Research Article Electroacupuncture Reduces Cocaine-Induced Seizures and Mortality in Mice

14 Pages·2015·3.19 MB·English

HindawiPublishingCorporation
Evidence-BasedComplementaryandAlternativeMedicine
Volume2013,ArticleID134610,13pages
http://dx.doi.org/10.1155/2013/134610
Research Article
Electroacupuncture Reduces Cocaine-Induced Seizures and
Mortality in Mice
Yi-HungChen,1BorisIvanic,1,2Chieh-MinChuang,1Dah-YuuLu,3andJaung-GengLin4
1GraduateInstituteofAcupunctureScience,ChinaMedicalUniversity,Taichung40402,Taiwan
2Children’sUniversityHospital,Limbova1,Bratislava,Slovakia
3GraduateInstituteofNeuralandCognitiveSciences,ChinaMedicalUniversity,Taichung40402,Taiwan
4SchoolofChineseMedicine,ChinaMedicalUniversity,Taichung40402,Taiwan
CorrespondenceshouldbeaddressedtoYi-HungChen;[email protected]
Jaung-GengLin;[email protected]
Received9January2013;Accepted17February2013
AcademicEditor:GerhardLitscher
Copyright©2013Yi-HungChenetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Theaimsofthisstudyweretocharacterizetheprotectiveprofileofelectroacupuncture(EA)oncocaine-inducedseizuresand
mortalityinmice.MiceweretreatedwithEA(2Hz,50Hz,and100Hz),ortheyunderwentneedleinsertionwithoutanesthesiaatthe
Dazhui(GV14)andBaihui(GV20)acupointsbeforecocaineadministration.EAat50HzappliedtoGV14andGV20significantly
reducedtheseizureseverityinducedbyasingledoseofcocaine(75mg/kg;i.p.).Furthermore,needleinsertionintoGV14and
GV20andEAat2Hzand50Hzatbothacupointssignificantlyreducedthemortalityrateinducedbyasinglelethaldoseofcocaine
(125mg/kg;i.p.).Intheshamcontrolgroup,EAat50HzappliedtobilateralTianzong(SI11)acupointshadnoprotectiveeffects
againstcocaine.Inaddition,EAat50HzappliedtoGV14andGV20failedtoreducetheincidenceofseizuresandmortalityinduced
bythelocalanestheticprocaine.Inanimmunohistochemistrystudy,EA(50Hz)pretreatmentatGV14andGV20decreasedcocaine
(75mg/kg;i.p.)-inducedc-Fosexpressionintheparaventricularthalamus.WhilethedopamineD3receptorantagonist,SB-277011-
A(30mg/kg;s.c),didnotbyitselfaffectcocaine-inducedseizureseverity,itpreventedtheeffectsofEAoncocaine-inducedseizures.
TheseresultssuggestthatEAalleviatescocaine-inducedseizuresandmortalityandthatthedopamineD3receptorisinvolved,at
leastinpart,intheanticonvulsanteffectsofEAinmice.
1.Introduction Seizuresareconsideredtobeamajordeterminantofcocaine-
related lethality in humans [4] and animals [9]. Sudden
1.1.Cocaine-InducedSeizuresandDeath. Cocaineisawidely death in cocaine abuse may also be attributed to cardiac
abused psychomotor stimulant [1, 2]. In addition to its arrhythmiaandintracerebralhemorrhage[10,11].Currently,
addiction liability, the abuse of cocaine is associated with no specific treatment modalities offer protection against
an array of medical complications [3]. Generalized tonic- cocaine-inducedseizuresanddeathinhumans.
clonic seizures and status epilepticus are well-documented
neurologic sequelae of cocaine abuse [3, 4]. Seizures may
be induced by cocaine after an accidental overdose or after 1.2.Acupuncture. Acupuncturehasbeenusedintraditional
recreationaluseofrelativelylowdosesofcocaine[5,6].The Chinesemedicineforover2,500years[12].Thepracticehas
incidence of seizures in cocaine users has been reported to recently gained popularity and is becoming an alternative
be 2–10% [7]. High doses of cocaine have been associated and/or complementary treatment modality for a variety of
withadisturbinglyhighnumberofsuddendeathsinadults; disordersworldwide[13].In1997,theUSNationalInstitutes
a recent study documented that 3.1% of all sudden deaths of Health stated that acupuncture is a useful method for
in Southwest Spain were related to the use of cocaine [8]. treating many conditions and has fewer side effects as
2 Evidence-BasedComplementaryandAlternativeMedicine
comparedwithothercontemporarymedicaltreatments,such mortality in a mouse model, (2) determine the frequency-
as surgery or drugs [13]. Two different strategies are used dependent antiseizure activity of EA, and (3) correlate the
in acupuncture therapy: manual acupuncture (MA) and efficacyofEAwiththeexpressionofFosproteininthebrain.
