research article interaction of acupuncture and...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 670858 9 pageshttpdxdoiorg1011552013670858

Research ArticleInteraction of Acupuncture and Electroacupunctureon the Pharmacokinetics of Aspirin and the Effect of BrainBlood Flow in Rats

Ming-Tsang Wu1 Lee-Hsin Shaw1 Yu-Tse Wu2 and Tung-Hu Tsai134

1 Institute of Traditional Medicine School of Medicine National Yang-Ming University 155 Li-Nong Street Section 2Taipei 112 Taiwan

2 School of Pharmacy Kaohsiung Medical University 100 Shih-Chuan 1st Road Kaohsiung 80708 Taiwan3Graduate Institute of Acupuncture Science China Medical University No 91 Hsueh-Shih Road Taichung 404 Taiwan4Department of Education and Research Taipei City Hospital No 145 Zhengzhou Road Datong Dist Taipei 103 Taiwan

Correspondence should be addressed to Yu-Tse Wu ytwukmuedutw and Tung-Hu Tsai thtsaiymedutw

Received 7 May 2013 Revised 12 October 2013 Accepted 21 October 2013

Academic Editor Tzeng-Ji Chen

Copyright copy 2013 Ming-Tsang Wu et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Acupuncture and electroacupuncture have been used to improve the brain and motor functions of poststroke patients and aspirinis used for the prevention of stroke recurrence Our hypothesis is that acupuncture and electroacupuncture treatments may interactwith aspirin in terms of pharmacokinetics via affecting the brain blood flow The aim of this study is to investigate the potentialinteractions of acupuncture and electroacupuncture on the pharmacokinetics of aspirin The effects of acupuncture treatmentson brain blood flow were measured by the laser Doppler blood flow imager The parallel pharmacokinetic study design includedthree groups control acupuncture and electroacupuncture groups Two acupoints namely Quchi (LI 11) and Zusanli (ST 36)were needled and stimulated electronically in anaesthetized rats The concentrations of aspirin and its metabolite salicylic acidwere determined by microdialysis and HPLC analysis after aspirin administration (30mgkg iv) The brain blood flow respondedto electroacupuncture treatments but the pharmacokinetic parameters of aspirin and salicylic acid in blood and brain were notsignificantly changed by acupuncture and electroacupuncture treatments This study may in part offer some evidence to supportthe contention that there is no significant interaction for the combination of aspirin with acupuncture or electroacupuncture

1 Introduction

Acupuncture as a part of traditional Chinese medicine hasbeen used internationally for treatment of many specificdiseases such as hemiplegia and other sequels of braindisease headache hypertension and insomnia according toWorld Health Organization (WHO) reports [1] Strokes andtheir sequelae are another major indication of acupunctureand early treatment of paresis after stroke has been provedhighly effective [2] Clinically electroacupuncture has beenapplied to conventional acupoints such as Baihui (GV20)Yanglingquan (GB34) Hegu (LI4) Quchi (LI 11) and Zusanli(ST 36) for poststroke patient rehabilitation [3 4] Two acu-pointsQuchi (LI 11) andZusanli (ST 36) have been suggestedto have potential benefits for post-stroke conditions such asspastic paralysis [5] and improved stroke patientsrsquo symptoms

and signs [6] Experimental animal data also support thatelectro-acupuncture treatment at Quchi (LI 11) and Zusanli(ST 36) have neuroprotective function [7] and cerebral pro-tective function [8] Acupuncture and electro-acupuncturehave clearly shown benefits for post-stroke patients [9 10]It is suggested that acupuncture can increase blood flow ofthe peripheral mesenteric and retrobulbar arteries [11 12]This blood flow is an important supporting factor to drugabsorption distribution metabolism and excretion so if theblood flow changes the pharmacokinetic parameters of drugmight be affected

Aspirin an acetylated salicylic acid is classified as one ofthe nonsteroidal anti-inflammatory drugs (NSAIDs) Aspirinis the most commonly used drug for relieving pain inflam-matory symptoms and fever It also has established efficacyfor preventing myocardial infarction and ischemic stroke

2 Evidence-Based Complementary and Alternative Medicine

as well as for treating acute myocardial infarction [13] Ina previous study low-dose aspirin has been shown usefulfor the prevention of cardiovascular diseases and strokerecurrence [14] Furthermore low-dose aspirin can alsoreduce mortality rates of cardiovascular disease and strokepatients [15] For post-stroke patients it may be beneficialto undergo treatment by electro-acupuncture combined withaspirin [16] But adverse reactions of which there is a widespectrum frequently accompany anti-inflammatory doses ofaspirin Aspirin and related derivatives of salicylic acid havebeen reported to have a wide range of drug interactions butrelatively few seem to be clinically important Many of theseinteractions are pharmacokinetic in nature [17] Cyclooxyge-nase is irreversible inhibition by aspirin and dose-related sideeffects of aspirin on gastrointestinal symptoms (eg bleedingcomplications) have been reported [18 19] In additionaspirin exhibits a nonlinear pharmacokinetics meaning thatthe binding of drugs to plasma components blood cellsand extravascular tissue may demonstrate concentrationdependence Therefore if acupuncture changes the pharma-cokinetics of aspirin the side effects may occur

