Effects of treatment of treadmill combined with electro-acupuncture on tibia bone mass and

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Effects of treatment of treadmill combined with
electro-acupuncture on tibia bone mass and
substance PExpression of rabbits with sciatic nerve
injury
Yan Wang
Heilongjiang University of Chinese Medicine, Harbin
Qiang Tang
Heilongjiang University of Chinese Medicine, Harbin
Luwen Zhu
Heilongjiang University of Chinese Medicine, Harbin
Ruyi Huang
Heilongjiang University of Chinese Medicine, Harbin
Lei Huang
Heilongjiang University of Chinese Medicine, Harbin
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Recommended Citation
Wang, Yan; Tang, Qiang; Zhu, Luwen; Huang, Ruyi; Huang, Lei; Koleini, Melanie; and Zou, Dequan, ,"Effects of treatment of
treadmill combined with electro-acupuncture on tibia bone mass and substance PExpression of rabbits with sciatic nerve injury." PLoS
One.11,11. e0164652. (2016).
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Authors
Yan Wang, Qiang Tang, Luwen Zhu, Ruyi Huang, Lei Huang, Melanie Koleini, and Dequan Zou
This open access publication is available at Digital Commons@Becker:http://digitalcommons.wustl.edu/open_access_pubs/5429
RESEARCHARTICLE
Effects of Treatment of Treadmill Combined
with Electro-Acupuncture on Tibia Bone Mass
and Substance PExpression of Rabbits with
Sciatic Nerve Injury
YanWang1,2*,QiangTang1,2,LuwenZhu1,2¤,RuyiHuang2,LeiHuang2¤,MelanieKoleini3,
DequanZou4
1 The2ndaffiliatedhospitalofHeilongjiangUniversityofChineseMedicine,Harbin,China,2 Heilongjiang
UniversityofChineseMedicine,Harbin,China,3 HRPO,WashingtonUniversitySchoolofMedicine,
a11111 St.Louis,Missouri,UnitedStatesofAmerica,4 PrograminPhysicalTherapy,WashingtonUniversitySchool
ofMedicine,St.Louis,Missouri,UnitedStatesofAmerica
¤ Currentaddress:RehabilitationCentreofthe2ndaffiliatedhospitalofHeilongjiangUniversityofChinese
Medicine,411GuogeliStreetHarbinCity,HeilongjiangProvince,150001,China
*[email protected]
Abstract
OPENACCESS
Citation:WangY,TangQ,ZhuL,HuangR,Huang
Theperipheralnervoussystemmayplayanimportantroleinnormalbonemaintenanceand
L,KoleiniM,etal.(2016)EffectsofTreatmentof
TreadmillCombinedwithElectro-Acupunctureon remodeling.SubstanceP(SP)isaneuropeptideassociatedwithbonelossandformation
TibiaBoneMassandSubstancePExpressionof thatmaymediatetheeffectsofthenervoussystem.Thepurposeofthisstudyistodeter-
RabbitswithSciaticNerveInjury.PLoSONE11
mineiftreadmillrunningcombinedwithelectro-acupunctureatJiajiacupoints(Jiaji-EA)
(11):e0164652.doi:10.1371/journal.
affectstibialbonemassandSPexpressioninrabbitswithsciaticnerveinjury.Twenty-four
pone.0164652
juvenilemaleNewZealandwhiterabbitswererandomlyassignedtooneof4groups:sham
Editor:SilvanaAllodi,FederalUniversityofRiode
injurycontrol(sham),sciaticnevercrushcontrol(SNCr),treadmillrunning(treadmill),and
Janeiro,BRAZIL
Jiaji-EAcombinedwithtreadmillrunning(ETgroup).TheSNCr,treadmill,andETgroups
Received:April20,2016
allhadaninducedsciaticnevercrushinjuryofapproximately2mm.Controlgroupsreceived
Accepted:September28,2016
nointervention;thetreadmillandETgroupsweretrainedbytreadmill;theETgroupalso
Published:November23,2016 receivedJiaji-EA.Afterthe4weeksoftreatment,toe-spreadingindex(TSI),BMD,bone
Copyright:©2016Wangetal.Thisisanopen strength,andSPexpressioninthetibiaweresignificantlylowerinthenerveinjurygroups
accessarticledistributedunderthetermsofthe (SNCr,treadmill,andET)comparedtotheshamgroups(p<0.05).Treatment(treadmilland
CreativeCommonsAttributionLicense,which
ETgroups)increasedallmeasurescomparedtotheSNCrgroup(p<0.05).Further,TSI,
permitsunrestricteduse,distribution,and
BMD,bonestrength,andSPexpressionintheETgroupwerehigherthanthetreadmill
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. group(p<0.05).Ourresultsindicatethattreadmilltherapycombinedwithelectro-acupunc-
tureatJiajiacupointspreventsbonelossinrabbittibiasaftersciaticnerveinjury.Thismay
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation occurintwoways:indirectlyinassociationwithaxonregenerationanddirectlyvialoading
files. onthebonemediatedthroughincreasedSPexpression.Thisstudyprovidesimportantevi-
Funding:ThisstudywassupportedbytheFundof dencefortheclinicaltreatmentofbonelossafterperipheralnerveinjury.