electroacupuncture(EA).EAisamodifiedformoftraditional
MA. The advantage of EA is in its combined therapeutic
effects of transcutaneous electric nerve stimulation (TENS) 2.Methods
and MA. Most studies use EA because the latter can be
standardizedbyfrequency,voltage,waveform,length,andso 2.1.LaboratoryAnimals. MaleICRmice(28–35g;BioLasco
forth[14].Studiesonanimalsandhumanshavedemonstrated Taiwan Co., Ltd., Taiwan) were housed under a 12:12h
thatacupunctureresultsinmultiplebiologicalresponses[15, light/dark cycle with food and water available ad libitum in
16].Thebestcharacterizedmechanismisviatheendogenous ouranimalfacilityforatleast4dayspriortotheexperiments,
opioidpeptidesandtheirreceptors[17]. whichwereconductedbetween10:00and17:00h.Theexper-
imental procedures were approved by the China Medical
UniversityInstitutionalAnimalCareandUseCommittee,in
1.3. Treating Epilepsy with Acupuncture. Acupuncture has accordance with the Chinese Taipei Society of Laboratory
beenusedtotreatepilepsyinChinaforthousandsofyears. Animal Sciences guidance on care and use of laboratory
Many reports have suggested that acupuncture, TENS and animals.Experimentsweredesignedtokeepthenumberof
other alternative therapies may produce positive effects in miceataminimumandcarewastakentominimizesuffering.
epilepsy[19],althoughsomestudiesdisagree[20].Themajor
advantage of acupuncture is the absence of side effects.
In the clinic, acupuncture at specific acupoints such as 2.2.Electroacupuncture. FortheEAtreatment,animalswere
the Zusanli (ST36), Dazhui (GV14), and Baihui (GV20) covered by paraffin film and restrained by tapes without
has significantly ameliorated symptoms of epilepsy [21–23]. anesthesia. A pair of stainless steel acupuncture needles
Although some evidence supports this contention [24–26], (Tianjin HaingLimSou Won Medical CO., Ltd.; Gauge 40)
the precise mechanism remains unknown. It is noted that were inserted 3-4mm deep into the murine equivalent of
acupuncture to GV20 point has been used to treat loss of the human GV14 and GV20, that is, (1) the skin at the
consciousness and tinnitus, in addition to relief of mental location between the last cervical and the first thoracic
abstraction,sluggishspeech,andhysteria[27].Recentstudies vertebralspinousprocessesatthemidlineoftheback,which
have shown that EA inhibits seizures in experimental rat is equivalent to the human GV14 and (2) at the vertex of
models.Theseprotectiveeffectsmayberelatedtoincreased the parietal bone, that is, the midpoint of the connecting
concentrations of inhibitory amino acids [28], decreased linebetweentheauricularapices,whichisequivalenttothe
levels of nitric oxide in the central nervous system (CNS) humanGV20acupoints[42,43].EAstimuliweredelivered
[26, 29], or increased cellular glutamic acid decarboxylase- byanEATrio300stimulator(Ito,Japan)at1mAintensities
67 mRNA expression, thereby increasing the production of fora15mindurationatafrequencyof2,50,or100Hz,with
𝛾-aminobutyric acid (GABA) [30]. The possibility that EA apulsewidthof150𝜇s.Thetwoelectrodeswereconnectedto
protectsagainstcocaine-inducedseizuresanddeathhasnot theneedles,whichwereinsertedintoGV20andGV14.Inthe
yetbeenexplored. controlgroup,animalswerealsocoveredbyparaffinfilmand
restrainedbytapefor15min[44].AshamEAwasperformed
bybilateralinsertionofapairofstainlesssteelacupuncture
1.4. Acupuncture and Abused Drugs. Acupuncturehasbeen
needlesapproximately3-4mmdeepintothemiddleofeach
widely used throughout Asian countries for treating many
scapula,whichisequivalenttothehumanTianzong(SI11).
functionaldisorders,suchassubstanceabuseandmentalill-
ness[31].Somepreclinicaldatahaveshownthatacupuncture
canmodifythemorphinewithdrawalsyndromeandsuppress 2.3. Behavioral Study. Following the injection of cocaine
alcoholdrinkingbehaviorinrats[32, 33]. Furtherevidence or saline, animals were placed in a plastic observation box
suggests that acupuncture at a specific acupoint (Shenmen; and seizure scores were assessed by the Itzhak scale, which
HT7)attenuatestheethanol-induceddopaminereleaseinthe categorizes five stages according to their severity over a
nucleus accumbens through the GABAB receptor [34] and 30minperiod[18].TheItzhakstagesofseizureareasfollows.
suppresses c-Fos expression in the nucleus accumbens and
ventromedialstriatumfollowinganicotinechallengeinrats Stage 1. Normal behavior (moving about the cage, sniffing,
sensitizedtonicotine[35]. andrearing).
Stage2. Hyperactivity(runningmovementcharacterizedby
1.5.c-FosExpression. Expressionofc-fosgeneorFosprotein rapidchangesinposition).
is widely used as a marker to identify neuronal pathways
involvedintheintegrationofnoxiousinputs[36–40].High Stage3. Animalremainsinthesameplaceforseveralseconds
doses of cocaine have been reported to induce c-fos mRNA with fast repetitive movements of the head, face, mouth, or
expressioninmanybrainregions[41]. forelimb,aswellasheadnodding.
Inthisstudy,EAexperimentswereconductedto(1)estab-
lish the applicability of EA to cocaine-induced seizure and Stage4. Forelimbclonusandrearing.
Evidence-BasedComplementaryandAlternativeMedicine 3
Stage 5. Full motor seizures, characterized by clonus of [50].SB-277011-A(30mg/kg;s.c.)waspurchasedfromTocris
forelimbsandhindlimbs,flexionofheadorentirebody,and Bioscience (Ellisville, MO, USA) and was used to block the
completelossofposturalcontrol. dopamineD3receptor[51].