A survey from PubMed with the key words of electro-acupuncture and pharmacokinetics of aspirin found noitems Accordingly this study develops a technique usingmicrodialysis coupled to liquid chromatography withphotodiode-array detection to monitor protein-unboundaspirin and its metabolites in blood and brain of the ratTo investigate potential interactions between acupunctureand electro-acupuncture on the pharmacokinetics ofaspirin microdialystates of the blood and brain on thegroups of control acupuncture and electro-acupuncturewere individually collected and measured to explore thepharmacokinetic interaction of aspirin To monitor the ratbrain blood flow a laser Doppler blood flow imager wasused to measure the brain blood flow on the groups ofcontrol acupuncture and electro-acupuncture The studymay in part provide important information regarding theeffects of acupuncture and electro-acupuncture as they areinfluenced by the pharmacokinetic and pharmacodynamicinteraction of aspirin Physiological factors affecting thedistribution of a drug include plasma protein bindingblood perfusion and membrane permeability Previouspapers have discovered that acupuncture changes bloodflow [12 20] which may affect directly the distribution of adrug In this work we assessed the effects of acupunctureon blood flow to confirm the reported results and thenperformed pharmacokinetic experiments We decided to usea rat model by microdialysis sampling to examine the effectsof acupunctureelectro-acupuncture on drug distributionbecause microdialysis provides several advantages such assampling protein-unbound drug providing high temporalresolution data and minimizing physiological disturbance ofthe subject for a pharmacokinetic evaluation [21] Aspirinhad poor oral bioavailability which may lead to fluctuatedpharmacological effects [22] and we chose intravenousroute for drug administration to minimize the fluctuatedpharmacological effects caused by inter-subject absorptionvariations The aim of our work is to evaluate the possibleinteractions between acupuncture and pharmacokinetics of

aspirin The use of acupuncture and electro-acupuncturemay result in various biological effects on the subject so weattempted to find the relationship between acupuncture andpharmacokinetics from a distribution viewpoint

2 Materials and Methods

21 Chemicals and Reagents Aspirin salicylic acid 120572-chloralose andurethanewere purchased fromSigma-AldrichChemicals (St Louis MO USA) Acetic acid sodium citratedextrose sodium chloride potassium dihydrogen phosphate(KH2PO4) and orthophosphoric acid (85 ww) were pur-

chased from E Merck (Darmstadt Germany) Acetonitrileof analytical grade was purchased from ECHO ChemicalCo (Taiwan) Deionized water fromMillipore (Milford MAUSA) was used for all aqueous solutions in this study

22 HPLC Instrumentation HPLC-UV instrumentationconsisted of a Shimadzu chromatographic pump (LC-20AT)a DGU-20A5 degasser an autosampler (SIL-20AC) anda photodiode array detector (SPD-M20A) (ShimadzuKyoto Japan) A reverse-phase C18 column (MerckPurospher STAR 250mm times 4mm id particle size 5 120583mDarmstadt Germany) was used to separate the analytesThe UV absorbance wavelength was set at 240 nm to detectaspirin and salicylic acid The mobile phase consisted ofacetonitrile 10mM KH

2PO4(29 71 vv pH 25 adjusted

by orthophosphoric acid) for analysis of blood and brainmicrodialysates A Millipore (022120583m) filter (Bedford MAUSA) was used to filter the mobile phase and we used asonicator (Branson CT USA) to degas before the mobilephase was used The flow rate was set at 1mLmin

23 Experimental Animals Adult male pathogen-freeSprague-Dawley rats (200ndash260 g) were obtained fromthe Laboratory Animal Center at National Yang-MingUniversity (Taipei Taiwan) Rats were housed in cages with12 h lightdark cycle food (Laboratory RodentDiet 5001 PMIFeeds Inc Richmond IN USA) and water were availablead libitum The animals were received at 6-7 weeks of ageand acclimated for at least one week All animal experimentsfollowed the National Yang-Ming University guidelinesand procedures for the care of laboratory animals andthe protocol listed above has been reviewed and approvedby the Institutional Animal Care and Use Committee(IACUC approval number 1011203) by the InstitutionalAnimal Experimentation Committee of National Yang-MingUniversity The animals had free access to food and waterThe rat was anaesthetized with urethane 10 gmL and120572-chloralose 01 gmL (1mLkg ip) before surgery Thefemoral vein was cannulated for further drug administrationand the ratrsquos body temperature was maintained by a heatingpad during the experiment and they were euthanized byoverdose CO

2under the anesthetic after the experimental

endpoint

24 Microdialysis Experiments The microdialysis systemincluded a CMA100 microinjection syringe pump a

Evidence-Based Complementary and Alternative Medicine 3

CMA140 microfraction collector (CMA StockholmSweden) and corresponding microdialysis probes locatedat sampling sites The microdialysis probes for blood andbrain sampling were made in our laboratory Briefly thedialysis membranes (150120583m outer diameter with a nominalmolecular weight cutoff of 13000 Spectrum Co LagunaHills CA USA) for blood and brain are 10mm and 3mmin length respectively [23] All unions were cemented withepoxy adhesive and the probes were made at least 24 h priorto use to allow for adequate time for the epoxy adhesive tohardenThe bloodmicrodialysis probewas locatedwithin thejugular veinright atrium and perfused with an anticoagulantdextrose solution (ACD citric acid 35mM sodium citrate75mM dextrose 136mM) The brain microdialysis probewas implanted in the right striatum (coordinates APminus02mm ML minus30mm DV minus75mm) and perfused withRingerrsquos solutionThe flow rates of ACD andRingerrsquos solutionwere set at 20 120583Lmin by a microinjection syringe pump forblood and brain microdialysis The implantation positions ofthe probes were verified by standard histological procedureat the end of experiments After different pretreatment inthree groups the dialysates were collected every 15 minutesfor 6 hours and preserved under minus20∘C refrigeration Avalidated HPLC-UV system was applied to determine theconcentration of aspirin and salicylic acid in the dialysatesof blood and brain A retrodialysis (119877dial) method was usedto estimate in vivo recovery following the method in ourprevious report [24]

25 Acupuncture and Electro-Acupuncture for Pharmacoki-netic Study The study design was divided into the threegroups of control acupuncture and electro-acupuncturegroups Each group included six SD rats (119873 = 6) In thecontrol group aspirin was administered alone (30mgkgiv) injected via the femoral vein The acupuncture andelectro-acupuncture groups were respectively treated withacupuncture and electro-acupuncture for 15 minutes andthen aspirin was administered (30mgkg iv) via the femoralvein respectively