InnovativeTalentsoftheHeilongjiangUniversityof
ChineseMedicine(grantnumber051290).
Additionalfundingforthisstudywasprovidedbya
postdoctoralresearchawardfromthe
AdministrationOfficeofHeilongjiangPostdoctoral
Program(grantnumberLBH-Z13202).
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 1/12
EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Injuriestoperipheralnervesdifferfrominjuriestomostothertissuetypesbecausefurther
neuronandtargetorgandegenerationoftenfollows[1].Fortunately,theperipheralnervous
system(PNS)hasaninnatecapacitytoregrowtotheirtargets,includingmuscle,bone,and
skin.However,theregenerationprocessisslowandincomplete[2]andisoftenaccompanied
bydisturbingmotor,autonomicandsensoryconsequences[3].Theperipheralnervoussystem
mayplayanimportantroleinbonehealingfollowingfractureorothertrauma[4–6].Bone
fracturesaccompaniedbyperipheralnerveinjurieshealslower[7]andsensorydenervation
negativelyaffectslong-termprognosis[8].SubstanceP(SP),aneuropeptidebelongingtothe
tachykininfamily,iswidelydistributedinthebody,particularlyinthecentralandperipheral
nervoussystemswhereitactsasaneurotransmitterorneuromodulator[9].Studiessuggest
thattheabsenceofSPreducesboneformationrateassociatedwithfracturehealing[10,11].
Exerciseaffectsbonesinbothhumansandanimals.Dynamicexerciseissuperiortostatic
exerciseinincreasingbonemassandgreaterintensitytrainingleadstogreaterincreasesin
bonemass[12].Increasedmechanicalloadingisalsobeneficialtobonegrowth.Running-
generatedimpactandloadonbonearebelievedtoincreasecorticalbonemassthrough
remodeling.
Acupuncture,atherapyoriginatinginChina,hasbeenwidelyusedforthetreatmentof
neurologicaldisorders,includingspinalcordinjury(SCI)[13].Electro-acupuncture(EA)at
theacupointsofGovernorVessel(DuMeridian)andJiajihasproventohaveatherapeutic
effectinthetreatmentofspinalcordinjury(SCI)bothinclinicalcareandanimalexperiments
[14].Jiajiacupointsarelocatedonthebilateralspinousprocessesoflumbarspine[15],5mm
lateraltotheposteriormidlineinrabbits.Thesciaticnerveandlumbosacralplexusarelocated
beneaththeseacupoints.Somestudies[16]suggestthesciaticnerveoftherabbitislocated
betweenL andS inthespinalcord.However,duetotherelationshipbetweensectionsof
7 2
lumbosacralspinalcordandtheirlumbosacralvertebrasinrabbit,webelievethatthesciatic
nerveislocatedbetweenL andL oftheoflumbosacralvertebras.Neuronsofthesciatic
4 6
nerveandlumbosacralplexusarebeneaththeseacupoints.Sinceadequateconnectivityinspi-
nalcircuitsandperipheralnervoussystemintegrationarealsoimportantfactorsinnerve
regrow[17],Jiaji-EAmayhelptofacilitatesciaticnerveregenerationafterthenerveinjuryvia
stimulationofthesciaticnerveandlumbosacralplexus.
