponse with inflammatory pain The LBT-pain threshold ofthe control group did not display any significant difference

at different time point the LBT of formalin group increasedin general and the LBT of contra- and ipsilateral Moxigroups decreased in general (Figure 2(a)) We calculatedthe total LBT of phase I and phase II the total LBT ofphase I and phase II of the formalin group significantlyincreased while the total LBT of phase I and phase IIof contra- and ipsilateral Moxi groups reduced and therewas no obvious difference between contra- and ipsilateralMoxi neither in phase I nor in phase II (Figure 2(b))These data suggested that ipsi- and contralateral moxibus-tion had a similar effect on formalin-induced inflammatorypain


5 60555045403530252015100

50

100

150

Time after Formalin Injection (5-Minute Interval)

Paw

Lic

king

Biti

ng T

ime (

sec)

Ipsi-Moxi n=8Contra-Moxi n=8

Formalin n=8Control n=8

phase I (0-10min)

phase II (10-45min)

(a)

phase I (0-10min)

phase II (11-45min)

0

100

200

300

Formalin n=8

Contra-Moxi n=8Ipsi-Moxi n=8

Paw

Lic

king

Biti

ng T

ime (

sec)

lowastlowastlowast

(b)

Figure 2 Analgesic effect of ipsi- and contralateral Moxi on formalin-induced pain model (a) LBT of each timepoint from 0 min to 60minwith 5 min interval compared with formalin group paw LBT decreased in control ipsilateral and contralateral groups (control salineinjection group CFA formalin injection group ipsi-Moxi ipsilateral moxibustion group contra-Moxi contralateral moxibustion group)(b) Total LBT of phase I and phase II F (2 21) = 3253 (intervention between columns) contraipsi-Moxi versus formalin of phase I lowast lowast lowastplt 0001 F (2 21) = 4945 (intervention between columns) contraipsi-Moxi versus formalin of phase II p lt 005

Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)

CFA n=8Ipsi-Moxi n=8Ipsi-smoke-free Moxi n=8

Control n=8

Moxa smoke n=8

lowast

lowast

lowast

lowast

lowast

lowast

lowast

∙∙∙∙∙

Figure 3 Analgesic effect of ipsi-Moxi ipsi-smoke-free Moxi and moxa smoke groups on CFA-induced pain model In total F (6 210) =4184 Plt00001 (time) F (4 35) = 2773 Plt00001 (group) and F (24 210) = 3355 Plt00001 (interaction) CFA versus control Plt001ipsi-Moxi versus CFA ipsi nonsmoking Moxi versus CFAlowastPlt005 ipsi-Moxi versus ipsi nonsmoking Moxi 998771Plt005 ipsi-Moxi versusmoxa smoke ePlt005 (control saline injection group CFA CFA injection group ipsi-Moxi ipsilateral moxibustion group ipsi-smoke-freeMoxi ipsilateral moxibustion with moxa smoke elimination group moxa smoke inhale moxa smoke group)There is no statistical differencebetween moxa smoke and CFA groups

32 Pain reshold of CFA-Induced Inflammatory Pain MiceThe pain threshold-thermal withdrawal latency (TWL) ofCFA-induced inflammatory pain mice was detected on thefifth day and immediately followed with moxibustion inter-vention for 30 min TWL baseline of each group displayedno significant difference and after CFA injection TWL ofCFA and moxibustion groups decreased remarkably There

was no statistical difference between moxa smoke and CFAgroups but TWL of ipsi-Moxi and ipsi-smoke-free Moxigroups increased immediately after intervention reached apeak after moxibustion for 30 min and began to declineand closed to baseline after 2 h (Figure 3) On the wholemoxa smoke failed to generate analgesic effect and therewas no big difference between ipsi-Moxi and ipsi-smoke-free


Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)

CFA group n=8Ipsi-Moxi group n=8Contra-Moxi group n=8Saline+ipsi-Moxi n=8Saline+contra-Moxi n=8