After the microdialysis experimental rat model was setup the stainless steel acupuncture needles (outer diameter of028mm) were sterilized with 75 alcohol before treatmentThen the needles were inserted into the rat at bilateralacupoints corresponding to the Quchi acupuncture point (LI11) and the Zusanli acupuncture point (ST 36) in humans[25] In the acupuncture group the acupuncture needlesremained inserted for 15 minutes before aspirin was injectedvia the femoral vein In the electro-acupuncture group theacupuncture needles were connected to an electrotherapeuticapparatus (Model-058 Ching-Ming Taiwan) and treatmentcontinued for 15 minutes before aspirin was injected viafemoral vein The electro-acupuncture parameters were setas a disperse-dense wave frequency of 2 and 50Hz and anintensity of 1mA [26]

26 Measurement of Brain Blood Flow The skull of theanaesthetized was exposed and a laser Doppler blood flowimager (moorLDI2 Moor Instruments UK) was used to

monitor the rat brain blood flow To obtain the baselinecontrol data the rat brain blood flow was measured beforeacupuncture or electro-acupuncture stimulationThen the ratbrain blood flow was continually measured during acupunc-ture or electro-acupuncture stimulation for 6 hours The ratblood flow data was analyzed and calculated by MoorLDIVersion 5 Research Software

27 Pharmacokinetic Data Calculation The concentrationsof aspirin and salicylic acid in the dialysate (119862

119898) were

converted to protein-unbound concentrations (119862119906) by the

following equation 119862119906= 119862119898119877dial Each individual set of

datawas used to calculate the pharmacokinetic parameters bythe pharmacokinetic program WinNonlin Standard EditionVersion 11 (Scientific Consulting Apex NC USA) Phar-macokinetic parameters of elimination half-life (119905

12) area

under the concentration-time curve (AUC) clearance (Cl)and apparent volume of distribution (119881

119889) were used in this

study

28 Statistical Analysis All data are presented as mean plusmnstandard error of mean (SEM) One-way ANOVA andpost hoc analysis were carried out for statistical comparisonbetween the control acupuncture and electro-acupuncturegroup using the statistical software The version of SPSS is1007 (SPSS Chicago USA) and the 119875 lt 005was consideredstatistical significantly

3 Results

31 Analytical Method Aspirin and salicylic acid were sepa-rated by acidic mobile phases which were adjusted to aciditywith 10mMKH

2PO4acetonitrile (71 29 vv pH 25 adjusted

by orthophosphoric acid) [24] Typical chromatograms ofaspirin and salicylic acid in rat blood and brain dialysates areshown in Figures 1 and 2 The retention times of aspirin andsalicylic acid were 7 and 98min respectively

The chromatogram of a blank blood dialysate is shown inFigure 1(a)The chromatogram of standard aspirin (1 120583gmL)and salicylic acid (1 120583gmL) is shown in Figure 1(b) The realblood samples containing aspirin (21 120583gmL) and salicylicacid (56 120583gmL) were collected at 30ndash45min dialysate afteraspirin administration (30mgkg iv) Figure 2(a) showsthe chromatogram of a blank brain dialysate Figure 2(b)shows the chromatogram of standard aspirin (15120583gmL) andsalicylic acid (15 120583gmL) Figure 2(c) shows the real braindialysate containing only salicylic acid (13 120583gmL) collectedat 105ndash120min after aspirin administration (30mgkg iv)

32 Pharmacokinetics of Aspirin and Salicylic Acid in Bloodand Brain Figures 3 and 4 show the concentration-timecurves of aspirin and salicylic acid in blood and brainrespectively for the groups of control acupuncture andelectro-acupuncture after aspirin administration (30mgkgiv) Tables 1 and 2 show the pharmacokinetic parametersof aspirin and salicylic acid in the control acupunctureand electro-acupuncture group in rat blood and brainrespectively However aspirin could not be observed in the


Table 1 The pharmacokinetic parameters of aspirin and salicylic acid for the rat blood The control group (aspirin administration 30mgkgonly) the acupuncture group (acupuncture stimulation 15min before aspirin administration 30mgkg) the electro-acupuncture group(electro-acupuncture stimulation 15min before aspirin administration 30mgkg)

Parameter Aspirin (30mgkg) Aspirin (30mgkg) + acupuncture Aspirin (30mgkg) + electro-acupunctureAspirin11990512

(min) 12 plusmn 2 11 plusmn 2 12 plusmn 4

AUC (min 120583gmL) 113 plusmn 33 95 plusmn 10 92 plusmn 5

Cl (mLminkg) 2918 plusmn 624 3330 plusmn 705 3430 plusmn 501

Vd (mLkg) 36 plusmn 08 38 plusmn 19 39 plusmn 05

Salicylic acid11990512





Data expressed as mean plusmn SEM (119899 = 6) 11990512 elimination half-life AUC area under the concentration-time curve Cl clearance Vd apparent volume ofdistribution

Table 2 The pharmacokinetic parameters of salicylic acid for the rat brain The control group (aspirin administration 30mgkg only)the acupuncture group (acupuncture stimulation 15min before aspirin administration 30mgkg) the electro-acupuncture group (electro-acupuncture stimulation 15min before aspirin administration 30mgkg)

Parameter Aspirin (30mgkg) Aspirin (30mgkg) + acupuncture Aspirin (30mgkg) + electro-acupuncture11990512






(min)00 25 50 75 100

(mAU

)

minus2

0

2

4

6

8

10

12

14

(a)

(min)

(mAU

)

00 25 50 75 100

21

minus2

0

2

4

6

8

10

12

14

(b)

(min)

(mAU

)

00 25 50 75 100

2

1

minus2

0

2

4

6

8

10

12

14

(c)