Inourpreviousanimalexperimentsandclinicalpractices,weobservedthatbonesthat
weredenervatedduetoperipheralinjurywereapttosufferbonelossfollowedbypathological
fracture.Previousstudiesfocusedprimarilyonthedamagedperipheralnervesthemselvesand
denervatedmuscle[18,19].Althoughbonehealingcombinedwithperipheralnerveinjuryhas
beeninvestigated[4,5,7,8],interventionsfordenervatedboneshavenotbeenextensivelystud-
ied.ThisstudymeasurestheeffectsofcombiningtreadmillrunningwithJiaji-EAontibia
bonemass,bonestrengthandSPexpressioninrabbitswithsciaticnerveinjury.Wehypothe-
sizethattreadmillrunningcombinedwithJiaji-EAwilldecreasebonelossandweakening,and
increaseSPexpression,reducingpathologicalfractureofdenervatedbones.
MaterialsandMethods
ThisstudywascarriedoutinstrictaccordancewiththerecommendationsintheGuidefor
theCareandUseofLaboratoryAnimalsofChineseAcademyofMedicalSciences.Thepro-
tocolwasapprovedbytheCommitteeontheEthicsofAnimalExperimentsoftheSecond
AffiliatedHospitalofHeilongjiangUniversityofChineseMedicine,China(PermitNumber:
ABX20140105A).Allsurgerywasperformedundersodiumpentobarbitalanesthesia,andall
effortsweremadetominimizesuffering.
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 2/12
EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
Subjects
Twenty-four11–12weeks-oldmaleNewZealandwhiterabbitswithameanweightof1.78
±0.12kgwererandomlyassignedtooneof4groups:sham(n=6),SNCr(n=6),treadmill
(n=6),orJiaji-EAcombinedwithtreadmill(ET,n=6).Allanimalswereanesthetizedwith
1.5±0.5ml/kgof10%chloralhydrateviatheearmarginalvein.IntheSNCr,treadmill,andET
groups,alateralapproachwascreatedonthelefthindlimb.Theincisionwaslocatedmid-way
betweentheattachmentpointofrectusfemoristendononkneejointandtherectusfemoris
betweentheupperonethirdandthemidline.Thetipofhemostaticforcepscoveredbythin
plastictubingwereusedtocrushthenervetrunkbyfullclampingthenervefor5minutes
[20,21];thiscauseddamageacrossapproximately2mmofthenerve.Theshamgroupwassub-
jectedtothesamesurgicalprocedurewithsciaticnerveexposurebutwithoutcrushinjury.
InterventionGroups
Theinterventionmethodsforallgroupswereasfollows:(1)Controlgroups(shamandSNCr):
nointervention,rabbitswereremovedfromcageandplacedonatreadmillwhiletreadmill
wasnotoperatingfor20mineachday,asashamprocedure;(2)Treadmillgroup:beforesur-
gery,therabbitsweremadetoadaptivelyrunonatreadmillon3consecutivedays.Threedays
post-surgery,theanimalsweremadetorunfor20minutesadayatarateof10m/minfor3
days,atarateof15m/minfor20min/dayfromthefourthtosixthday,andthenatarateof
20m/minfor20min/dayfromtheseventhdayonward,6days/weekforatotaltreatmentcycle
durationof4weeks;(3)ETgroup:rabbitsreceivedtreadmillexerciseusingthesameprotocol
asthetreadmillgroup.Inaddition,3dayspost-surgery,6Jiajiacupoints(3acupointsoneach
side)wereidentifiedbilateralofthespinousprocessesofL toL ofspineapproximately5mm
4 6
lateraltotheposteriormidline.ThelengthoftheJiajiregionisapproximate30mm.Thefour
limbsofeachanimalintheETgroupwerefixedontheexperimentaltableintheproneposi-
tionbyathickcottonband.Thefurintheacupointregionwasshavedandsterilizedusing
iodophor,thenasterileneedle(0.20mmindiameterand25mminlength)wasinsertedinto
eachacupointtoadepthof10-15mm.Thentheneedleswereconnectedtoa3Hz/selectrical
currentfor30min/day,6days/weekforatotaltreatmentdurationof4weeks(Fig1).Therab-
bitsinSNCrandTreadmillgroupsweregivenashamprocedure,withshavingofbackand
confinementbutwithoutneedleinsertionandapplicationofelectricalcurrent.Noanesthesia
wasusedduringtheelectro-acupunctureprocedureandtherabbitsdidnotdisplayany
increasedpainbehaviors.