Control group n=8

lowast

lowast

lowast

lowast

lowast

Figure 4 Analgesic effect of ipsi- and contralateralMoxi on normal and CFA-induced pain mice In total F (6 252) = 1142 Plt00001 (time)F (5 42) = 2542 Plt00001 (group) and F (30 252) = 2439 Plt00001 (interaction)There is no statistical difference among control saline +ipsi-Moxi and saline + contra-Moxi groups (control saline injection group saline + ipsi-Moxi saline injection with ipsilateral moxibustiongroup saline + contra-Moxi saline injection with contralateral moxibustion group) CFA versus control Plt001 ipsi-Moxi versus CFAcontra-Moxi versus CFA lowastPlt005 (control saline injection group CFA CFA injection group ipsi-Moxi ipsilateral moxibustion groupcontra-Moxi contralateral moxibustion group) The difference between ipsi-Moxi and contra-Moxi groups has no statistical significance

Moxi groups which indicated that the analgesic effect mainlyproduced by heat stimulation instead of moxa smoke Ipsi-and contra-Moxi were also applied with saline injected miceThe result showed that there was no statistical differenceamong control saline + ipsi-Moxi and saline + contra-Moxi groups which meant the analgesic effect inducedmainly in inflammatory states (Figure 4) while with CFAinjection TWL of ipsi- and contra-Moxi groups increasedremarkably and reached a peak after moxibustion for 30min and then they began to decline and closed to baselineafter 2h (Figure 4) These data suggested that both ipsi-and contralateral moxibustion generated a similar analgesiceffect on CFA-induced inflammatory pain However ipsi-and contra-Moxi failed to induce analgesic effect in the micewithout inflammatory pain syndrome

4 Discussion

In this study we mainly found that ipsi- and contralat-eral moxibustion achieved similar analgesic effect in bothformalin-induced acute inflammatory pain andCFA-inducedchronic inflammatory pain In formalin-induced pain exper-iment the similar analgesic effect of ipsi- and contralateralmoxibustion could be found in phase I and phase II InCFA-induced pain experiment the similar analgesic effectof ipsi- and contralateral moxibustion could be lasted for120 min As we know formalin-induced inflammatory painincludes two different phases phase I is mainly due to directchemical stimulation of peripheral nociceptors and phaseII is generated by the ongoing stimulation of nociceptorsby inflammatory mediators andor by a first phase-induced

spinal cord hyperexcitability ie central sensitization [15 16]CFA-induced inflammation consists of a persistent unilateralinflammation generating hyperactivity of primary afferentsfrom dorsal root ganglia and hyperexcitation of spinal dorsalhorn interneurons [17] Hence our results implied thatcontralateral moxibustion alleviated pain symptom mainlythrough the central nervous system instead of local analgesia

In this experiment we also tested the local skin temper-ature of ST36 to exclude potential influencing factors suchas stress-induced analgesia Then we found that the highestskin temperature of moxibustion site was 375∘C which wasconsidered as a warm temperature and could not inducewhole-body heat stress [18 19] Given that moxibustionwas used for both preventing and treating pain symptomin clinic ipsi- and contralateral moxibustion interventionswere applied with saline injection and it seemed that bothipsi- and contralateral moxibustion failed to induce paintolerance of mice in physiological states It also indicatedthat contralateral moxibustion though giving moxibustiontreatment at the unaffected side could merely increase painthreshold of mice in pathological conditions On the otherhand it is known that moxa smoke and thermal stimulationare produced simultaneously during moxibustion and therewas research suggested that moxa smoke contributed tothe therapeutic effect of moxibustion in some diseaseslike wound infections common warts anal fistula and soforth [20] Since moxa smoke was produced in both ipsi-and contralateral moxibustion moxa smoke and smoke-freemoxibustion were applied separately and the results showedthat giving moxa smoke could not affect the pain thresholdof mice Therefore these results confirmed that contralateral