Figure 1 Typical chromatograms of (a) blank blood dialysate (b) blank blood dialysate spiked with ASA (1 120583gmL) and SA (1 120583gmL) (c)blood sample containing ASA (21 120583gmL) and SA (56 120583gmL) after administration of aspirin (30mgkg iv) at 30ndash45min (1) Acetylsalicylicacid (ASA) (2) salicylic acid (SA)


00 25 50 75 100minus5

(mAU

)

0

5

10

15

20

25

(min)

(a)

00 25 50 75 100

2

1

minus5

(mAU

)0

5

10

15

20

25

(min)

(b)

00 25 50 75 100

2

minus5

(mAU

)

0

5

10

15

20

25

(min)

(c)

Figure 2 Typical chromatograms of (a) blank brain dialysate (b) blank brain dialysate spiked with ASA (15 120583gmL) and SA (15 120583gmL) and(c) brain sample containing SA (13 120583gmL) collected at 105ndash120min after administration of aspirin (30mgkg iv) (1) Acetylsalicylic acid(ASA) (2) salicylic acid (SA)

brain at the dosage of aspirin administration (30mgkg)The in vivo hydrolysis of aspirin occurs very rapidly inhuman which makes clinicians dependent majorly on thedetermination of salicylate to assess the therapeutic progress[27] In our study aspirin can be only observed in the bloodmicrodialysis samples and salicylic acid can be detected inthe blood and brain microdialysis samples (Figures 3 and 4)The pharmacokinetic data (Tables 1 and 2) demonstrate thatacupuncture and electro-acupuncture did not significantlyinteract with the concentration of aspirin and salicylic acidin the blood and brain after aspirin administration (30mgkgiv)

33 Rat Brain Blood Flow Figure 5 shows the rat brain bloodflow images In Figures 5(a) and 5(b) increased blood flowwas found after electro-acupuncture stimulation for 5minFigure 5(c) shows the brain blood flow for consecutive 6 hafter electro-acupuncture stimulation Figure 6 and Table 3demonstrated the integrated brain blood flow of controlacupuncture and electro-acupuncture groups A 15-minuteperiod before treatment (minus15ndash0min) was used as the baselinefor brain blood flow measurement Then during the timeof 0ndash15min there was treatment with different stimulationfor the acupuncture and electro-acupuncture groups Afterstimulation (15min) the needles were removed The datademonstrate that the brain blood flow increased in theelectro-acupuncture groupmore than in the groups of controland acupuncture The time to reach the highest brain bloodflow is around 15min after electro-acupuncture stimulation

Table 3 The numeric descriptions of brain blood flow in the threegroups

Time (min) Control group Acupuncturegroup

Electro-acupuncture

groupminus15 942 plusmn 37 938 plusmn 47 948 plusmn 86

0 938 plusmn 42 935 plusmn 28 985 plusmn 54













Each group contained six rats (119873 = 6)Results were expressed as mean plusmn standard deviation and the unit of bloodflow is BPU

4 Discussion

According to the formula for dose translation based onthe body surface area [28] the human equivalent dose is486mgkg which equals approximately 291mg aspirin for a


100

10

1

01

001

00010 30 60 90 120 150 180 210 240 270 300 330 360

ASA (ASA 30mgkg)ASA (ASA 30mgkg) + AASA (ASA 30mgkg) + EA

SA (ASA 30mgkg)SA (ASA 30mgkg) + ASA (ASA 30mgkg) + EA

Bloo

d as

pirin

and

salic

ylic

acid

(120583g

mL)

Time (min)

Figure 3 Concentration-time curve of protein-unbound aspirinand salicylic acid in the blood after aspirin administration at30mgkg The control group (aspirin administration only) concen-tration of ASA (∙) concentration of SA (I) The acupuncture group(acupuncture stimulation 15min before aspirin administration)concentration of ASA (◼) concentration of SA (◻) The electro-acupuncture group (electro-acupuncture stimulation 15min beforeaspirin administration) concentration of ASA (998771) concentration ofSA (998779) Acetylsalicylic acid (ASA) salicylic acid (SA) Acupuncture(A) electro-acupuncture (EA)

10

1

01

0010 30 60 90 120 150 180 210 240 270 300 330 360

Time (min)


Brai

n sa

licyl

ic ac

id (120583

gm

L)

Figure 4 Concentration-time curve of protein-unbound salicylicacid in the brain after aspirin administration 30mgkg The con-trol group (aspirin administration only) concentration of SA (∙)The acupuncture group (acupuncture stimulation 15min beforeaspirin administration) concentration of SA (◼) The electro-acupuncture group (electro-acupuncture stimulation 15min beforeaspirin administration) concentration of SA (998771) Salicylic acid (SA)Acupuncture (A) electro-acupuncture (EA)

60 kg adultThedose (30ndash325mg) of aspirin has been used forthe secondary prevention of vascular events after ischaemicstroke [29] Patients with recent symptomatic lacunar infarctsidentified by magnetic resonance imaging have received325mg of aspirin daily to evaluate the reduction of the riskof recurrent stroke and the risk of bleeding and death [30]The dose selection for aspirin is acceptable and reasonable

The absorption distribution metabolism and elimina-tion of a drug are influenced by various physiological factorssuch as plasma protein binding blood perfusion membranepermeability enzymatic metabolism and membrane trans-ports [31] In our current study we only focus on the effects ofaltered blood perfusion caused by electro-acupuncture on thepharmacokinetics of aspirin However electro-acupuncturemay have additional biological effects on the subject Theeffects of electro-acupuncture on metabolic enzymes trans-porter activities and plasma protein expression have to beevaluated in the future studies

