Research Article The Possible Neuronal Mechanism of Acupuncture ... - Semantic Scholar

13 Pages·2013·2.48 MB·English

HindawiPublishingCorporation
Evidence-BasedComplementaryandAlternativeMedicine
Volume2013,ArticleID429186,13pages
http://dx.doi.org/10.1155/2013/429186
Research Article
The Possible Neuronal Mechanism of Acupuncture:
Morphological Evidence of the Neuronal Connection between
Groin A-Shi Point and Uterus
Chun-YenChen,1Rey-ShyongChern,2Ming-HueiLiao,2Yung-HsienChang,3
Jung-YuC.Hsu,1andChi-HsienChien2
1DepartmentofCellBiologyandAnatomy,CollegeofMedicine,NationalCheng-KungUniversity,Tainan,Taiwan
2GraduateInstituteandDepartmentofVeterinaryMedicine,CollegeofVeterinaryMedicine,NationalPingtungUniversityof
ScienceandTechnology,1,ShuefuRoad,Neipu,Pingtung912,Taiwan
3GraduateInstituteofChineseMedicalScience,ChinaMedicalUniversity,Taichung,Taiwan
CorrespondenceshouldbeaddressedtoChi-HsienChien;[email protected]
Received8November2012;Accepted25January2013
AcademicEditor:LixingLao
Copyright©2013Chun-YenChenetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Somatovisceralreflexsuggestedthatthesomaticstimulationcouldaffectvisceralfunctionlikeacupuncturewhichtreatsdiseases
bystimulatingacupoints.Theneuronalconnectionbetweensomaticpointandvisceralorganwasnotclear.Uterinepainreferred
tothegroinregionhaslongbeenrecognizedclinically.Wesselmann,usingneurogenicplasmaextravasationmethod,showedthat
uterinepainwasreferredtothegroinregionthroughaneuronalmechanism(WesselmannandLai1997).Thisconnectioncould
beconsideredthroughthesomatovisceralreflexpathway.However,therelaycenterofthispathwayisstillnotclearlyidentified.In
thepresentstudy,beevenomwasinjectedinthegroinregiontoinducecentralFosexpressiontomapthesensoryinnervationof
groinregion.Pseudorabiesvirus(PrV),atransneuronaltracer,wasinjectedintheuterustoidentifythehighermotorcontrolof
theuterus.ImmunohistochemistrystainingrevealedtheFosexpressionandPrV-infecteddouble-labeledneuronsinthenucleusof
solitarytract(NTS),thedorsalmotornucleusofvagus(DMX),andtheparaventricularhypothalamicnucleus(PVN).Theseresults
suggestasomatoparasympatheticneuronalconnection(groin-spinaldorsalhorn-NTS/DMX-uterus)andasomatosympathetic
neuronalconnection(groin-spinaldorsalhorn-NTS-PVN-uterus).Thesetwoneuronalconnectionscouldbetheprerequisitesto
theneuronalbasisofthesomatovisceralreflexandalsotheneuronalmechanismofacupuncture.
1.Introduction myocardial ischemia triggered by sympathetical excitation
[7]. Other studies have shown that electroacupuncture-like
The somatovisceral reflex was mentioned by Sato in 1995 stimulation can activate a sympathetic inhibitory system in
andsuggestedthatsomaticstimulationcouldevokesympa- thebraintoregulatecardiovascularresponses[5,10,11].Both
theticreflexresponseand,thereby,modulatefunctioningof the somatovisceral reflex and acupuncture stimulation sug-
visceral organ [1]. This phenomenon is in some way alike gesttheneuronalconnectionbetweensomaticacupointand
acupuncturethatstimulatesspecificsomaticpointstorelieve itscorrespondingorgan.However,theneuronalconnection
painandtreatmanydifferentdiseases[2].Manystudieshave ofthesomatovisceralreflexoracupunctureisstillnotclear.
shown that acupuncture can significantly modulate visceral Pervious report demonstrated that gynecological pain
function by stimulating specific acupoints [3–8]. Previous induced by dysmenorrhea, ascending genital infection, or
research suggested that the activation of the somatosen- cystic or hemorrhagic ovarian pathology usually refer pain
sory pathway played an important role in the physiolog- to the low back, thighs, and abdominal wall [12]. Referred
ical effects of acupuncture [9]. Li et al.’s research showed paininthelowbackandabdominalwallwasalsoreportedby
thatelectroacupuncture-likestimulationdiminishesregional womeninlabor[13].Thesereportssuggestedthatthegroin
2 Evidence-BasedComplementaryandAlternativeMedicine
region can account to the pain of uterine inflammation or intracardially,followedby1000mLof4%paraformaldehyde
diseases. According to traditional Chinese medicine, some in0.1Mphosphatebuffersolution(PBS).T10–S1segmentsof
acupoints, called A-shi points, do not have fixed specific spinalcord,brainstem,andbrainwereremoved.
locations and are usually pain-associated points [14–16].