moxibustion had a similar analgesic effect with ipsilateralmoxibustion

Current data implied that applying moxibustion at anyside of acupoint ST36 relieved inflammatory pain symptomand this found might be very useful for a more widespreadapplication ofmoxibustion in different conditions Contralat-eral moxibustion is able to be called Jujiu just like contralat-eral acupuncture which is also named Juci or Miuci [21 22]one of the traditional acupuncture techniques recorded inthe classic textbook Huang Di Nei Jing [23] For instancesome of the acupoints are located at the thin and tender skinplaces with many capillaries and these points are not allowedto be stimulated by moxibustion for a long time Switchingthe side acupoints could reduce the potential damage to thelocal skin and capillaries but still guarantee the treatmenteffect Many classic TCM books also pointed out that it wasnot suitable to apply moxibustion at the acupoints with localskin lesion such as local infection trauma anomaly andpostamputation most of which were at the affected bodyside (ipsilateral side) for the reason that moxibustion at theaffected side might increase the damage of local surface Inthis case it is more wisely to carry on moxibustion treatmentat the alternative side which had a similar effect with theaffected side However the current study was only an animalexperiment and further clinical trials are needed to providemore powerful supporting data for the use of contralateralmoxibustion

Why contralateral and ipsilateral moxibustion could gen-erate a similar effect Our results indicated that the centralnerve systemmight be crucial for analgesic effect of contralat-eral moxibustion Since no similar study has been presentedit should be considered in our next study On the other handunderlying contralateral acupuncture and electroacupunc-ture (EA) have demonstrated that both peripheral and cen-tral nerve systems including spinal interneurons [24 25]endogenous opioids and diffuse noxious inhibitory controls(DNIC) were involved in the analgesic effects of acupuncture[26 27] These observations suggested that spinal interneu-rons were crucial in producing the contralateral acupunctureeffect [28 29] It seemed that there were differences onthe responses of subnucleus reticularis dorsalis (SRD) neu-rons between ipsilateral and contralateral acupuncture Thegreater activation of total nociceptive convergence (TNC)than of partial nociceptive convergence (PNC) neurons wasrepeatedly observed when the ipsilateral part of the bodywas stimulated and the responses of TNC neurons wereslightly weaker from ipsilateral parts of the body than fromtheir contralateral counterparts [24] Another study showedthat up-regulated expression of TRPV1 in dorsal root ganglia(DRG) and dorsal horn of the spinal cord (SCDH) after CFAinjection was to be found on both ipsi- and contralateralEA stimulation [4] The interactions between the two sidesand projections of trigeminal afferents to the contralateralmedullary horn also contribute to the contralateral stimula-tion [21 30] With functional magnetic resonance imaging(fMRI) researchers found that unilateral acupuncture stim-ulation was able to generate bilateral neuronal modulation[31 32] Additionally a study indicated that the anteriorcingulate cortex (ACC) could be one of the major regions for

pain affection which completely abolished analgesic effects ofcontra-EA while the decrease of analgesic effects on ipsi-EAwas still present [33] Does contralateral moxibustion effectshare the same mechanism with contralateral acupuncture orelectroacupuncture It is still far from clear Therefore it isquite necessary to clarify the mechanism of the antinocicep-tive effect induced by contralateral moxibustion in the future

5 Conclusion

In conclusion the present study indicated a similar analgesiceffect between ipsi- and contralateral Moxi These datasupported the traditional acupuncture theory that stimula-tion could also be applied at the contralateral side in painsituation However to clarify the mechanism of the analgesiceffect of Moxi further dedicated investigation on this issue isrequired

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interestand no financial interests related to the material of thismanuscript

Authorsrsquo Contributions

Chuan-Yi Zuo Peng Lv and Cheng-Shun Zhang contributedequally to this work

Acknowledgments

This work has been supported by grants from 973 Program(no 2015CB544504) National Natural Science Foundation ofChina (81774437 and 81373735) Sichuan Provincial Innova-tive Research Team Program (2014TD0018 and 2015TD0010)and Innovative Research Team in University of SichuanProvince (16TD0015)

References

[1] Y Tang H Y Yin P Rubini and P Illes ldquoAcupuncture-inducedanalgesia a neurobiological basis in purinergic signalingrdquoNeu-roscientist A Review Journal Bringing Neurobiology Neurology ampPsychiatry vol 22 no 6 p 563 2016