Urethane and 120572-chloralose were used as anesthesia agentsin this study Anaesthesia using urethane-chloralose is acommonly used combination for pharmacokinetic studiesand has been considered acceptable for pharmacokinetic-pharmacodynamic studies [32] Our study included thecontrol group to exclude the effects caused by interactionsbetween anesthesia agents and aspirin Although the combi-nation of acupuncture electro-acupuncture herbal medicineand Western drug therapy has been used in clinical appli-cations for several decades there has been little research onthe interaction between acupuncture or electro-acupunctureand the pharmacokinetics of Western drugs In 2004 wedeveloped a microdialysis system to explore the interactionof acupuncture on the acupoints of Taichong (LR3) andYanglingquan (GB34) for the pharmacokinetics of geniposidein rats [33] The results indicated that these two acupointsdid not affect the pharmacokinetics of geniposide in ratblood liver and bile in that experimental model HoweverZhou et al (2009) showed that electro-acupuncture at theacupoints of Jizhong (GV6) Dazhui (GV14) and Zhongwan(CV12) increases the absorption of baicalin from extracts ofScutellaria baicalensis Georgi in normal rats [25] Howeverbased on the previous results there is no consensus forthe effects of acupuncture or electro-acupuncture on thepharmacokinetics of herbal medicine In this study thepharmacokinetic data demonstrate that stimulation withacupuncture and electro-acupuncture had little significanteffect on the concentration of aspirin and salicylic acid in theblood and brain after aspirin administration (30mgkg iv)One potential explanation is that several factors may affectthese results such as the anesthetized experimental animalmodel the acupoints selected the dose of aspirin or theprotein-unbound form of analytes collected bymicrodialysisFurthermore acupuncture or electro-acupuncture stimula-tion of physiological functions for example the autonomicnervous system or organ blood flow can indirectly affect theabsorption distribution metabolism and excretion of drugs[34]

Another possible explanation is that the short-termand long-term stimulation of acupuncture and electro-acupuncture may have different results In our study we


(a)

(b)

Pre EA During EA1 min During EA5 min During EA10min During EA15min Post EA1 min

Post EA5 min Post EA10min Post EA20min Post EA30 min Post EA40 min Post EA50 min

Post EA60 min Post EA120min Post EA180min Post EA240 min Post EA300min Post EA360 min

(c)

Figure 5 Rat brain blood flow image (a) Before electro-acupuncture stimulation (b) after electro-acupuncture stimulation for 5min (c)continuous monitoring for 6 h

try to confirm the short-term acupuncture and electro-acupuncture stimulation interaction on the pharmacokinet-ics of aspirin in rats However long-term stimulation ofacupuncture and electro-acupuncture points would be a veryinteresting subject for future studies

Recent studies have found that the mechanism ofacupuncture is partly related to the nervous and vascularsystem [35] Thus the effect of acupuncture and electro-acupuncture stimulation in awake or anesthetized subjectsmight be different After having been under anesthesia fora period of time a subjectrsquos physiological responses toacupuncture or electro-acupuncture might be concealed Toperform acupuncture and electro-acupuncture stimulation

on laboratory animals and for experimental feasibility con-siderations an anesthetized animal model for blood andbrain sampling was used To avoid the anesthesia effecton acupuncture a well-designed clinical trial should beperformed

5 Conclusions

In this study our data demonstrate that acupuncture andelectro-acupuncture did not significantly interact with thepharmacokinetics of aspirin (30mgkg iv) in rat bloodand brain According to our results in this work the useof acupuncture and electro-acupuncture did not change the


Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200

Control groupAcupuncture groupElectroacupuncture group

minus15

Figure 6 Rat brain blood flow control group (∙) acupuncturegroup (◼) electro-acupuncture group (998771) EA electro-acupuncturegroup

distribution and pharmacokinetics of single-dose aspirin inrats which might suggest in part the safety of combination ofacupuncture and electro-acupuncture and aspirin Howeverfurther studies are necessary to clarify other potential mecha-nisms of acupuncture and electro-acupuncture that influencepharmacokinetics

Conflict of Interests

The authors declare that they have no conflict of interests inthe publication of this paper

Acknowledgments

Funding for this study was provided in part by researchGrants from the National Research Institute of ChineseMedicine Taipei Taiwan the National Science Council(NSC102-2113-M-010-001-MY3) Taiwan and TCH 102-0210102-62-084 from Taipei City Hospital Taipei Taiwan

References

[1] WHO Neurological Disorders Public Health Challenges WorldHealth Organization 2006

[2] F-P Chen Y-Y Kung T-J Chen and S-J Hwang ldquoDemo-graphics and patterns of acupuncture use in the Chinesepopulation the Taiwan experiencerdquo Journal of Alternative andComplementary Medicine vol 12 no 4 pp 379ndash387 2006

[3] Z Fang J Ning C Xiong and Y Shulin ldquoEffects of elec-troacupuncture at head points on the function of cerebralmotor areas in stroke patients a PET studyrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID902413 9 pages 2012

[4] N Li F-W Tian C-WWang et al ldquoDouble-center randomizedcontrolled trial on post-stroke shoulder pain treated by elec-troacupuncture combined with Tuinardquo Zhongguo Zhen Jiu vol32 no 2 pp 101ndash105 2012

[5] Z H Yue L Li X R Chang et al ldquoComparative study oneffects between electroacupuncture and acupuncture for spasticparalysis after strokerdquoZhongguo Zhen Jiu vol 32 no 7 pp 582ndash586

[6] Y-Z He B Han S-F Zheng et al ldquoEffect of different acupunc-ture needle-retaining time on hemorheology in ischemic strokepatientsrdquo Zhen Ci Yan Jiu vol 32 no 5 pp 338ndash341 2007

[7] A Chen Z Lin L Lan et al ldquoElectroacupuncture at the Quchiand Zusanli acupoints exerts neuroprotective role in cerebralischemia-reperfusion injured rats via activation of the PI3KAktpathwayrdquo International Journal of Molecular Medicine vol 30no 4 pp 791ndash796 2012