2.4. Immunohistochemistry Procedure. The immunohisto- 2.6. Statistical Analysis. Data were expressed as means and
chemistry procedure was similar to that described by Inan the standard error of the mean (SEM). Group comparisons
etal.[45].Twohoursaftertheinjectionofsalineorcocaine, forseizurescoresandthenumberofcocaine-inducedc-Fos
animalsweredeeplyanesthetizedwithurethane(1.2g/kg;i.p.) positivenucleiwereevaluatedbyone-wayANOVA,followed
and perfused intracardially with ice-cold 0.1M phosphate- by Tukey’s post-hoc test. Group comparisons for mortality
buffered saline (PBS) followed by 4% paraformaldehyde ratesandseizureincidencewereevaluatedbychi-squaretest
in 0.1M PBS. The brains were removed, postfixed for 2h, orFisher’sexacttest.Significancewasconsideredat𝑃<0.05
and kept in 30% sucrose solution overnight. Brain sections foralltests.StatisticalanalysiswasperformedbySPSSversion
of 50𝜇m thickness were cut with a cryostat (LEICA CM 18.0(SPSSInc.,Chicago,IL,USA).
∘
3050).Free-floatingsectionswerestoredinPBSat4 Cuntil
immunohistochemistrywasperformed.Threebrainsections, 3.Results
from −2.255mm to −1.856mm caudal to bregma, were
randomly selected for immunohistochemistry procedures. 3.1. Positioning of Acupuncture Needles. We used X-ray
Tissues were processed for c-Fos immunoreactivity by the images to ensure accurate positioning of acupuncture nee-
avidin-biotincomplexprocedure[46].Tissueswereinitially dles.AsshowninFigure1(a),amousewasanesthetizedwith
treated with 3% H2O2 to reduce endogenous peroxidase urethaneandtwoacupunctureneedleswereinsertedintothe
activity,thenwashedtwicefor10minwithPBSandblocked GV14 and GV20 acupoints. Figure1(b) shows the position
with 20% normal goat serum (1:20) for 2h at room tem- oftheacupunctureneedles,asrevealedbyX-ray.Figure1(c)
perature. The sections were then incubated on a shaker for showsthepositionsofGV14,GV20,andSI11inmice.
∘
2dat4 Cwitharabbitc-Fosantibody(1:1000dilution)(sc-
52;SantaCruz,USA).Afterthoroughrinsing,sectionswere 3.2. Effects of EA on Cocaine-Induced Seizures. Firstly, the
incubatedinbiotinylatedanti-rabbitimmunoglobulinGsec- effectsofEAwereexaminedonseizuresinducedbyasingle
ondaryantibody(1:300dilution;VectorLaboratories,USA) dose of cocaine (75mg/kg) in six groups of mice (a control
for2hatroomtemperature.Followingtwo10minPBSrinses, group,threeEAtreatmentgroups,ashamEAgroup,anda
the sections were incubated in a complex of avidin-biotin- needleinsertiongroup).Inthecontrolgroup,animalswere
peroxidasein1:300solutionatroomtemperaturefor90min restrained with the same procedure as that used with the
(Vectastain ABC Elite kit, Vector Laboratories). Following EA groupbut withoutEA. In the EA treatmentgroups,EA
three10minwashesinTris-bufferedsaline,thesectionswere (2Hz,50Hz,and100Hz)wasappliedfor15mintoGV14and
placed in a 0.05% diaminobenzidine (Sigma)/0.001% H2O2 GV20acupointspriortococaineinjection.IntheshamEA
solution for 4-5min and washed again with Tris-buffered group,micereceivedEA(50Hz)appliedtobilateralSI11.In
saline 3 times for 10min each. Sections were mounted on theneedleinsertiongroup,needleswereinsertedintoGV14
slides with 0.25% gel alcohol, air-dried, and dehydrated andGV20acupoints,butwithoutelectricalstimulation.After
with graded alcohol (50%, 70%, 95%, and 100%, for 6min undergoingcontrol,EA,sham,orneedleinsertiontreatment,
each) followed by xylene (3 timesfor 10min each), and the all animals received an intraperitoneal injection of cocaine
coverslip was placed using Permount. c-Fos positive nuclei (75mg/kg). Seizure severity was measured by the 5-stage
wereobservedunderalightmicroscopeandcountedat200x cocaineseizurescalepublishedbyItzhak[18].
magnification.c-Fosimmunoreactivenucleiwerecountedon As shown in Figure2, the average seizure score was
the captured images using MetaMorph (Universal Imaging significantlylowerinanimalstreatedwithEA(50Hz)com-
Corp.) software. Bilateral calculations were performed on pared with those in the control and sham EA groups. No
theparaventricularthalamus,amygdalaarea,andcaudoputa- such effects were seen with EA 2Hz, EA 100Hz, or needle
men.Thenumberofimmunoreactivenucleiwasaveragedfor insertion. It appears that EA 50Hz at the GV14 and GV20
eachmouse. acupoints significantly reduced seizure scores induced by a
singlecocaineadministration(75mg/kg),whereasEAat2Hz
and100Hzhadnosucheffect.Furthermore,cocaine-induced
2.5.DrugsandChemicals. Cocaine-HClwaspurchasedfrom
seizures were not significantly affected by either 15min of
the National Bureau of Controlled Drugs, Department of
needleinsertionintoGV14andGV20orbyEAatSI11.