Therefore,thegroinregioncouldbetheA-shipointrelated 2.3. Pseudorabies Virus Injection in Left Uterine Horn. The
totheuterus.In1997,WesselmannandLaifoundthatuterine rats were anesthetized with ketamine (1mL/kg) intraperi-
inflammationinratspretreatedwithEvansBlueDyeresulted toneally.Alaparotomywasperformedand40𝜇LofPingtung
in neurogenic plasma extravasation of dye in the skin over strainpseudorabiesvirus[22]wasinjectedintotheleftuterus
theabdomen,lowerback,thighs,andgroin,afterantidromic horn (𝑛 = 9). After the injections, the abdominal wall was
stimulation of peripheral nerves [17]. This result suggested sutured,theskinclosed.Theanimalsweresacrificedat6to8
thepossibilityofasomatovisceralneuralconnectionbetween daysafterPrVinjection(inthesamewayasdescribedabove).
theuterusandgroinareas.Althoughthesefindingsconfirm The spinal cord, dorsal root ganglion of T10–S2 segment,
the existence of a neural connection between the uterus brainstem,andbrainwereremoved.
andgroinregion,theexactlocationofthiscentralneuronal
connectionremainsunknown.
2.4. Bee Venom Injection after PrV Injection. The rats were
TheFosproteinisanimmediate-earlygenetranscription
anesthetized and PrV was injected into the left uterus horn
factor induced by short-term signals and alters target gene
(in the same way as described above) (𝑛 = 9). After the
expression causing long-term change in cellular phenotype
injections,theabdominalwallwassutured,theskinclosed,
[18].Ithasbeenusedtomaptheactivatedneuralcellsafter
and the animals allowed to survive for 6 to 8 days. Before
differenttypesofstimulationandshowscorrelatedanatom-
theratsweresacrificed,50𝜇Lof1%beevenomwasadmin-
ical neural pathways [19–21]. Pseudorabies virus (PrV) is
istrated subcutaneously into midpoint of left groin region
a swine neurotropic herpes virus that has been used for
and saline was injected as the control. After 90 minutes,
transneuronaltracinginmanystudies[22–26].ThePingtung
the rats were sacrificed and perfused with 250mL of saline
(PT) strain of PrV has been demonstrated to label sympa-
intracardially,followedby1000mLof4%paraformaldehyde
theticpre-andpostganglionicneuronsafterinjectioninthe
in 0.1M phosphate buffer solution (PBS). The spinal cord,
specific auricular kidney point [22]. The study showed that
dorsalrootganglionofT10–S1segment,brainstem,andbrain
the PT strain of PrV was a useful transneuronal tracer in
wereremoved.
somatovisceralresearch. To establish the neural connection
betweenthegroinregionanduterus,beevenomwasinjected
in the groin region to induce c-Fos expression neurons 2.5. Immunohistochemistry. Tissues of groin region-bee
innervating the groin region and PrV was injected in the venom injection group and uterine horn-pseudorabies
uterus to infect the hierarchical motorneuronsinnervating virus injection group were postfixed up to 4hr in para-
the uterus. Furthermore, to evaluate central doubled Fos formaldehydePBSandthencryoprotectedin10,20,and30%
expression and PrV-infected neurons in order to identify sucroseinPBsolution.Serial30𝜇mthicktransversesections
the neuronal connection between the somatic point (groin of all dorsal root ganglia, spinal cord, brainstem, and brain
region)anditsrelatedvisceralorgan(theuterus). werecutwithacryomicrotome.Allsectionsfromtheganglia
andeveryfivesectionsfromothersampleswerecollectedin
0.01Mphosphatebuffersaline(PBS).Floatingsectionswere
2.MaterialsandMethods
washed30min(10min,3times)andincubatedwithblocking
solution (5% normal goat serum, 0.05% Triton X-100, and
The study protocol was approved by Animal Care and
3%BSAin0.1MPB)for1hr.Thesectionswerewashedand
Use Committee, and all experiments were conducted in
incubatedwiththeprimaryantibody(IgGofrabbitanti-FOS
accordance with the animal care guidelines of the National
in 1:2000 or IgG of swine anti-PrV in 1:1000) in blocking
InstitutesofHealthandtheInternationalAssociationforthe ∘
solution for 72hr at 4 C. After incubation, the sections
StudyofPain. ∘
were rinsed and incubated for 1hr at 25 C with secondary
antibody(biotin-conjugatedIgGofgoatanti-rabbitin1:500
2.1.Animals. Sprague-Dawleyadultvirginfemalerats(250– or goat anti-swine in 1:200) in blocking solution. The
350g)wereused.Animalswerehousedona12h-12hlight- sectionswere washedthreetimesfor30min andincubated
darkcycle,andallanimalshadfreeaccesstostandardfood using ABC kit (Vector) for 1hr. After rinsing, the sections