[8] L Ren Y-K Wang Y-N Fang A-W Zhang and X-L LildquoEffect of electroacupuncture therapy on the expression ofNa(v)11 and Na(v)16 in rat after acute cerebral ischemiardquoNeurological Research vol 32 no 10 pp 1110ndash1116 2010

[9] M W Kim Y C Chung H C Jung et al ldquoElectroacupunc-ture enhances motor recovery performance with brain-derivedneurotrophic factor expression in rats with cerebral infarctionrdquoAcupuncture in Medicine vol 30 no 3 pp 222ndash226 2012

[10] H Zheng S-W Zhu F Yang et al ldquoEfficacy observation ofThoroughfare Vessel theory in acupuncture for post-strokedysphasiardquo Zhongguo Zhen Jiu vol 31 no 12 pp 1067ndash10702011

[11] S Uchida and H Hotta ldquoAcupuncture affects regional bloodflow in various organsrdquo Evidence-based Complementary andAlternative Medicine vol 5 no 2 pp 145ndash151 2008

[12] S Uchida F Kagitani A Suzuki and Y Aikawa ldquoEffect ofacupuncture-like stimulation on cortical cerebral blood flow inanesthetized ratsrdquo Japanese Journal of Physiology vol 50 no 5pp 495ndash507 2000

[13] K K Wu ldquoAspirin and salicylate an old remedy with a newtwistrdquo Circulation vol 102 no 17 pp 2022ndash2023 2000

[14] D Bennett B Yan LMacGregor D Eccleston and SM DavisldquoA pilot study of resistance to aspirin in stroke patientsrdquo Journalof Clinical Neuroscience vol 15 no 11 pp 1204ndash1209 2008

[15] H C Diener J Bogousslavsky L M Brass et al ldquoAspirinand clopidogrel compared with clopidogrel alone after recentischaemic stroke or transient ischaemic attack in high-risk patients (MATCH) randomised double-blind placebo-controlled trialrdquoThe Lancet vol 364 no 9431 pp 331ndash337

[16] S H Shah R Engelhardt and B Ovbiagele ldquoPatterns ofcomplementary and alternative medicine use among UnitedStates stroke survivorsrdquo Journal of the Neurological Sciences vol271 no 1-2 pp 180ndash185 2008

[17] S P Clissold ldquoAspirin and related derivatives of salicylic acidrdquoDrugs vol 32 no 4 pp 8ndash26 1986

[18] ldquoA comparison of two doses of aspirin (30 mg versus 283 mga day) in patients after a transient ischemic attack or minorischemic stroke The Dutch TIA Trial Study Grouprdquo The NewEngland Journal of Medicine vol 325 no 18 pp 1261ndash1266

[19] J Hirsh E Salzman L Harker et al ldquoAspirin and other plateletactive drugs Relationship among dose effectiveness and sideeffectsrdquo Chest vol 95 no 2 pp 12Sndash18S 1989

[20] M Backer M G Hammes M Valet et al ldquoDifferent modesof manual acupuncture stimulation differentially modulatecerebral blood flow velocity arterial blood pressure and heart


rate in human subjectsrdquoNeuroscience Letters vol 333 no 3 pp203ndash206 2002

[21] T-H Tsai ldquoAssaying protein unbound drugs using microdial-ysis techniquesrdquo Journal of Chromatography B vol 797 no 1-2pp 161ndash173 2003

[22] A K Pedersen and G A FitzGerald ldquoDose-related kinetics ofaspirin Presystemic acetylation of platelet cyclooxygenaserdquoTheNew England Journal of Medicine vol 311 no 19 pp 1206ndash12111984

[23] R Pirola S R Bareggi andG De Benedittis ldquoDetermination ofacetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatographyrdquo Journal of Chromatogra-phy B vol 705 no 2 pp 309ndash315 1998

[24] L-H Shaw and T-H Tsai ldquoSimultaneous determination andpharmacokinetics of protein unbound aspirin and salicylic acidin rat blood andbrain bymicrodialysis an application to herbal-drug interactionrdquo Journal of Chromatography B vol 895-896pp 31ndash38 2012

[25] J Zhou F Qu E Burrows Y Yu and R Nan ldquoAcupuncturecan improve absorption of baicalin from extracts of Scutellariabaicalensis Georgi in ratsrdquo Phytotherapy Research vol 23 no10 pp 1415ndash1420 2009

[26] X-BWang J Chen T-J Li et al ldquoEffect of electroacupuncturein different frequencies on electromyography and ambulationin stroke patients with lower-extremity spasticity a randomizedcontrolled studyrdquo Zhongguo Zhen Jiu vol 31 no 7 pp 580ndash5842011

[27] E J Eyring and P C Ford ldquoComparison of acetyl-salicylicacid (aspirin) hydrolase activities in various tissues of severalspeciesrdquoComparative Biochemistry and Physiology B vol 43 no2 pp 333ndash337 1972

[28] S Reagan-Shaw M Nihal and N Ahmad ldquoDose translationfrom animal to human studies revisitedrdquo FASEB Journal vol22 no 3 pp 659ndash661 2008

[29] E S Group P H Halkes J van Gijn et al ldquoAspirin plusdipyridamole versus aspirin alone after cerebral ischaemia ofarterial origin (ESPRIT) randomised controlled trialrdquo TheLancet vol 367 no 9523 pp 1665ndash1673

[30] S P S Investigators O R Benavente R G Hart et al ldquoEffectsof clopidogrel added to aspirin in patients with recent lacunarstrokerdquoTheNew England Journal of Medicine vol 367 no 9 pp817ndash825 2012

[31] C-Y Wu and L Z Benet ldquoPredicting drug disposition viaapplication of BCS transportabsorption elimination interplayand development of a biopharmaceutics drug disposition clas-sification systemrdquo Pharmaceutical Research vol 22 no 1 pp11ndash23 2005