After4weeksoftreatment,allanimalswereanesthetizedwith1.5±0.5ml/kgof10%chloral-
hydrateviaearmarginalveinandtheirheartswerecuttobleedoutaftertheanimalsbecame
deeplyunconscioustoeuthanizethem.Theirlefttibiaswereharvestedbydisarticulationatthe
kneeandankle.First,allligamentsaroundthekneejointandthetendonsofgastrocnemius
attachedatmedialandlateralcondylesoffemurwereseveredandthetibiawasdislocated
fromthefemur.TheAchillestendonwasthencut.Next,theentiretricipitalmusclewas
excisedfromthesofttissuesconnectedtothetibia.Thentheanteriortibialmusclewas
removedfromtibia.Theligamentsconnectingtheanklejointweresheared,separatingthe
tibiaandfoot.Finally,oftheremainingsofttissueonthetibiawasremoved.
(1)FunctionalAnalysis(FA). Themotorfunctionofthelefthindlimbwasassessed
beforethecrushinjuryand4weekspostinjuryusingthetoe-spreadingreflex.Thetoe-spread-
ingreflexisareliability,sensitivityandnon-invasivemethodforassessingrecoveryofperoneal
nervefunctionafterinjury[22,23].Theprocedurewasasfollows:eachrabbitwasheldbythe
looseskinofitsbackthenwassuddenlyloweredthroughtheairwithoutlettingthemcontacta
surface.Rabbitswithfunctionalnerveinnervationreflexivelyspreadtheirsecond,thirdand
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 3/12
EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
Fig1.ThetherapyofElectro-acupuncture.ThefourlimbsofeachrabbitintheETgroupwerefixedonthe
experimentaltableinthepronepositionbyathickcottonband.Sixsterileneedleswereinsertedintoeach
acupointtoadepthof10-15mm.Thentheneedleswereconnectedtoa3Hz/selectricalcurrent.
doi:10.1371/journal.pone.0164652.g001
fourthtoes.Themovementofthesetoeswascapturedusingadigitalvideocamera(I-phone5,
USA)andthenwasgradedfollowingthetoe-spreadingindex(TSI)describedbyE.Gutmann
(Table1)[22,23].
(2)Bonestrength. Tibiaswerewrappedinsalinesoakedgauzeandfrozen(-20˚C)after
excision.Bonestrengthwasmeasuredby3-pointbendingtestingusingaUniversalTesting
Machine(ZwickZ010,Germany)thesameday.Thetibiawasputonthetestingapparatus
withanterioraspectup.The3-pointbendingtestwasperformedwithasupportspanof60
mmandstrainrateof1mm/min.TheUltimateLoadofthetibiawasmeasuredatbonefailure
[24].
(3)BoneMineralDensity(BMD). Thebonemineraldensityofeachtibiawasmeasured
usingDual-EnergyX-rayabsorptiometry(DXA)(Noland,USA).Afterstrengthtesting,the
bonewasdividedintotwosections.Theproximalsectionwasplacedonwhitepaperinhori-
zontalpositionwithanteriorsideupundertheDXA.Arectangularareaof1.53cm2approxi-
mately1cmbelowthetibialplateauwasscannedtomeasureBMD.
(4)SPexpression. Afterbonestrengthtesting,a10mmthinksampleofcorticalbone
fromapproximatelythemiddleofthetibialshaftwassectionedperpendiculartothelongaxis
ofeachbone(approximatecylinder)andfixedinaformaldehydesolution.Asecondbone
samplewasusedforBMDtesting.ThebonesweredemineralizedusingEDTA-NaOH.Forty
to50gofEDTA(cid:1)2Nawasaddedto500mlofdistilledwaterand0.2gofNaOH.Another350ml
ofdistilledwaterwasaddedandmixed,andthenadditionalwaterwasaddedtobringthefinal
volumeupto1000ml.Thebonetissuespecimenswereputintothesolutionandplacedina
4˚Crefrigeratorandcheckeddaily.Thesolutionwasreplacedeverysecondday.Boneswere
considereddemineralizedwhenthetissuespecimenscouldbeeasilystabbedbyapin.Once
demineralized,thetissuespecimenswerewashedwithdistilledwaterandthenembeddedin
Table1. Thetoe-spreadingindexforbehavioralanalyses.