[2] S Hayhoe ldquoAcupuncture for episodic cluster headache Atrigeminal approachrdquo Acupuncture in Medicine vol 2015 no 1pp 55ndash58 2016

[3] K-W Lu C-K Hsu C-L Hsieh J Yang and Y-W Lin ldquoProb-ing the effects and mechanisms of electroacupuncture at ipsi-lateral or contralateral ST36-ST37 acupoints on CFA-inducedinflammatory painrdquo Scientific Reports vol 6 Article ID 221232016

[4] H Zhang J Sun C Wang et al ldquoRandomised controlled trialof contralateral manual acupuncture for the relief of chronicshoulder painrdquo Acupuncture in Medicine vol 34 no 3 pp 164ndash170 2016


[5] H Gao X Gao G Liang and B-X Ma ldquoContra-lateral nee-dling in the treatment of hemiplegia due to acute ischemicstrokerdquo Acupuncture amp Electro-erapeutics Research vol 37no 1 pp 1ndash12 2012

[6] Y-F Zhang and L-P Chen ldquoTreatment of 40 cases of femalelumbago and pain of the lower abdomen by contralateralacupuncture at Xialiao (BL 34)rdquo Chinese Acupuncture amp Moxi-bustion vol 32 no 12 p 1085 2012

[7] B Cheng ldquo206 cases of spinogenic dizziness treated by con-tralateral acupuncturerdquo Journal of Traditional ChineseMedicinevol 16 no 1 pp 35-36 1996

[8] J PMWhiteM Cibelli A Rei Fidalgo et al ldquoRole of transientreceptor potential and acid-sensing ion channels in peripheralinflammatory painrdquo Anesthesiology vol 112 no 3 pp 729ndash7412010

[9] K Nakamoto T Nishinaka N Sato et al ldquoHypothalamicGPR40 signaling activated by free long chain fatty acids sup-presses CFA-induced inflammatory chronic painrdquo PLoS ONEvol 8 no 12 p e81563 2013

[10] S Hunskaar and K Hole ldquoThe formalin test in mice dissocia-tion between inflammatory andnon-inflammatory painrdquoPAINvol 30 no 1 pp 103ndash114 1987

[11] N R C C f t U o t G f t Care and U o L Animals ldquoGuidefor the Care and Use of Laboratory Animalsrdquo Publication No85-23(rev) Vol 327 No 3 pp 963-965 2011

[12] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo PAIN vol 16 no 2pp 109-110 1983

[13] C C Long K E Sadler and B J Kolber ldquoHormonal and mole-cular effects of restraint stress on formalin-induced pain-likebehavior in male and female micerdquo Physiology amp Behavior vol165 pp 278ndash285 2016

[14] W Zhou R Lei C Zuo et al ldquoAnalgesic effect of moxibustionwith different temperature on inflammatory and neuropathicpain mice a comparative studyrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2017 Article ID 4373182 8pages 2017

[15] T J Coderre A L Vaccarino andRMelzack ldquoCentral nervoussystem plasticity in the tonic pain response to subcutaneousformalin injectionrdquo Brain Research vol 535 no 1 pp 155ndash1581990

[16] M Cui S Khanijou J Rubino and K R Aoki ldquoSubcutaneousadministration of botulinum toxin a reduces formalin-inducedpainrdquo PAIN vol 107 no 1-2 pp 125ndash133 2004

[17] A Benani C Vol T Heurtaux et al ldquoUp-regulation of fattyacid metabolizing-enzymes mRNA in rat spinal cord duringpersistent peripheral local inflammationrdquo European Journal ofNeuroscience vol 18 no 7 pp 1904ndash1914 2003

[18] R Almasi G Petho K Bolcskei and J Szolcsanyi ldquoEffect ofresiniferatoxin on the noxious heat threshold temperature in therat A novel heat allodynia model sensitive to analgesicsrdquo BritishJournal of Pharmacology vol 139 no 1 pp 49ndash58 2010