Health, Taipei, Taiwan. Freshly dissolved in 0.9% NaCl, all
Table1depictstheincidenceofseizures(higherorequal
testagentswereadministeredinadose/volumeof10mg/mL.
tostage3)inducedbycocaine(75mg/kg;i.p.).Pretreatment
Cocaine at the dose of 75mg/kg causes clonic seizures in
of animals with EA (50Hz) significantly reduced seizure
mice[48].Cocaineat125mg/kgisconsideredtobealethal
incidence.
dose [49]. In this study, cocaine at 75mg/kg and 125mg/kg
was injected intraperitoneallyto induce seizures and death.
ProcaineHClwaspurchasedfromSigmaChemicalCo.(St. 3.3. Effects of EA on Cocaine-Induced Death. The effects of
Louis,MO,USA).Procaine(250mg/kgand400mg/kg;i.p.) EA on death induced by single administrations of a high
wasusedtoinduceseizuresanddeathinmice,respectively dose of cocaine (125mg/kg) were investigated in a series
4 Evidence-BasedComplementaryandAlternativeMedicine
∙GV20
∙GV14
∙ ∙
SI11 SI11
(a) (b) (c)
Figure1:Photograph(a)andX-ray(b)ofamouseunderanesthesiawithacupunctureneedlesinsertedintoGV14andGV20acupoints.(c)
Mouseschematicshowingthelocationoftheacupointsusedinthestudy.
5 Cocaine75mg/kg ∗∗ Cocaine 125 mg/kg
∗∗∗
∗∗ ∗ 80 ∗ ∗
4
60
es 3
or
Seizure sc 2 ortality (%) 40
M
1
20
0
Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine
𝑛=21 + + + + + 0
needle EA EA EA sham Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine
insertion 2 Hz 50 Hz 100 Hz EA 𝑛=34 + + + + +
𝑛=20 𝑛=20 𝑛=23 𝑛=20 𝑛=20 needle EA EA EA sham
insertion 2 Hz 50 Hz 100 Hz EA
Figure2:EffectsofEAatdifferentfrequencies(2Hz,50Hz,and 𝑛=18 𝑛=18 𝑛=32 𝑛=18 𝑛=17
100Hz),needleinsertionexertedatGV14andGV20acupoints,and
Figure3:EffectsofEAatdifferentfrequencies(2Hz,50Hz,and
shamEA(50Hz;atbilateralSI11)oncocaine-inducedseizures.Mice
100Hz),needleinsertionexertedatGV14andGV20acupoints,and
wereadministeredwithcocaine(75mg/kg;i.p.)at1minafterEAor
sham EA (at bilateral SI11; 50Hz) on cocaine-induced mortality
needleinsertion.SeizureseveritywasmeasuredbytheItzhakfive-
rates.Micewereadministeredwithcocaine(125mg/kg;i.p.)at1min
stagescale[18].Between-groupcomparisonsforeachgroupwere
performedbyone-wayANOVAfollowedbyTukey’stest(∗𝑃<0.05; afterEAorneedleinsertionandmortalitywasmonitoredfor1hour.
∗∗𝑃<0.01;n:thenumberofanimals). Bsqeutwareeente-sgtr(o∗u𝑃p<com0.0p5a;ri∗s∗o𝑃ns<fo0r.0ea1c;∗h∗∗g𝑃rou<p0w.0e0re1;pne:rftohremneudmbbyecrhoi-f
animals).
of six groups, that is, control group, three EA treatment
groups,ashamEAgroup,andaneedleinsertiongroup.After at 2Hz and 50Hz applied to GV14 and GV20, significantly
undergoingtreatment,allanimalsreceivedanintraperitoneal reducedthemortalityrateinducedbyasingledoseofcocaine
injectionofcocaine(125mg/kg). (125mg/kg).However,EAat100Hzhadnosucheffect.EAat
As shown in Figure3, mortality rates were significantly SI11asashamcontrolreducedthemortalityrateinducedby
lower in the needle insertion and EA (2, 50Hz) groups as cocainebutdidnotreachastatisticallysignificantlevel.
compared to mortality in the control group. The EA (2Hz)
groupmortalityratewasalsolowerthanthatoftheshamEA 3.4. Effects of EA on Procaine-Induced Seizure and Death.
group.NeedleinsertionintoGV14andGV20,aswellasEA In addition to inhibiting dopamine uptake, cocaine acts as
Evidence-BasedComplementaryandAlternativeMedicine 5
Procaine 400 mg/kg
100
80
%) 60
y (
alit
Mort 40
20
0
Procaine Procaine Procaine
+ +
𝑛=20
EA 2 Hz EA 50 Hz
𝑛=21 𝑛=20
Figure 4: Effects of EA at 2Hz and 50Hz exerted at GV14 and
Figure5:Schematicrepresentationofthebrainregionsinwhichthe
GV20 acupoints on procaine-induced (400mg/kg, i.p) mortality
c-Fosimmunoreactiveneuronswerecounted.Theparaventricular
rates. At 1min after EA, procaine was injected and the mortality
thalamus (in red), the caudoputamen (blue), and the amygdala
was monitored for 1 hour. Between-group comparisons for each
area (including lateral amygdalar nucleus, basolateral amygdalar
groupwereperformedbychi-squaretestorFisher’sexacttest.No
nucleus,andbasomedialamygdalarnucleus;green)areindicated.
significantbetween-groupdifferenceswereobserved(n:thenumber
ThedrawinghasbeenmodifiedfromtheAllenBrainAtlasonline
ofanimals).
database(http://www.brain-map.org/;[47]).