andwater. were developed with GOD method followed by mounting
on gelatin-coated slides and overslipped with mounting
2.2. Bee Venom Injection in the Left Groin Region. The medium.
rats were anesthetized with ketamine (95mg/kg) intraperi-
toneally. 50𝜇L of 1% bee venom (Sigma) were dissolved in 2.6. Immunofluorescence. Tissues of groin-uterus injection
normal saline and administrated subcutaneously into the group were postfixed and sectioned in the same way
midpoint between genital pore and apex of the left groin described above. Floating sections were washed 30min
region(𝑛 = 6)accordingtoWesselmannandLai’sresearch (10min,3times)andincubatedwithblockingsolution(5%
[17]. Saline was injected as the control. After 90 minutes, normalgoatserum,0.05%TritonX-100,and3%BSAin0.1M
the rats were sacrificed and perfused with 250mL of saline PB) for 1h. The sections were washed and incubated with
Evidence-BasedComplementaryandAlternativeMedicine 3
(a) (b)
L2spinal segmen(cid:289)t
neurons/ ±SEM) 233505 ∗ ∗ n neurons/ ±SEM)n11110246
pression (meann 112050 ∗ xpressioon (mea 468
Fos exsectio 05 Fos esecti 02
T10 T12 L1 L3 L5 S1 S3 Lamina I Lamina II Lamina III Lamina IV Lamina V
Spinal segments Spinal laminas
Ipsilateral Ipsilateral
Contralateral Contralateral
(c) (d)
Figure1:Fosexpressionneuronsinthespinalcordafterbeevenominjectionintheleftgroinregion(𝑛=9).(a)NeuronsexpressFosprotein
(arrow)inipsilateralL2spinaldorsalhorn.(b)HighermagnificationofFosexpressionneuronsin(a)(scalebar:100um).(c)Meannumber
ofFosexpressionneuronsinT10toS3spinalsegments(±SEM).∗𝑃<0.05.(d)MeannumberofFosexpressionneuronsinlaminasItoVof
L2spinalsegment.
two kinds of primary antibody (IgG of rabbit anti-FOS in region induces central Fos expression and the contralateral
1:2000 and IgG of swine anti-PrV in 1:1000) in blocking side as the control. In the spinal cord, Fos protein is pre-
solutionfor72hrat4∘C.Afterincubation,thesectionswere dominantly(70%)apparentinipsilateralT13(14.5±1.1),L1
rinsed and incubated for 1hr at 25∘C with two secondary (23.5±1.5),andL2(26.6±2.5)spinaldorsalhorn(Figures
antibodies(FITC-conjugatedIgGofgoatanti-swinein1:200 1(a),1(b),and1(c)).Mostofthec-Fosexpressionneuronsare
and TRITC-conjugated IgG of goat anti-rabbit in 1:500) in residedinlaminasI(12.6 ± 1.2)andII(10.9 ± 1.4)ofthe
blockingsolution.Thesectionswerewashedfor30minand dorsalhorn(Figure1(d)).
mounted on gelatin-coated slides followed by coverslipping In the supraspinal area, c-Fos expression neurons
withmountingmedium. appearedinnumerousnucleiofthethalamus,hypothalamus,
pons, and medulla. The c-Fos expression nuclei include the
nucleus of solitary tract (NTS) (Figure2(a)), parabrachial
2.7.DataandStatisticalAnalysis. FosandPrVimmunoreac-
nucleus (PB), locus coeruleus (LC) (Figure2(b)), raphe
tivity neurons developed with GOD method in dorsal root
pallidus nucleus (RPa) (Figure2(c)), paraventricular
ganglia, spinal cord, and brain were counted with bright
thalamic nucleus (PVT) (Figure2(f)), lateral hypothalamic
fieldmicroscope.FosandPrVdoublelabeledneuronswere
area (LH) (Figure2(e)), and paraventricular hypothalamic
observed with fluorescent microscope. Anatomical identifi-
nucleus(PVN)(Figure2(d)).Table1listedthefosexpression
cation of brain structures was essentially based on the Rat
neuronsinsupraspinalareasofsalineandbeevenomgroups.
BrainatlasofPaxinosandWatson[27].Alldatawereanalyzed
The NTS of bee venom group expressed significantly the
by𝑡-test.
differencebetweenthefosexpressionneuronsandthesaline
group.