[32] F M Bertera C A Di Verniero M A Mayer G F BramugliaC A Taira and C Hocht ldquoIs urethane-chloralose anaesthe-sia appropriate for pharmacokinetic-pharmacodynamic assess-ment Studies with carvedilolrdquo Journal of Pharmacological andToxicological Methods vol 59 no 1 pp 13ndash20 2009

[33] T-Y Tseng and T-H Tsai ldquoMeasurement of unbound geni-poside in blood liver brain and bile of anesthetized ratsan application of pharmacokinetic study and its influence onacupuncturerdquo Analytica Chimica Acta vol 517 no 1-2 pp 47ndash52 2004

[34] H Boxenbaum ldquoInterspecies variation in liver weight hepaticblood flow and antipyrine intrinsic clearance extrapolation ofdata to benzodiazepines and phenytoinrdquo Journal of Pharmacoki-netics and Biopharmaceutics vol 8 no 2 pp 165ndash176 1980

[35] J Johansson L Manneras-Holm R Shao et al ldquoElectrical vsmanual acupuncture stimulation in a rat model of polycysticovary syndrome different effects on muscle and fat tissueinsulin signalingrdquo PLoS One vol 8 no 1 Article ID e543572013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


as well as for treating acute myocardial infarction [13] Ina previous study low-dose aspirin has been shown usefulfor the prevention of cardiovascular diseases and strokerecurrence [14] Furthermore low-dose aspirin can alsoreduce mortality rates of cardiovascular disease and strokepatients [15] For post-stroke patients it may be beneficialto undergo treatment by electro-acupuncture combined withaspirin [16] But adverse reactions of which there is a widespectrum frequently accompany anti-inflammatory doses ofaspirin Aspirin and related derivatives of salicylic acid havebeen reported to have a wide range of drug interactions butrelatively few seem to be clinically important Many of theseinteractions are pharmacokinetic in nature [17] Cyclooxyge-nase is irreversible inhibition by aspirin and dose-related sideeffects of aspirin on gastrointestinal symptoms (eg bleedingcomplications) have been reported [18 19] In additionaspirin exhibits a nonlinear pharmacokinetics meaning thatthe binding of drugs to plasma components blood cellsand extravascular tissue may demonstrate concentrationdependence Therefore if acupuncture changes the pharma-cokinetics of aspirin the side effects may occur

A survey from PubMed with the key words of electro-acupuncture and pharmacokinetics of aspirin found noitems Accordingly this study develops a technique usingmicrodialysis coupled to liquid chromatography withphotodiode-array detection to monitor protein-unboundaspirin and its metabolites in blood and brain of the ratTo investigate potential interactions between acupunctureand electro-acupuncture on the pharmacokinetics ofaspirin microdialystates of the blood and brain on thegroups of control acupuncture and electro-acupuncturewere individually collected and measured to explore thepharmacokinetic interaction of aspirin To monitor the ratbrain blood flow a laser Doppler blood flow imager wasused to measure the brain blood flow on the groups ofcontrol acupuncture and electro-acupuncture The studymay in part provide important information regarding theeffects of acupuncture and electro-acupuncture as they areinfluenced by the pharmacokinetic and pharmacodynamicinteraction of aspirin Physiological factors affecting thedistribution of a drug include plasma protein bindingblood perfusion and membrane permeability Previouspapers have discovered that acupuncture changes bloodflow [12 20] which may affect directly the distribution of adrug In this work we assessed the effects of acupunctureon blood flow to confirm the reported results and thenperformed pharmacokinetic experiments We decided to usea rat model by microdialysis sampling to examine the effectsof acupunctureelectro-acupuncture on drug distributionbecause microdialysis provides several advantages such assampling protein-unbound drug providing high temporalresolution data and minimizing physiological disturbance ofthe subject for a pharmacokinetic evaluation [21] Aspirinhad poor oral bioavailability which may lead to fluctuatedpharmacological effects [22] and we chose intravenousroute for drug administration to minimize the fluctuatedpharmacological effects caused by inter-subject absorptionvariations The aim of our work is to evaluate the possibleinteractions between acupuncture and pharmacokinetics of

aspirin The use of acupuncture and electro-acupuncturemay result in various biological effects on the subject so weattempted to find the relationship between acupuncture andpharmacokinetics from a distribution viewpoint

2 Materials and Methods

21 Chemicals and Reagents Aspirin salicylic acid 120572-chloralose andurethanewere purchased fromSigma-AldrichChemicals (St Louis MO USA) Acetic acid sodium citratedextrose sodium chloride potassium dihydrogen phosphate(KH2PO4) and orthophosphoric acid (85 ww) were pur-

chased from E Merck (Darmstadt Germany) Acetonitrileof analytical grade was purchased from ECHO ChemicalCo (Taiwan) Deionized water fromMillipore (Milford MAUSA) was used for all aqueous solutions in this study

22 HPLC Instrumentation HPLC-UV instrumentationconsisted of a Shimadzu chromatographic pump (LC-20AT)a DGU-20A5 degasser an autosampler (SIL-20AC) anda photodiode array detector (SPD-M20A) (ShimadzuKyoto Japan) A reverse-phase C18 column (MerckPurospher STAR 250mm times 4mm id particle size 5 120583mDarmstadt Germany) was used to separate the analytesThe UV absorbance wavelength was set at 240 nm to detectaspirin and salicylic acid The mobile phase consisted ofacetonitrile 10mM KH

2PO4(29 71 vv pH 25 adjusted

by orthophosphoric acid) for analysis of blood and brainmicrodialysates A Millipore (022120583m) filter (Bedford MAUSA) was used to filter the mobile phase and we used asonicator (Branson CT USA) to degas before the mobilephase was used The flow rate was set at 1mLmin