Table 1: EA (50Hz) applied to GV14 and GV20 reduced the
incidenceofcocaine-inducedseizuresbutdidnotaltertheincidence
ofprocaine-inducedseizures. EA on cocaine-induced (75mg/kg) c-Fos expression in the
mousebrain.Miceweredividedinto4groups:salinecontrol,
Control EA50Hz
EAalone,cocaine,andcocaineplusEA.
∗
Cocaine(75mg/kg;i.p.) 20/21 14/21
All mice in the saline control group and cocaine group
Procaine(250mg/kg;i.p.) 8/9 8/9 wererestrainedwithoutEA.MiceintheEAaloneandcocaine
The proportion of mice exhibiting seizures is shown here. The effects of plus EA groups received EA (50Hz) at GV14 and GV20
cocaine,aloneandincombinationwithEA,werecomparedbyFisher’sexact acupoints. Following the restraining procedure or EA, the
test(∗P<0.05).Similarly,theeffectsofprocaine,aloneandincombination
micewereallowedtorecoverfor1min,beforebeinginjected
withEA,werecompared.
withsaline(salinecontrolandEA-alonegroups)orcocaine
(cocainegroupandcocaineplusEAgroups).
+ Figure5 shows the brain regions in which the c-Fos
alocalanestheticbyblockingvoltage-dependentNa chan-
immunoreactive neurons were counted. Results are shown
nels [52, 53]. Local anesthetics, such as procaine, can cause
in Figures 6, 7, and 8. c-Fos expression was examined in
CNSandcardiovasculartoxicitywhenplasmaconcentrations
the three brain areas, including paraventricular thalamus
areincreasedbyaccidentalintravenousinjectionofalethal
(Figure6), amygdala area (Figure7), and caudoputamen
dose of procaine [54]. However, procaine has no CNS
(Figure8).Fewc-Fospositivenucleiwerefoundintheseareas
stimulanteffectscomparedwiththoseofcocaine.Therefore,
inthesalinecontrolgroup.IntheEA-alonegroup,c-Fospos-
theeffectsofEAonprocaine-inducedseizuresanddeathwere
itivenucleiwerefoundonlyintheparaventricularthalamus
examined.
(Figure6).ItappearsthatEA50Hzalonehadnosignificant
Theseizureincidenceofprocaine-treatedmice(250mg/
effect on c-Fos expression, except in the paraventricular
kg; i.p.) is shown in Table1. When animals were pretreated
thalamus. In the cocaine group, c-Fos positive nuclei were
withEA(50Hz)atacupointsGV14andGV20,theincidence
foundinallthreeareasandatsignificantlyhigherlevelsthan
wasnotaffected.Furthermore,asshowninFigure4,mortal-
thoseseeninthesalinecontrolgroup.InthecocaineplusEA
ityratesdidnotdiffersignificantlybetweenthecontrolgroup
group,althoughc-Fospositivenucleiwerenotedinallthree
andEA(2and50Hz)groups.ItappearsthatEAat2Hzand
brainareas,c-Fos-positivenucleiweresignificantlydecreased
50Hz, when applied to GV14 and GV20, fails to affect the
in the paraventricular thalamus (Figure6) as compared to
mortality rate associated with procaine (400mg/kg; i.p.) in
thoseinthecocainegroup.Itappearsthatpretreatmentwith
mice.
EA50Hzsignificantlyreducedthenumberofc-Fospositive
cells induced by cocaine in the paraventricular thalamus,
3.5. EA Attenuation of Cocaine-Induced c-Fos Expression. but not in the amygdala area (Figure7) and caudoputamen
This series of studies was undertaken to test the effects of (Figure8).
6 Evidence-BasedComplementaryandAlternativeMedicine
Saline EA+saline Cocaine EA+cocaine
(a)
Paraventricular thalamus
∗∗∗
∗∗∗
50 ∗∗∗
∗∗∗
∗
ei
cl 40
u
n
e
v
ositi 30
p
os
F
c- 20
of
er
b
m 10
u
N
0
Saline Saline+EA Cocaine Cocaine+EA
𝑛=5 𝑛=5 𝑛=9 𝑛=14
(b)
Figure6:(a)Representativephotomicrographsofc-Fosexpressioninducedbycocaineintheparaventricularthalamusandtheeffectsof
EA.c-Fosexpressionwasnotobservedamongsalinecontrols,whereasc-Fosexpressionwasevidentintheparaventricularthalamusamong
animalsintheEA-alone,cocaine-alone(75mg/kg;i.p.),andcocaineplusEAgroups.(b)EffectsofEAoncontrolsandcocaine-inducedc-Fos
expressionintheparaventricularthalamus.Thenumbersofc-Fospositivenucleiwerecountedandaveragedfromthreerandomlychosen
sectionsfromeachanimalineachgroup.EAat50HzwasappliedtotheGV14andGV20acupoints.EAincreasedc-Fosexpressioninthe
paraventricularthalamus,whileEAdecreasedthenumberofc-Fospositivenucleiinducedbycocaine.Between-groupcomparisonsforeach
groupwereperformedbyANOVA,followedbyTukey’stest(∗𝑃<0.05;∗∗∗𝑃<0.001;n:thenumberofanimals).