Grade Symptoms
1 Justvisiblespreadingofthe4thtoealone(also2ndand3rd)
2 Slightspreadingofallthreetoes
3 Spreadingofallthreetoes(lessforcefulthannormal)
4 Fullspreadingofallthreetoes(equaltonormal)
doi:10.1371/journal.pone.0164652.t001
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 4/12
EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
paraffin.Five-micronthicksectionsofbone(5sectionsforeachrabbit)werecutusingimmu-
nohistochemistrymethodforSPexpression.Afterdeparaffinization,thesectionsweresub-
mergedinasolutionofphosphate-bufferedsaline(PBS)with0.3%Triton-X100(pH7.4)for
30minandasolutionof0.3%H2O2for1hinordertoblockendogenousperoxidase.Thesec-
tionswerethenincubatedwithpolyclonalSPrabbitantiratantibodydiluted1:2000inasolu-
tionconsistingof1%bovineserumalbuminand0.05%sodiumazidein0.1MPBSfor24hat
4˚C.AfterthreewashingsinPBS,thespecimenswerethenexposedtobiotinylatedgoatanti-
rabbitor-mouseIgGdiluted1:200inPBSfor4hatroomtemperature.ThreewashingsinPBS
werefollowedbytreatmentwithABCdiluted1:100inPBSfor4hatroomtemperature.The
peroxidasereactionwasthendevelopedfor10minin0.05MTRISbuffer(pH7.6)containing
0.02%3,3-diaminobenzidinetetrahydrochlorideand0.006%H2O2.[25].ThedensityofSP-
positivestainingwasanalyzedusingaDigitalmedicalimageanalysissystem(MoticMed6.0)
[26].
Analysis
StatisticalcalculationswereperformedusingSPSS19.0forWindows.Alldataarepresentedas
means±SD.AllvariablesweretestedfornormaldistributionusingtheKolmogorov-Smirnov
test(p>0.05).One-wayanalysisofvariance(ANOVA)followedbytheLSDpost-hoctestwas
usedforthestatisticalanalysis.Asignificantdifferencewasdefinedasp(cid:20)0.05.
Results
1.FunctionalAnalysis(FA)
MotorfunctionrecoverywasassessedbyTSIat4weeksoftreatment.Beforethecrushinjury,
allofTSImeasuresweregrade4.After4weeksoftreatment,TSIininjurygroups(SNCr,
treadmillandET)werereducedcomparedtotheshamgroup(p<0.05),TheTSIinthetread-
millandETgroupswassignificantlyhighercomparedtotheSNCr(p<0.05).Further,theTSI
intheETgroupswassignificantlyhigherthanthetreadmillgroup(p<0.05)(Fig2).
2.BMDofthetibia
InjurytothesciaticnerveledtosignificantreductionsintheBMDoftherabbits’tibias.The
SNCr,treadmill,andETgroupsallhaddecreasedBMDcomparedtotheshamgroup(p<0.05).
TheBMDofthetibiainthetreadmillandETgroupswassignificantlyhighercomparedtothe
SNCr(p<0.05).Further,theBMDofthetibiasintheETgroupwassignificantlyhigherthan
thetreadmillgroup(p<0.05)(Table2).
3.Bonestrengthofthetibia
SignificantreductionsinthebonestrengthofthelefttibiawerenotedintheSNCr,treadmill,
andETgroupscomparedtotheshamgroup(p<0.05).BonestrengthinthetreadmillandET
groupswassignificantlyhigherthantheSNCrgroup(p<0.05).ThebonestrengthintheET
groupwassignificantlyhigherthanthetreadmillgroup(p<0.05)(Table2).
4.SPexpressioninthetibia
SignificantreductionsintheSPexpressioninthetibiawerefoundintheSNCr,treadmill,and
theETgroupscomparedtotheshamgroup(p<0.05).SPexpressionwaspartiallyrestoredby
treadmillrunning.BoththetreadmillandETgroupsweresignificantlyhighercomparedto
theSNCrgroup(p<0.05).SPexpressionintheETgroupwassignificantlyhighercomparedto
thetreadmillgroup(p<0.05)(Table2andFig3).