3.Result
3.2. The Appearance of PrV Infection Neurons after Virus
3.1.FosExpressionNeuronsafterBeeVenomStimulationinthe InjectionintheUterus. PrV-infectedneuronsappearedinthe
GroinRegion. Injectingbeevenomintheleftsomaticgroin centralnuclei(Figures3and4)6–8daysafterPrVinjectionin
4 Evidence-BasedComplementaryandAlternativeMedicine
PB
scp
d LC
d
m l
v
(a) (b)
RPa
PVN
3V
(c) (d)
ic
D3V
f
PV
opt
(e) (f)
Figure2:Fosexpressionneurons(arrow)inthesupraspinalareaafterbeevenominjectionintheleftgroinregion(𝑛 = 9).(a)Nucleusof
solitarytract(NTS).(b)Locuscoeruleus(LC),parabrachialnucleus(PB).(c)Raphepallidusnucleus(RPa).(d)Paraventricularhypothalamic
nucleus(PVN).(e)Lateralhypothalamicarea.(f)Paraventricularthalamicnucleus(PV).3V:thirdventricle;4V:fourthventricle;D3V:dorsal
thirdventricle;d:dorsal;f:fornix;ic:internalcapsule;l:lateral;m:medial;opt:optictract;scp:superiorcerebellarpeduncle;v:ventral(scale
bar:100um).
theleftuterushorn.Inthespinalcord,themostPrV-infected preoptic area (MPA), and PVN (Figure4(e)). All PrV-
neuronswerespottedinlaminasIVandVofT11–L1andL5– infectedneuronsinsupraspinalareawerelistedinTable2.
S2spinalsegments(Figures3(a),3(b),and3(c)),rarelyinthe
superficiallaminas(laminasI,II).
Inthesupraspinalarea,PrV-infectedneuronswerefound 3.3.FosExpressionandPrV-InfectedDoubleLabeledNeurons
inthehypothalamus,pons,andmedulla,includingtheNTS, in the NTS, DMX, and PVN. After uterine PrV injection
dorsalmotornucleusofvagus(DMX)(Figure4(a)),interme- andc-Fosexpressionofgroinbeevenomstimulation,double
diatereticularnucleus(IRt),ambiguusnucleus(Amb),lateral labeled neurons appeared in the hypothalamus, and specif-
reticular nucleus (LRt), A5 noradrenaline cell group (A5) icallyinthePVN(Figure5(c1)).Someotherdoublelabeled
(Figure4(b)), raphe pallidus nucleus (RPa) (Figure4(c)), neurons are apparent in the NTS and DMX (Figures 5(c2),
gigantocellular reticular nucleus (Gi) (Figure4(d)), medial 5(c3),and5(c4)).Comparingsalineandbeevenominjection
Evidence-BasedComplementaryandAlternativeMedicine 5
(a) (b)
7
ed M) 6
V-infect±nSE 45
er of Prns (mea 23
bo
umeur 1
Nn
0
T10 T11 T12 T13 L1 L2 L3 L4 L5 L6 S1 S2
Spinal segments
Ipsilateral
Contralateral
(c)
Figure3:PrV-infectedneuronsinspinalcordafter6to8dPrVinjectionintheleftuterinehorn(𝑛=8).(a)PrV-infectedneuron(arrow)
inT12spinalsegment.(b)HighermagnificationofPrV-infectedneuronin(a)(arrow)(scalebar:100um).(c)MeannumberofPrV-infected
neuronsinT10toS2spinalsegments(±SEM).
groupsinPrV-infectedneurons,thepercentageofthedouble [28]. Pain, as one of the dechi sensations, is a relatively
labeledneuronsinPVNofbeevenominjectiongroupwere strongandeasilyinducedsensorymodalityinanimalstudy.
significantlypredominant(𝑃<0.1)(Figure5(d)). Panic stimulation can induce neurons expressing the Fos
proteinwhichcouldbeusedtoinvestigateeithersomatic[29–
31]orvisceral[31–33]noxiousafferentpathways.Takahashi
4.Discussion
andNakajima[34]intravenouslyinjectedintheEvansBlue
Dyeandobservedextravasationinthegroinafterelectrical
After the application of bee venom to the left groin region,
stimulation of the spinal nerves. This result proves that the
the c-Fos protein expression neurons were presented in
sensoryinnervationofgroinregioncomesfromT13,L1,and
the left spinal dorsal horn and certain supraspinal nuclei.
L2spinalnerves.Theinjectionofbeevenomintheleftgroin
PrV-infected uterine supraspinal neurons resided in the A5
regioninourstudyinducedc-Fosexpressionintheipsilateral
noradrenalinecellgroup(A5),raphepallidusnucleus(RPa),
spinal dorsal horn of T13, L1, and L2, and particularly in
and gigantocellular reticular nucleus (Gi). Double labeled
superficial laminas I and II (Figures 1(c) and 1(d)). This
neuronslocatedintheNTS,motornucleusofvagus(DMX),
suggeststhatthespinalFosexpressionneuronsinourstudy
andPVN(Figure5).Theneuronalconnectionbetweengroin
werespecificallyactivatedbynoxiousstimulationofthegroin
regionanduterussuggeststhatthenucleiofPVN,NTS,and
region.