3.6. EA Effects on Cocaine-Induced Seizures Blocked by the [56]andwefoundthatEAinducedc-Fosexpressioninthe
Dopamine D3 Receptor Antagonist SB-277011-A. Cocaine paraventricularthalamus.Wethereforetestedthepossibility
enhancesmonoaminesystem activitythroughtheblockade thatdopamineD3receptorsmaymediatetheeffectsofEAon
of dopamine and serotonin reuptake. However, the mecha- cocaine-inducedseizures.
nismofseizuresinducedbycocaineiscomplexandinvolves Four groups were included: a control group, an SB-
interactionofthedrugwithseveralneurotransmittersystems 277011-A group, an EA group, and an SB-277011-A plus EA
as well as with voltage-dependent sodium channels [55]. group. In the SB-277011-A group and EA group plus SB-
It is believed that dopamine receptors play an important 277011-A,thecompoundSB-277011-A(30mg/kg)wasadmin-
roleincocaine-inducedseizuresanddeath.Itisknownthat istered subcutaneously for 30min prior to the restraining
theparaventricularthalamusexpressesdopamineD3mRNA procedureorEAtreatment.Aftertherestrainingprocedure
Evidence-BasedComplementaryandAlternativeMedicine 7
Saline EA+saline Cocaine EA+cocaine
(a)
Amygdala
∗∗∗
60 ∗∗∗
∗∗∗
∗∗∗
ei
cl 50
u
n
e
v
ositi 40
p
Fos 30
c-
of
er 20
b
m
u
N 10
0
Saline Saline+EA Cocaine Cocaine+EA
𝑛=5 𝑛=5 𝑛=9 𝑛=14
(b)
Figure7:(a)Representativephotomicrographsofc-FosexpressioninducedbycocaineintheamygdalaandtheeffectsofEA.Whereasc-Fos
expressionwasabsentamonganimalsinthesalinecontrolandEAplussalinegroups,c-Fosexpressionintheamygdalawasobservedin
animalstreatedwithcocaine(75mg/kg;i.p.)andcocaineplusEA.(b)EffectsofEAoncontrolsandcocaine-inducedc-Fosexpressioninthe
amygdala.Thenumbersofc-Fospositivenucleiwerecountedandaveragedfromthreerandomlychosensectionsfromeachanimalineach
group.EAat50HzwasappliedtotheGV14andGV20acupoints.Cocaineadministrationinducedamarkedlevelofc-Fosexpression,while
EAdidnotsignificantlydecreasethenumberofc-Fospositivenucleiinducedbycocaine.Between-groupcomparisonsforeachgroupwere
performedbyANOVA,followedbyTukey’stest(∗∗∗𝑃<0.001;n:thenumberofanimals).
orEAtreatment,allanimalsreceivedanintraperitonealinjec- 4.Discussion
tionofcocaine(75mg/kg).Seizureseveritywasmeasuredby
theItzhakfive-stagecocaineseizurescale[18]. 4.1. EA Decreases Cocaine-Induced Effects. The psychostim-
AsshowninFigure9,theEA(50Hz)grouphadsignif- ulant and euphoric effects of cocaine are considered to be
icantlyloweraverageseizurescorescomparedwiththoseof associated with the blockade of dopamine uptake in the
theremainingthreegroups.Notably,seizurescoresintheSB- CNS. Acupuncture has been widely used throughout Asian
277011-AgroupandSB-277011-AplusEAgroupdidnotdiffer countriesfortreatingvariousfunctionaldisorders,including
significantly from the control group but were significantly substance abuse [31]. Immunohistochemical investigations
higherthanthoseintheEAgroup.Itappearsthatwhilethe by Lee et al. (2009) [57] reported that acupuncture can
dopamine D3 receptor antagonist, SB-277011-A (30mg/kg; reduce repeated cocaine-induced locomotor activity in rats
s.c.), did not affect cocaine-induced seizure severity, it did and the expression of tyrosine hydroxylase (TH) in the rat
preventtheeffectsofEAoncocaine-inducedseizures. brain,whichsuggeststhatacupuncturemayeffectivelyinhibit
8 Evidence-BasedComplementaryandAlternativeMedicine
Saline EA+saline Cocaine EA+cocaine
(a)
Caudoputamen
∗∗∗
∗∗∗
∗∗∗
60 ∗∗∗
ei
cl
u 50
n
e
v
ositi 40
p
Fos 30
c-
of
er 20
b
m
u
N 10
0
Saline Saline+EA Cocaine Cocaine+EA
𝑛=5 𝑛=5 𝑛=9 𝑛=14
(b)
Figure8:(a)Representativephotomicrographsofc-FosexpressioninducedbycocaineinthecaudoputamenandtheeffectsofEA.Whereas
c-FosexpressionwasnotobservedinthesalinecontrolandEAplussalinegroups,c-Fosexpressioninthecaudoputamenwasobservedamong
animalstreatedwithcocaine(75mg/kg;i.p.),andcocaineplusEA.(b)EffectsofEAoncontrolsandcocaine-inducedc-Fosexpressioninthe
caudoputamen.Thenumbersofc-Fospositivenucleiwerecountedandaveragedfromthreerandomlychosensectionsfromeachanimalin
eachgroup.EAat50HzwasappliedtotheGV14andGV20acupoints.Cocaineadministrationinducedamarkedlevelofc-Fosexpression,
whileEAdidnotsignificantlydecreasethenumberofc-Fospositivenucleiinducedbycocaine.Between-groupcomparisonsforeachgroup
wereperformedbyANOVA,followedbyTukey’stest(∗∗∗𝑃<0.001;𝑛:thenumberofanimals).