[19] V Kartik M Craig and A Biochem ldquoTrp Channelsrdquo 2012[20] H Xu B Zhao Y Cui et al ldquoEffects of moxa smoke on mono-

amine neurotransmitters in SAMP8 micerdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID178067 6 pages 2013

[21] K Miura T Ohara J L Zeredo Y Okada K Toda and KSumikawa ldquoEffects of traditional ldquoJucirdquo (contralateral acupunc-ture) on orofacial nociceptive behavior in the ratrdquo Journal ofAnesthesia amp Clinical Research vol 21 no 1 pp 31ndash36 2007

[22] X Wang ldquoTextual research on Miu Ci (Is it contralateralcollateral needling)rdquo Zhonghua yi shi za zhi vol 39 no 3 p173 2009

[23] P U Unschuld Huang Di Nei Jing Su WenNature KnowledgeImagery in an Ancient Chinese Medical Text With an Appendixe Doctrine of the Five Periods and Six Qi in the Huang Di NeiJing Su Wen University of California Press 2003

[24] Z Bing L Villanueva and D Le Bars ldquoAcupuncture-evokedresponses of subnucleus reticularis dorsalis neurons in the ratmedullardquo Neuroscience vol 44 no 3 pp 693ndash703 1991

[25] Z Bing L Villanueva andD Le Bars ldquoAcupunctureand diffusenoxious inhibitory controls Naloxone-reversible depression ofactivities of trigeminal convergent neuronsrdquo Neuroscience vol37 no 3 pp 809ndash818 1990

[26] L F He ldquoInvolvement of endogenous opioid peptides in acu-puncture analgesiardquo PAIN vol 31 no 1 pp 99ndash121 1987

[27] B Pomeranz and D Chiu ldquoNaloxone blockade of acupunctureanalgesia endorphin implicatedrdquoLife Sciences vol 19 no 11 pp1757ndash1762 1976

[28] I Bileviciute-Ljungar and T Lundeberg ldquoContralateral treat-ment with xylocaine reduces nociceptive behaviour in mono-neuropathic ratsrdquoNeuroReport vol 11 no 2 pp 291ndash295 2000

[29] I Bileviciute-Ljungar and T Lundeberg ldquoContralateral but notsystemic administration of bupivacaine reduces acute inflam-mation in the rat hindpawrdquo Somatosensory andMotor Researchvol 17 no 3 pp 285ndash293 2000

[30] M F Jacquin N L Chiaia and R W Rhoadest ldquoTrigeminalprojections to contralateral dorsal horn Central extent periph-eral origins and plasticityrdquo Somatosensory amp Motor Researchvol 7 no 2 pp 153ndash183 1990

[31] K K S Hui J Liu N Makris et al ldquoAcupuncture modulatesthe limbic system and subcortical gray structures of the humanbrain evidence from fMRI studies in normal subjectsrdquoHumanBrain Mapping vol 9 no 1 pp 13ndash25 2015

[32] J D Lee J S Chon H K Jeong et al ldquoThe cerebrovascularresponse to traditional acupuncture after strokerdquo Neuroradiol-ogy vol 45 no 11 pp 780ndash784 2003

[33] M Yi H Zhang L Lao et al ldquoAnterior cingulate cortex iscrucial for contra- but not ipsi-lateral electro-acupuncture inthe formalin-induced inflammatory pain model of ratsrdquoMole-cular Pain vol 7 no 1 p 61 2011

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Evidence-Based Complementary andAlternative Medicine

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investigate inflammatory pain CFA-induced inflammatorypain is a well-characterized model of chronic inflammatorypain [9] The formalin-induced acute inflammatory paincould be divided into two phases the first phase (phase I0ndash5 min) is caused by the initial tissue injury and a directactivation of peripheral small afferent fibers by formalinthe second phase (phase II 15ndash60 min) is considered tobe mediated by a low level of peripheral nerve activitywhose effect is then enhanced in the spinal level by cen-tral sensitization [10] In this study both formalin- andCFA-induced inflammatory pain models were establishedto compare the analgesic effect between contra-Moxi andipsi-Moxi