DMXnotonlyreceivesomaticstimulationfromgroinregion
Fos expression neurons appeared in the NTS, giganto-
butalsomodulatethefunctionofuterus.Thosenucleimay
cellularreticularnucleus(Gi),raphepallidusnucleus(RPa),
playimportantrolesinsomatovisceralreflex[1]andcouldbe
and PVN after bee venom stimulated groin region (Figures
theresultoftheneuronalmechanismofacupuncture.
3 and 6). The NTS can be not only activated by somatic
noxious stimulation but also triggered by vagal afferent
4.1. Sensory Innervation of the Left Groin A-Shi Point. The activation as a physiological adaptation to pain [35]. Pre-
stimulatingacupointselicitacompositeofuniquesensations vious studies showed that Gi can be activated by noxious
called dechi. Dechi sensations including pressure, soreness, stimulation related to the activation of the descending
heaviness, and dull pain are essential for clinical efficacy antinociceptive pathway [36–39]. Electrically stimulating
6 Evidence-BasedComplementaryandAlternativeMedicine
Gr AP
NTS
DMX
(a) (b)
RMg
RPa
(c) (d)
PVN
3V
(e)
Figure4:PrV-infectedneurons(arrow)inthesupraspinalareaafter7-8dPrVinjectionintheleftuterushorn(𝑛 = 8).(a)PrV-infected
neuronsintheNTSandDMX.(b)PrV-infectedneuronsintheA5noradrenalinecellgroup.(c)PrV-infectedneuronsintheRPaandRMg.
(d)PrV-infectedneuronsintheGi.(e)PrV-infectedneuronsinthePVN.3V:thirdventricle;AP:areapostrema;DMX:motornucleusof
vagus;Gi:gigantocellularreticularnucleus;Gr:gracilenucleus;NTS:nucleusofsolitarytract;PVN:paraventricularhypothalamicnucleus;
RPa:raphepallidusnucleus;RMg:raphemagnusnucleus(scalebar:100um).
the raphe nucleus could induce analgesia, proving that the 4.2. Hierarchical Innervation of the Uterus. The PrV trans-
raphenucleusplaysacrucialroleinpaininhibitionresponse neuronaltracingmethodiswidelyusedtodetectthehierar-
[40, 41]. The thermal stimulation from hind feet inducing chicallycentralinnervationofurethra,clitoris,penis,urinary
FosexpressioninthePVNshowedthatthePVNcanreceive bladder, and uterus [23, 24, 44–51]. In our previous study,
the noxious input [42]. The activation of PVN initiates the Pingtung strain of PrV applied to the auricular kidney
the hypothalamus-pituitary-adrenal hormone cascade by point transneuronally and specifically infected sympathetic
releasing corticotropin-releasing factor (CRF) and arginine pre-andpostganglionicneurons[22].Inordertoinvestigate
vasopressinfromitsparvocellularcells [43]. Fosexpression the highest central control of uterus, the survival time was
neuronsinthosenucleisuggestthatinjectingbeevenomin proportionally extended to 6–8 days in this study. All PrV-
thegroinregionactivatesnotonlypaintransmissionpathway infected nuclei in our study (Table2) could be found in
but also the nuclei regulating physiological responses and thesenucleireportedbyotherstrainsofPrVtransneuronal
inhibitingpaininthecentralnervoussystem. studies[23,24].ThisresultconfirmsthatthePingtungstrain
Evidence-BasedComplementaryandAlternativeMedicine 7
Table1:Fosexpressionneuronsinsupraspinalareabetweensalineandbeevenominjectiongroups.