the behavioral effects of cocaine by modulating the central reduced the seizure severity induced by a single cocaine
dopaminergicsystem. (75mg/kg; i.p.) administration. Moreover, needle insertion
As mentioned earlier, cocaine abuse is associated with into GV14 and GV20 as well as EA at 2Hz and 50Hz
a risk of various medical complications, including seizures exerted at GV14 and GV20 acupoints significantly reduced
and death [3]. It has previously been reported that cocaine the mortality rate induced by a single cocaine (125mg/kg)
at doses ranging from 2.5mg/kg to 20mg/kg (i.p.) causes administration.Conversely,EAat50Hzappliedtobilateral
conditioned place preference, representing the rewarding SI11 acupoints had no such effects. In addition, EA failed
effects of cocaine [58]. At a dose as high as 40mg/kg to protect against procaine-induced seizure incidence and
(i.p.), cocaine caused seizures in mice [59]. The calculated lethality in mice. We report here for the first time that EA
ED50valueforcocaine-inducedseizuresis58.84mg/kg[59]. reduced seizures and mortality induced by a high dose of
Cocaine at 75mg/kg (i.p.) produces seizures in more than cocaine.
90%ofmice[48].Cocaineat125mg/kgisconsideredtobe
a lethal dose [49]. In the present study, we found that EA 4.2. Cocaine-Induced c-Fos Expression and Effects of EA.
at 50Hz exerted at GV14 and GV20 acupoints significantly Expression of the c-fos gene or Fos protein is commonly
Evidence-BasedComplementaryandAlternativeMedicine 9
Cocaine (75 mg/kg) paraventricularthalamushasbeenimplicatedinstressreac-
6 ∗
∗ ∗ tivity[68],reward-seekingbehavior[69],andgeneralarousal
activity[70].Furthermore,inactivationoftheparaventricular
5
thalamuspreventscontext-inducedreinstatementofalcohol-
seeking [71] and cocaine-primed reinstatement in rats [72].
4
es Paraventricularthalamuslesionsblockcocainesensitization
or [67].
c
e s 3 Theparaventricularthalamusisrelatedtoseizuresactiv-
ur
z ity.MraovitchandCalando[73]usedimmunocytochemistry
ei
S 2 to determine the regional and temporal distribution of Fos
proteinexpressioninawakeandunrestrainedratsafterauni-
1 lateral stereotaxic microinjection of the cholinergic agonist
carbacholinthethalamicventroposterolateralandreticular
0 nuclei, previously shown to cause limbic and generalized
Cocaine Cocaine Cocaine Cocaine
+ + + seizures[73].Theyfoundthatparaventricularthalamusacti-
𝑛=10
EA EA SB-277011-A vationoccurredafter15minafteradministrationofepileptic
𝑛=11 + 𝑛=9
agents.
SB-277011-A
Theamygdalaisinvolvedinbothtemporallobeepilepsy
𝑛=12
and in cocaine mechanisms in the brain. In particular, the
Figure 9: Effects of the dopamine D3 receptor antagonist, centralnucleusoftheamygdalaisahighlyepileptogenicbrain
SB-277011-A, on the anticonvulsant effects of EA. SB-277011-A area and, of the amygdaloid nuclei, responds most rapidly
(30mg/kg) was administered subcutaneously 30min prior to the
to a kindling stimulus [74]. The caudoputamen is a part of
control restraining procedure or EA treatment. EA (50Hz) was
the striatum area and plays an important role in control of
appliedfor15mintotheGV14andGV20acupointspriortococaine
movementinanimals[75–77].
injection. After the restraining procedure or EA treatment, all
As shown by our results and the published literature
animalsreceivedanintraperitonealinjectionofcocaine(75mg/kg).
[41],acutecocaineadministrationincreasesc-Fosexpression
Seizure severity was measured by the Itzhak five-stage scale [18].
Between-groupcomparisonsforeachgroupwereperformedbyone- in many brain areas. Among these areas, the paraventric-
wayANOVA,followedbyTukey’stest(∗𝑃<0.05;n:thenumberof ular thalamus and amygdala are structures associated with
animals). seizures, while the caudoputamen is associated with move-
mentcontrol.Wethereforeanalyzedc-Fosexpressioninthese
three specific regions. Our results implicate involvement of
theparaventricularthalamusintheeffectsofEAoncocaine-
used as a marker for neuronal activation, seizure pathways, inducedseizures.