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 5/12
EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
Fig2.Comparisonoftoe-spreadingindexforbehavioralanalyses.After4weeksoftreatment,
comparedwithShamgroup,aP<0.05;ComparedwithSNCrgroup,bP<0.05;Comparedwithtreadmillgroup,
cP<0.05.
doi:10.1371/journal.pone.0164652.g002
Discussion
Thepurposeofthisstudywastodetermineifdynamic,weight-baringexercisecombinedwith
Jiaji-EAimprovestibialbonehealthinrabbitswithsciaticnerveinjury.Inthisstudy,wefound
thatTSI,BMD,bonestrength,andSPexpressioninthetibiaarereducedaftersciaticnerves
injury.TSI,BMD,bonestrength,andSPexpressioncanbepartiallyrestoredbyintervening
withtreadmillrunningalone.However,TSI,BMD,bonestrength,andSPexpressionlevels
werefurtherimprovedbyaddedJiaji-EAtothetreadmilltherapy.Inthecurrentstudy,thebet-
terTSI,BMDandbonestrengthfoundintheETgroupareassociatedwithincreasedexpres-
sionofSP.SP—immunoreactivenervefibersinnervatetheboneandadjacenttissues[27].SP-
immunoreactiveaxonshavebeenlocalizedinbone,andSPreceptorsarewidelydistributedin
osteoclastsandosteoblasts[28].
Thestatusofinnervationisimportantforboneunionandfunctionalrecovery[29].The
lackofneuralcontrolmaydelayfracturehealing[30].Consistentwithearlierresearch,we
observedthattheBMDandbonestrengthofthetibiaarereducedaftersciaticnervesinjury
[4.5.7.11.29].Post-traumaticbonelossmaybepartiallytheresultofdecreasedfunctionalload
bearing(disuseosteoporosis)[31].Thisosteopeniacanleadtoosteoporosisandincreasedfrac-
turerisk[32].Bonetissuesarephysiologicallyexposedtomechanicalloadingthroughphysical
Table2. ComparisonofBMD,bonestrengthandSP4weeksposttreatments.
Group(n=6) BMD(g/cm2) Bonestrength(N) SP
Sham 0.197±0.008a 199.043±16.123 0.035±0.004
SNCr 0.123±0.008 51.315±11.459a 0.010±0.005a
Treadmill 0.151±0.002a,b 86.507±20.942a,b 0.017±0.001a,b
ET 0.176±0.005a,b,c 158.539±41.292a,b,c 0.025±0.002a,b,c
ap<0.05vs.shamgroup;
bp<0.05vs.SNCrlgroup;
cp<0.05vs.treadmillgroup.
doi:10.1371/journal.pone.0164652.t002
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EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
Fig3.SubstancePsemiquantitationoflefttibia4weeksposttreatment.SPstainingisshowninbrown
(redarrow).Scalebar=50μm.Valuesaremeans±SDofsixrabbitsineachgroup.A.SPinshamgroups
werehigherthanallothers(p<0.05);B.SPinSNCrgroupwaslessthanallothers(p<0.05);C.SPintreadmill
groupwashigherthanSNCrgroup(p<0.05),lessthanETgroup(p<0.05);D.SPinETgroupwashigherthan
othergroups(p<0.05),exceptshamgroups,waslowerthanshamgroups(p<0.05).
doi:10.1371/journal.pone.0164652.g003
activityincludingexercise,whichcontributestodevelopmentofgrowingbone[33,34].Inthe
currentstudy,exercisetraining(treadmillrunning)increasedcorticalthicknessingrowing
bone[35].
Accordingly,loadbearing,activeexercisesarepredictedtobeamongthemostimportant
methodstopreventbonelossandtohelpmaintainBMD[36,37].Variousanimalmodelsand
trainingmodes,includingtreadmillrunning,jumping,freefalllanding,andpassiveresistant
training,indicateregularexerciseisbeneficialtobonedevelopmentandhealth[37–41].Inthis
study,wechosetreadmillrunningastheexerciseinterventionbecauseitisactive,fullloading,
andcanbequantified.Themaineffectsoftreadmillrunningmaybeduetothesuppressionof
bonemassreduction[38].BMDandbonestrengthoftibiainthetreadmillgroupweresignifi-
cantlyhighercomparedtotheSNCrgroupbutstillsignificantlylowerthantheshamcontrol,
indicatingdecreasesinloadbaringplayapartbutarenotsolelyresponsibleforboneloss.