c-fosexpressionneuronsinsupraspinalarea Saline Beevenom
Diencephalon
Thalamus
Paratenialthalamicnucleus(PT) 96.3±49.5 105.8±27.2
Precommissuralnucleus(PrC) 69.6±18.6 53.2±17.4
Rhomboidthalamicnucleus(Rh) 44.9±21.1 62.8±15.2
Reuniensthalamicnucleus(Re) 112.6±36.0 88.9±24.3
Pretectalnucleus 89.9±18.6 61.3±16.0
Lateraldorsalthalamicnucleus(LD) 83.4±25.9 72.6±21.9
Mediodorsalthalamicnucleus(MD) 134.7±56.9 163.5±47.6
Paraventricularthalamicnucleus(PV) 98.9±28.9 75.9±17.8
Lateralhabenularnucleus(LHb) 37.8±5.0 44.4±13.0
Lateralposteriorthalamicnucleus(LP) 82.9±30.6 89.5±29.9
Lateralgeniculatenucleus(LG) 82.7±23.5 62.9±13.3
Suprageniculatethalamicnucleus(SG) 97.0±40.4 72.1±18.2
Hypothalamus
Medialpreopticarea(MPA) 82.3±17.2 94.9±24.9
Suprachiasmaticnucleus(SCh) 67.0±15.8 72.3±13.7
Arcuatenucleus(Arc) 50.4±15.3 50.8±11.6
Supramammillarynucleus(SuM) 172.6±17.3 189.2±33.3
Lateralmammillarynucleus(LM) 94.3±13.2 92.6±29.0
Supraopticnucleus(SO) 41.0±19.5 86.7±20.6
Lateralhypothalamicarea(LH) 108.6±20.3 121.0±26.2
Premammillarynucleus(PM) 136.4±13.0 96.1±40.0
Posteriorhypothalamicarea(PH) 169.4±33.1 149.4±28.6
Ventromedialhypothalamicnucleus(VMH) 100.1±44.8 86.9±36.4
Dorsomedialhypothalamicnucleus(DM) 142.2±30.2 150.4±25.0
Anteriorhypothalamicarea(AHC) 100.1±30.6 73.9±15.6
Lateroanteriorhypothalamicnucleus(LA) 118.4±19.4 133.2±55.5
Paraventricularhypothalamicnucleus(PVN) 82.3±14.3 62.1±9.7
Anterodorsalpreopticnucleus(ADP) 60.3±20.3 60.9±8.8
Mesencephalon
Edinger-Westphalnucleus(EW) 30.7±4.2 29.9±3.1
Paranigralnucleus(PN) 51.6±14.9 61.0±13.2
Interfascicularnucleus(IF) 26.1±6.6 30.4±4.6
Pons
Parabrachialnucleus(PB) 70.1±15.9 52.6±13.1
Dorsalraphenucleusventrolateralpart(DRVL) 52.6±35.8 90.6±17.2
Dorsalraphenucleus(DR) 37.3±16.1 16.1±4.3
Pontinenuclei(Pn) 277.1±107.9 298.7±68.2
Nucleusoflaterallemniscus(LL) 58.0±22.7 64.8±12.0
Locuscoeruleus(LC) 73.4±14.8 31.4±5.7
Prepositusnucleus(Pr) 13.7±8.0 22.3±6.4
Areapostrema(AP) 21.8±8.3 31.4±11.9
Medulla
Gigantocellularreticularnucleus(Gi) 16.4±2.2 14.7±4.9
Raphemagnusnucleus(RMg) 20.6±1.7 11.9±3.3
Raphepallidusnucleus(RPa) 12.6±2.3 19.1±4.8
Medialvestibularnucleus(MVe) 33.5±3.9 45.7±7.1
Inferiorolivarynucleus(IO) 40.7±10.7 39.5±8.2
Caudoventrolateralreticularnucleus(CVL) 18.8±4.9 18.9±3.9
Nucleusofsolitarytract(NTS) 17.1±4.0 33.8±5.0∗
∗
Significantdifferencebetweensalineandbeevenominjectiongroups,𝑃<0.1.
8 Evidence-BasedComplementaryandAlternativeMedicine
Table2:PrV-infectedneuronsinsupraspinalarea. and it regulates the uterus through both hormonal and
neuronalinnervations.
PrV-infectedneuronsinsupraspinalarea
Hypothalamus 4.3. Somatovisceral Neuronal Connection Nuclei between the
Medialpreopticarea(MPA) + GroinA-ShiPointandtheUterusLocatedintheNTS,DMX,
Arcuatenucleus(Arc) + and PVN. Retrograde tracer injection in the NTS suggests
that the spinal superficial dorsal horn neurons (lamina I)
Ventromedialhypothalamicnucleus(VMH) +
directly project to the NTS [60]. Our result (Figure2(a))
Dorsomedialhypothalamicnucleus(DM) +
and a previous study [35] also showed that the NTS can be
Paraventricularhypothalamicnucleus(PVN) ++
activatedbysomaticnoxiousstimulation.Severalanatomical
Pons
and electrophysiological studies have shown the neuronal
Locuscoeruleus(LC) +
connectionbetweentheuterusandNTSandDMXthrough
Medulla thevagusnerve[23,24,50,54,55].ThePrV-infectedneurons
Gigantocellularreticularnucleus(Gi) +++ in the NTS and DMX (Figure4(a)) also confirmed the
Raphemagnusnucleus(RMg) ++ efferentinnervationoftheNTSandDMXtotheuterusasin
Raphepallidusnucleus(RPa) + apreviousstudy[55].NeuronswithcellbodiesintheDMX
Caudoventrolateralreticularnucleus(CVL) ++ send their dendrites into the gelatinosus solitary nucleus,
Nucleusofsolitarytract(NTS) +++ where they receive synaptic inputs from gastric sensory
afferents [61]. These researches suggested that the NTS and
Dorsalmotornucleusofvagus(DMV) +++
DMXplayimportantrolesinvisceralinnervation,including
A5noradrenalinecells(A5) ++
thedigestivefunctionsofthestomachandbaroreceptorreflex
Lateralreticularnucleus(LRt) ++
[61–66].ThedoublelabeledNTSandDMXneuronsinour
+:1–3infectedneurons,++:4–8infectedneurons,and+++:>9infected studysuggestthatthenucleimaybeoneoftherelaycentersof
neurons.
thesomatovisceralreflexofthegroinA-shipointanduterus.