23 Experimental Animals Adult male pathogen-freeSprague-Dawley rats (200ndash260 g) were obtained fromthe Laboratory Animal Center at National Yang-MingUniversity (Taipei Taiwan) Rats were housed in cages with12 h lightdark cycle food (Laboratory RodentDiet 5001 PMIFeeds Inc Richmond IN USA) and water were availablead libitum The animals were received at 6-7 weeks of ageand acclimated for at least one week All animal experimentsfollowed the National Yang-Ming University guidelinesand procedures for the care of laboratory animals andthe protocol listed above has been reviewed and approvedby the Institutional Animal Care and Use Committee(IACUC approval number 1011203) by the InstitutionalAnimal Experimentation Committee of National Yang-MingUniversity The animals had free access to food and waterThe rat was anaesthetized with urethane 10 gmL and120572-chloralose 01 gmL (1mLkg ip) before surgery Thefemoral vein was cannulated for further drug administrationand the ratrsquos body temperature was maintained by a heatingpad during the experiment and they were euthanized byoverdose CO

2under the anesthetic after the experimental

endpoint

24 Microdialysis Experiments The microdialysis systemincluded a CMA100 microinjection syringe pump a








119898) were




12) area



study


3 Results


























(min)00 25 50 75 100

(mAU

)

minus2

0

2

4

6

8

10

12

14

(a)

(min)

(mAU

)

00 25 50 75 100

21

minus2

0

2

4

6

8

10

12

14

(b)

(min)

(mAU

)

00 25 50 75 100

2

1

minus2

0

2

4

6

8

10

12

14

(c)



00 25 50 75 100minus5

(mAU

)

0

5

10

15

20

25

(min)

(a)

00 25 50 75 100

2

1

minus5

(mAU

)0

5

10

15

20

25

(min)

(b)

00 25 50 75 100

2

minus5

(mAU

)

0

5

10

15

20

25

(min)

(c)






Electro-acupuncture
















4 Discussion



100

10

1

01

001

00010 30 60 90 120 150 180 210 240 270 300 330 360



Bloo

d as

pirin

and

salic

ylic

acid

(120583g

mL)

Time (min)


10

1

01

0010 30 60 90 120 150 180 210 240 270 300 330 360

Time (min)


Brai

n sa

licyl

ic ac

id (120583

gm

L)







(a)

(b)




(c)





5 Conclusions



Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























119898) were




12) area



study


3 Results


























(min)00 25 50 75 100

(mAU

)

minus2

0

2

4

6

8

10

12

14

(a)

(min)

(mAU

)

00 25 50 75 100

21

minus2

0

2

4

6

8

10

12

14

(b)

(min)

(mAU

)

00 25 50 75 100

2

1

minus2

0

2

4

6

8

10

12

14

(c)



00 25 50 75 100minus5

(mAU

)

0

5

10

15

20

25

(min)

(a)

00 25 50 75 100

2

1

minus5

(mAU

)0

5

10

15

20

25

(min)

(b)

00 25 50 75 100

2

minus5

(mAU

)

0

5

10

15

20

25

(min)

(c)






Electro-acupuncture
















4 Discussion



100

10

1

01

001

00010 30 60 90 120 150 180 210 240 270 300 330 360



Bloo

d as

pirin

and

salic

ylic

acid

(120583g

mL)

Time (min)


10

1

01

0010 30 60 90 120 150 180 210 240 270 300 330 360

Time (min)


Brai

n sa

licyl

ic ac

id (120583

gm

L)







(a)

(b)




(c)





5 Conclusions



Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































(min)00 25 50 75 100

(mAU

)

minus2

0

2

4

6

8

10

12

14

(a)

(min)

(mAU

)

00 25 50 75 100

21

minus2

0

2

4

6

8

10

12

14

(b)

(min)

(mAU

)

00 25 50 75 100

2

1

minus2

0

2

4

6

8

10

12

14

(c)



00 25 50 75 100minus5

(mAU

)

0

5

10

15

20

25

(min)

(a)

00 25 50 75 100

2

1

minus5

(mAU

)0

5

10

15

20

25

(min)

(b)

00 25 50 75 100

2

minus5

(mAU

)

0

5

10

15

20

25

(min)

(c)






Electro-acupuncture
















4 Discussion



100

10

1

01

001

00010 30 60 90 120 150 180 210 240 270 300 330 360



Bloo

d as

pirin

and

salic

ylic

acid

(120583g

mL)

Time (min)


10

1

01

0010 30 60 90 120 150 180 210 240 270 300 330 360

Time (min)


Brai

n sa

licyl

ic ac

id (120583

gm

L)







(a)

(b)




(c)





5 Conclusions



Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 25 50 75 100minus5

(mAU

)

0

5

10

15

20

25

(min)

(a)

00 25 50 75 100

2

1

minus5

(mAU

)0

5

10

15

20

25

(min)

(b)

00 25 50 75 100

2

minus5

(mAU

)

0

5

10

15

20

25

(min)

(c)






Electro-acupuncture
















4 Discussion



100

10

1

01

001

00010 30 60 90 120 150 180 210 240 270 300 330 360



Bloo

d as

pirin

and

salic

ylic

acid

(120583g

mL)

Time (min)


10

1

01

0010 30 60 90 120 150 180 210 240 270 300 330 360

Time (min)


Brai

n sa

licyl

ic ac

id (120583

gm

L)







(a)

(b)




(c)





5 Conclusions



Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

10

1

01

001

00010 30 60 90 120 150 180 210 240 270 300 330 360



Bloo

d as

pirin

and

salic

ylic

acid

(120583g

mL)

Time (min)


10

1

01

0010 30 60 90 120 150 180 210 240 270 300 330 360

Time (min)


Brai

n sa

licyl

ic ac

id (120583

gm

L)







(a)

(b)




(c)





5 Conclusions



Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)




(c)





5 Conclusions



Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Time (min)15 45 75 105 135 165 195 225 255 285 315 345 375

Brai

n bl

ood

flow

(BPU

)

700

800

900

1000

1100

1200


minus15





Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article interaction of acupuncture and...

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