and sites of action of neuroactive drugs [60–64]. It has
beenreportedthatanacuteinjectionofcocaine(65mg/kg)
in rats increased expression of c-fos mRNA in the dentate 4.3.EAInducesc-FosExpressionintheParaventricularThala-
gyrus of the hippocampus and olfactory bulb and limbic mus and Dopamine D3 Receptors Are Involved in the Effects
cortical regions as well as the striatum and ventromedial of EA. Medeiros et al. (2003) reported higher levels of c-
hypothalamicnucleus[41]. FosexpressioninducedbyEAattheST36pointinanimals,
In this study, we focused on three brain areas: the par- thatis,inthedorsalraphenucleus,locuscoeruleus,posterior
aventricular thalamus, amygdala, and caudoputamen. We hypothalamus, and central medial nucleus of the thalamus
found that cocaine at a dose of 75mg/kg (i.p.) induced [78]. In the present study, we found that EA alone at GV14
markedc-Fosexpressioninallareas.Pretreatmentwith50Hz andGV20acupointsinducedsignificantc-Fosexpressionin
EA significantly reduced the number of c-Fos positive cells theparaventricularthalamus.
inducedbycocaineintheparaventricularthalamus,butnot Recent research indicates that dopamine D3 receptors
intheamygdalaorcaudoputamen. may play an important role in cocaine-induced seizures
Thethalamusisconsideredtobeanimportantinterface [51].TheD3 antagonistSB-277011-Ahasbeenusedtoblock
between the ventral pallidum and the dorsal medial pre- dopamine D3 receptors, at doses ranging from 3mg/kg to
frontalcortexwhichmaythereforecontributetothedevelop- 30mg/kg(s.c.)[18,79].SB-277011-Apreventedtheanticon-
mentofcompulsivedrug-seekingbehavior[65].Onethala- vulsanteffectsoftheD3/D2receptoragonist(+)-PD-128,907
micnucleusthatisofparticularinterestistheparaventricular oncocaine-inducedseizures.Notably,theprotectionafforded
thalamus,acomponentofthedorsalmidlinethalamicgroup by (+)-PD-128,907 was eliminated in D3 receptor-deficient
[66]. The thalamic paraventricular nucleus projects to the mice, whereas the anticonvulsant effects of (+)-PD-128,907
nucleusaccumbensandotherlimbicsites,includingthepre- werepreservedinD2receptorknockoutmice.
frontalcortex,amygdala,andhippocampus;theseprojections As mentioned above, the paraventricular thalamus ex-
are predominantly glutamatergic [67]. The paraventricular presses dopamine D3 mRNA, while EA alone induces c-
thalamus also receives a dopaminergic innervation, in part Fosexpressionintheparaventricularthalamus.Wetherefore
derivedfromtheventraltegmentalarea,andparaventricular soughttodeterminewhetherdopamineD3receptorsmediate
thalamusneuronsexpressingdopamineD3mRNA[56].The the effects of EA on cocaine-induced seizures. Our results
10 Evidence-BasedComplementaryandAlternativeMedicine
revealed that while the D3 receptor antagonist SB-277011-A otherfrequency[87].EAatafrequencyof60Hzinducedthe
didnotaffectcocaine-inducedseizureseverity,itprevented simultaneousreleaseofmet-enkephalin, 𝛽-endorphin,and
the effects of EA on cocaine-induced seizures. This finding dynorphin-Ainextensiveanalgesia-relatednucleiandareas
suggeststhattheD3 receptorisinvolved,atleastinpart,in oftheCNS,suchastheperiaqueductalgray,theparaventricu-
theanticonvulsanteffectsofEA. larnucleusofthehypothalamus,theventromedialnucleusof
Interesting topics that remain for future studies include thehypothalamus,thedorsalraphenucleus,andthenucleus
aninvestigationintowhetherintraparaventricularthalamus raphe magnus, amongst others. These results suggest that
injection of a D3 receptor antagonist prevents EA-induced EAat60Hzmaycontributetooptimalanalgesiceffects.The
anticonvulsiveeffectsandwhetheraD3 receptorantagonist effectsofEAat50or60Hzdeservetobestudiedfurther[87].
antagonizes EA-induced changes to c-Fos expression in No effective treatment currently exists for cocaine-in-
the paraventricular thalamus or has any effect on cocaine- ducedseizuresanddeath.OurresultssuggestthatEAreduces
inducedmortality. seizure severity and death caused by cocaine in an animal
modelofcocaineabuse.Wefoundevidenceforinvolvement
ofthedopamineD3receptor,atleastinpart,intheanticon-
4.4.AComplexMechanismUnderliesCocaine-InducedDeath.
vulsanteffectsofEA.
It is noted that EA at 50Hz only, when exerted at GV14
and GV20 acupoints, significantly reduced seizure severity
induced by a single cocaine (75mg/kg; i.p.) administration. Acknowledgments
However,needleinsertionintoGV14andGV20acupointsas
TheauthorsaregratefultoMs.IonaMacDonaldandMs.Ya-
wellasEAat2Hzand50Hzeffectivelyreducedthemortality
TingWufortheirhelpinproducingthefinalpaper.Thiswork
rateinducedbyasinglecocaine(125mg/kg)administration.
wassupportedbyGrantsNSC100-2320-B-039-018-andNSC
Asmentionedearlier,seizuresareconsideredtobeamajor
101-2320-B-039-035-MY3fromtheNationalScienceCouncil,
determinant of cocaine-related lethality in both humans
Taipei,Taiwan.
[4] and animals [9]. However, the mechanism of sudden
deathincocaineabusealsoincludescardiacarrhythmiaand
intracerebralhemorrhage[10,11].Cocaineproduceseupho- References
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