2 Materials and Methods

21 Animals Adult male C57BL6J mice weighing 20-25gwere purchased from Beijing Hua Fu Kang Bioscience CoLtd All mice were maintained on 12-h lightdark cycle24∘C and 40-50 humidity conditions with free access tofood and water After being adaptively fed for one weekmice for formalin-induced inflammatory pain research wererandomly assigned into control group formalin group ipsi-Moxi group and contra-Moxi group and mice for CFA-induced inflammatory pain research were randomly dividedinto control CFA ipsi-Moxi contra-Moxi saline + ipsi-Moxi saline + contra-Moxi ipsi-smoke-free Moxi and moxasmoke groups After all the experiments mice were inhaledwith isoflurane for anesthesia and then applied with cervicaldislocation All efforts were made to minimize sufferingAll animal experiments were conducted in accordance withthe National Institutes of Health (NIH) Guide for the Careand Use of Laboratory Animals [11] and the ethical guide-lines of the International Association for the Study of Pain[12] All experimental procedures were approved by theAnimal Care and Use Committee of Chengdu Universityof Traditional Chinese Medicine (No AECCDUTCM-2015-04)

22 Inflammatory PainModel Theformalin-induced inflam-matory pain models were prepared by injecting 10 120583l of 1 (vv) formalin solution (Sigma-Aldrich St Louis MOthe concentration was adjusted using sterile saline) into theplantar surface of the left hind paw of mice [10] Mice inthe control group were injected 10120583l sterile saline (KelunIndustry Group Sichuan China) instead of formalin TheCFA-induced inflammatory pain models were establishedby injecting 20 120583l of 05 mgml complete Freundrsquos adjuvant(CFA Sigma-Aldrich St Louis MO) into the plantar surfaceof left hind paws [9] In the control group mice were injected20 120583l normal saline

23 Moxibustion Intervention Moxibustion was carried outrespectively for 15 min on formalin-induced pain modelbefore formalin injection (Figure 1(a)) and for 30 min onthe fifth day on CFA-induced pain model after CFA injec-tion (Figure 1(b)) Before moxibustion intervention the furon moxibustion site was shaved to expose ST36 (Zusanliacupoint located 2 mm lateral to the anterior tubercle

of the tibia in the anterior tibial muscle and 4mm distalto the knee joint lower point Figure 1(c)) Ipsi-Moxi wasdone over the left ST36 while contra-Moxi was appliedat the right ST36 (Figure 1(c)) For ipsi-smoke-free Moxigroup moxa smoke was eliminated by C200 smoke purifier(Shenzhen Conyson Company Guangzhou China) Duringmoxibustion operation the distance between the skin of ST36and the lighted end of animal-used moxa sticks (length120 mm diameter 5 mm Nanyang Hanyi MoxibustionTechnology Development Co Ltd China Figure 1(e)) wascontrolled within 1-15 cm All procedures were performed atroom temperature (24∘C) The control and the model groupswithout moxibustion operation were under the same condi-tion

24 Painreshold Measurements

241 LickingBiting Times Calculation As previouslydescribed [13] spontaneous behaviors following intraplantarformalin injection were measured Mice were firstlyacclimatized for 60 min in an individual Plexiglas cageplaced atop a glass surface and then were videotapedfollowing intradermal formalin injection and analyzedfor nociceptive behaviors (defined as licking lifting andflinching of the injected paw sum of lickingbiting timeswere counted) in five min bins for 60 min following formalininjection The first phase of spontaneous behavior wasdefined as 0ndash10 min after injection and the second phaseof testing was defined as 10ndash45 min after injection Theprocedure was done at room temperature (24∘C)

242 ermal reshold Measurement As previouslydescribed [14] the thermal threshold was assessed bymeasuring the thermal withdrawal latency (TWL) with aPlantar Test Apparatus (Hargreaves method PL-200 TaiMeng China) Mice were placed in a transparent squareand bottomless acrylic chamber (9 cmtimes8 cmtimes6 cm) After15 min of habituation period the infrared source placedunder a glass plate was positioned near the operator directlybeneath the plantar surface of the left hind paw Withdrawalof the paw indicating the sensation of pain in the micecaused the infrared source to switch to the off position andthe reaction time counter to stop To avoid tissue damage aresting interval of at least 5 min was set between tests andthe maximum time of heat focus was 20 sec Each mouse wastested for three times and the average value was set as theTWL All experiments were performed at room temperature(approximately 24∘C) and the stimuli were applied onlywhen the animals were calm but not sleeping or grooming