Retrograde labeling study shows that the NTS receives
of PrV was a sustainable strain as a transneuronaltracer in spinal projections from the superficial dorsal horn [60]
hierarchicalinnervationstudying. and projects to the PVN [42]. Fos expression studies also
LeeandPapkadiscoveredPrV-infectedsympatheticand suggestedthatthePVNcouldbeactivatedbysomaticnoxious
parasympathetic preganglionic neurons at T11–13 and L6– stimulations [42, 67]. Swanson proved that the PVN, a
S1spinalsegments,afterPrVinjectionintheuterinecervix higher hormonal regulation center, projects neuronal fibers
[23, 24]. The results indicate the visceral efferent of the to the pituitary gland, medulla, and spinal cord [68]. The
uterusaremainlyfromT11–T13andL6–S1segments.Inour neuronal connection between the PVN and the uterus was
study,PrV-infectedpreganglionicneuronsaremainlyinthe also proved by previous indicators [23, 24, 50] and our
intermediolateralnucleus(IML)andsacralparasympathetic studies (Figure4(e)). Noxious stimulation and retrograde
nucleusofT10–L2andL6–S1spinalsegments(Figure2).Our tracer injection in different visceral organs showed that the
resultisinaccordwithpreviousstudies[23,24,50]. PVNwasnotonlyreceivenoxiousafferentbutalsoinnervate
Supraspinal PrV-infected high hierarchical uterine neu- visceral organs as well [24, 69–72]. The reflex inhibition
rons are located in the NTS, dorsal motor nucleus of vagus of the heart rate elicited by acupuncture-like stimulation
(DMX), A5 noradrenaline cell group (A5), raphe pallidus likely occurs through the activation of the hypothalamic
nucleus (RPa), gigantocellular reticular nucleus (Gi), and nucleus,whichinhibitstheactivityofpremotorsympathetic
PVN (Figure4). The NTS is the major brainstem structure neurons in the rostral ventrolateral medulla (rVLM) [73,
receiving both general and special visceral sensory inputs, 74]. Although bee venom stimulation in groin A-shi point
including visceral pain [52, 53]. Electrophysiological and induced less neuronal activity in the PVN, comparing to
HRP studies have shown that the NTS contains neuronal saline group (Table1) in our study, there was higher per-
connection with the uterus [54, 55]. The DMX is generally centage of double labeled neurons in the PVN than saline
recognized as parasympathetic preganglionic neurons that injectiongroup(Figure5(d)).Thosepreviousresearchesand
innervate various visceral organs. Retrograde tracer studies ourresultsuggestthatthePVNmaybeanotherrelaycenter
haveshownthattheDMXinnervatesthececum,uterus,and ofthesomatovisceralreflexbetweenthegroinanduterus.
colondirectlyorindirectly[50,52,55–57].Theneuronsinthe AlthoughtheelectrophysiologicalstudiesshowlaminasI
A5cellgroup,RPa,andGiinnervatingtheuteruswerealso andVofthespinaldorsalhornreceiveafferentinformation
confirmed by previous PrV tracing researches [23, 24, 50]. from both somatic and visceral tissues [75–79], no double
Therefore, the PVN function is not only a uterus-related labeled spinal dorsal neurons can be detected in our study.
hormone regulation center [58], but also a direct neuronal Thissolidevidenceshowstheneuronalconnectionbetween
connection to the uterus [23, 24]. After PHA-L injection thegroinregionandtheuterusisnotadmittedthroughthe
in the PVN, terminal varicosities appeared in the IML of spinalcord.
thethoracicspinalcord[59].Retrogradetracerstudiesalso
showed a neuronal connection between the PVN and the 4.4. The Morphologic Evidence of Somatovisceral Reflex:
uterus[23,24,50].Insummary,alltheseresultssuggestthat A Possible Neuronal Pathway of Acupuncture. A previous
thePVNhaseitheradirectorindirectneuronalconnection study suggested that acupuncture may influence visceral
Evidence-BasedComplementaryandAlternativeMedicine 9
3V 3V 3V
(a1) (b1) (c1)
AP AP AP
CN CN CN
(a2) (b2) (c2)
(a3) (b3) (c3)
(a4) (b4) (c4)
(cid:289)ected %) 60 ∗
V-infea ( 50
ons/Prpinal ar 40
uras 30
nepr
ouble-labeled neurons in su 12000
D
PaAP MoV NTS
Saline+PrV
Bee venom+PrV
(d)
Figure5:Double-labeledneuronsofFosexpressionandPrVinfection(𝑛 = 8).Fosexpressionneurons((a),red,arrow)inthePVN(a1),
NTS((a2),(a3)),andDMX((a2),(a4)).PrV-infectedneurons((b),green,arrow)inthePVN(b1),NTS((b2),(b3)),andDMX((b2),(b4)).