25 Statistical Analysis Results are expressed as mean plusmnstandard error of mean (SEM) Statistical evaluation wascarried out by GraphPad Prism 6 (GraphPad Software SanDiego CA USA) Lickingbiting time calculation resultswere evaluated by One-way Analysis of Variance (ANOVA)followed by Tukey-Kramer test Thermal threshold measure-ment results were evaluated by repeated measures two-wayANOVA followed by Tukey-Kramer test P values lt 005 wereconsidered statistically significant



600 15

Formalin injection



Time (min)

(a)

(b)

(c)

(d)

(e)

(f)

Time (min)

CFA injection

Day 4


Ipsi-moxi

LocalInflammation

Zusanli(ST36)

Zusanli(ST36)

Contra-moxi


Moxa Stick

Zusanli(ST36)

05

10152025303540

0 5 110 115 220 225 330Time (min)

Tem

pera

ture

(C)∘


3 Results




5 60555045403530252015100

50

100

150


Paw

Lic

king

Biti

ng T

ime (

sec)



phase I (0-10min)

phase II (10-45min)

(a)

phase I (0-10min)

phase II (11-45min)

0

100

200

300

Formalin n=8


Paw

Lic

king

Biti

ng T

ime (

sec)

lowastlowastlowast

(b)


Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control n=8

Moxa smoke n=8

lowast

lowast

lowast

lowast

lowast

lowast

lowast

∙∙∙∙∙





Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control group n=8

lowast

lowast

lowast

lowast

lowast



4 Discussion









5 Conclusion


Data Availability






Acknowledgments


References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















600 15

Formalin injection



Time (min)

(a)

(b)

(c)

(d)

(e)

(f)

Time (min)

CFA injection

Day 4


Ipsi-moxi

LocalInflammation

Zusanli(ST36)

Zusanli(ST36)

Contra-moxi


Moxa Stick

Zusanli(ST36)

05

10152025303540

0 5 110 115 220 225 330Time (min)

Tem

pera

ture

(C)∘


3 Results




5 60555045403530252015100

50

100

150


Paw

Lic

king

Biti

ng T

ime (

sec)



phase I (0-10min)

phase II (10-45min)

(a)

phase I (0-10min)

phase II (11-45min)

0

100

200

300

Formalin n=8


Paw

Lic

king

Biti

ng T

ime (

sec)

lowastlowastlowast

(b)


Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control n=8

Moxa smoke n=8

lowast

lowast

lowast

lowast

lowast

lowast

lowast

∙∙∙∙∙





Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control group n=8

lowast

lowast

lowast

lowast

lowast



4 Discussion









5 Conclusion


Data Availability






Acknowledgments


References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















5 60555045403530252015100

50

100

150


Paw

Lic

king

Biti

ng T

ime (

sec)



phase I (0-10min)

phase II (10-45min)

(a)

phase I (0-10min)

phase II (11-45min)

0

100

200

300

Formalin n=8


Paw

Lic

king

Biti

ng T

ime (

sec)

lowastlowastlowast

(b)


Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control n=8

Moxa smoke n=8

lowast

lowast

lowast

lowast

lowast

lowast

lowast

∙∙∙∙∙





Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control group n=8

lowast

lowast

lowast

lowast

lowast



4 Discussion









5 Conclusion


Data Availability






Acknowledgments


References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Baseline CFA 30 60 90 120 1500

5

10

15

20

25

Time (min)

TWL

(sec

)


Control group n=8

lowast

lowast

lowast

lowast

lowast



4 Discussion









5 Conclusion


Data Availability






Acknowledgments


References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























5 Conclusion


Data Availability






Acknowledgments


References







































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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