MergeddoublelabelingFosexpressionandPrV-infectedneurons((c),yellow,arrow)inthePVN(c1),NTS((c2),(c3)),andDMX((c2),(c4))
(scalebar:1000um).(d)ThepercentageofdoublelabeledneuronsinPVN,DMX,andNTSbetweensalineandbeevenominjectiongroups
(∗𝑃<0.1).
10 Evidence-BasedComplementaryandAlternativeMedicine
NTS and DMX
Vagus nerve
DRG PVN
NTS
DRG
SPN
Uterus
Groin region
Uterus
Groin region
(a) (b)
Figure6:Schematicdrawingofthreeneuronalpathwaysofsomatovisceralreflex.(a)Somato-parasympatheticreflexpathwaythroughthe
vagusnerve.(b)Somato-sympatheticreflexpathwaysthroughtheNTSandPVN.DMX:dorsalmotornucleusofvagus;DRG:dorsalroot
ganglia;NTS:nucleusofsolitarytract;SPN:sympatheticpreganglionicneurons;PVN:paraventricularhypothalamicnucleus;blue:somatic
afferent;red:visceralefferent.
function via the activation of the somatosensory neurons Abbreviations
[9]. However, most researches focused on the physiological
PrV: Pseudorabiesvirus
responses induced by acupuncture [5, 10, 11]. The purpose
NTS: Nucleusofsolitarytract
ofourstudyistoinvestigateandprovidethemorphological
PVN: Paraventricularhypothalamicnucleus
evidence of somatovisceral reflex and possible neuronal
DMX: Dorsalmotornucleusofvagus
pathway of acupuncture. Our result suggests that the PVN,
PB: Parabrachialnucleus
NTS, andDMX couldbetherelaycenterofthesomatovis-
LC: Locuscoeruleus
ceral reflex. Thevisceralorgansusually receive sympathetic
RPa: Raphepallidusnucleus
and parasympathetic dual interacted and the interaction
PVT: Paraventricularthalamicnucleus
is antagonistic. Therefore, our study proved morphological
LH: Lateralhypothalamicarea
evidenceofbothsympatheticandparasympatheticpathways
IRt: Intermediatereticularnucleus
of somatovisceral reflex between the groin A-shi point and
Amb: Ambiguusnucleus
theuterus(Figure6).Thesomato-parasympatheticpathway
LRt: Lateralreticularnucleus
starts from the stimulation of the groin A-shi point, which
A5: A5noradrenalinecellsgroup
activates neurons in the spinal dorsal horn. The signal in Gi: Gigantocellularreticularnucleus
turn elicits noxious input to the NTS [60]. Neurons in the MPA: Medialpreopticarea
NTS relay the information and project to the DMX [80], CRF: Corticotropin-releasingfactor
which innervates the uterus through the vagus nerve [55] IML: Intermediolateralnucleus
(Figure6(a)). The somato-sympathetic pathways from the SPN: Sympatheticpreganglionicneurons.
neurons in the spinal dorsal horn project to the NTS [60]
then direct connection to the PVN [81]; it innervates the
Acknowledgment
visceral organ through the sympathetic pre- and postgan-
glionic neurons [59] (Figure6(b)). These complementary
This study is supported by research Grants (no. NSC-92-
somato-sympathetic and somato-parasympathetic systems
2320-B-006-078- and no. NSC-93-2320-B-006-075-) from
coincidentally match the concept of Yin-Yang theory in
theNationalScienceCouncil.
traditionalChinesemedicine[2,82].
In conclusion, the present study provides the morpho-
References
logicalevidenceoftheneuronalconnectionbetweensomatic
groin A-shi point and its corresponding visceral organ
[1] A.Sato,“Somatovisceralreflexes,”JournalofManipulativeand
uterus. Therefore, we come up to the conclusion that the
PhysiologicalTherapeutics,vol.18,no.9,pp.597–602,1995.
somato-sympathetic/somatoparasympathetic pathways are
[2] World Health Organization. Regional Office for the Western
themorphologicalbasisofsomatovisceralreflexandalsothe
Pacific,StandardAcupunctureNomenclature:ABriefExplana-
neuronalsubstrateofacupuncturepathways. tionof361ClassicalAcupuncturePointNamesandTheirMul-