Running-generatedimpactandloadonbonemaysuppressmetabolicturnoverandincrease
corticalbonemassthroughincreasedperiostealapposition[42].Ourresultsdemonstrate
thattreadmillrunningcaneffectivelypreservebonemass,consistentwithpreviousfindings
[43–46].
Severalneuropeptidesmaybelocalmodulatorsofbonemetabolism,influencingperiosteal
andmedullarybloodflow,angiogenesis,andnociception,inadditiontohavingdirecteffects
onosteoblastsandosteoclasts[4,47].Furthermore,arecentstudyshowedthatboneandperi-
osteumareinnervatedbysympatheticandsensorynervefibers,implicatingtheperipheral
nervoussysteminbonemetabolismandindicatingsensoryandsympatheticneurotransmit-
tershavecrucialtrophiceffectsessentialforproperboneformation[11].Studiesofnerve
regenerationinanimalsuseavarietyofelectrophysiologicalandmorphologicaltests,butthese
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 7/12
EffectsofTreatmentofTreadmillandEAonTibiaBoneLossafterSciaticNerveInjury
endpointsdonotnecessarilycorrelatewiththereturnofmusclefunction[23].Alternatively,
FAvaluecanbeusedtodirectlydeterminetheprognosisofmotorfunctionafterperipheral
nerveinjury[48].TSIisbelievedtobeanassessmentoffunctionthatassessestheendpointof
peronealnerveregeneration[23].WefoundthattheTSIsofrabbitsinSNCrgroupimproved
littleinthe4weeksafterinjury,similartoapreviousstudy[48].However,theTSIsofrabbits
inETgroupweresuperiortoallotherinjurygroups.ThisindicatesusingJiaji-EAcombine
withtreadmillrunningisbeneficialforsciaticneverregenerationafterinjury.
Nerveregenerationplaysacrucialroleinimprovingbonemass.Changesatthespinal
cordlevelmaypersistforalongtime,contributingtochronicdeficitsafterperipheralnerve
injuries[49,50].Thus,modulatingtheplasticchangesatthespinalcordlevelareimportant
toimprovefunctionalrecovery[51].Sincetheeffectivenessofelectro-acupunctureatJiaji
acupointstreatmentofSCIhasbeendemonstratedinbothhumansandanimals[14],we
believedthatelectricalstimulationoftheJiajiacupointsbetweenL andL ofthespine
4 6
stimulatesthesciaticnerveneurons.Inaddition,ourpreviousstudyshowsthatsecondary
degenerationofneuronsisreducedandaxonregenerationisfacilitatedbyJiaji-EAafter
peripheralnerveinjury[unpublisheddata].Inthisstudy,weobservedthattheTSI,BMD,
bonestrength,andSPoftibiaintheETgroupweresignificantlyincreasedcomparedtothe
SNCrandtreadmillgroups.Theseresultssuggestthatbonemetabolismmaybeimprovedby
Jiaji-EAcombinedwithtreadmillindirectlyinassociationwithaxonregenerationand
directlyvialoadingonthebone.
SubstanceP-immunoreactivenervefibersinnervatetheboneandadjacenttissues.SPfre-
quentlyco-localizeswithcalcitoningene-relatedpeptideintheperipheralsensoryaxonsof
bones[27].Theaxondensityvariesdependingonthestageofskeletaldevelopment,theloca-
tioninthebodyandpathologicalconditions[18],suggestingarelationshipbetweenneuropep-
tidesandbone.Inthisstudy,wefoundsignificantreductionsintheSPexpressioninthetibia
intheSNCr,treadmill,andETgroupscomparedtotheshamgroup.Ourdataisconsistent
withthefindingsofKingeryalet[52].Theyobservedrapid,widespreadlossoftrabecular
bonemassinbothhindlimbsafterunilateralsciaticnervetransectioninrats.Theypurposed
thatthecontralateralneurogenicextravasationresponsewasdiminishedandtherewasacon-
tralateralreductioninboneSPexpressionafternerveinjury,suggestinginhibitingSPsignaling
hasnegativeeffectsonbonemass[52].OtherstudiesreportthatSPstimulatesosteogenesis
andthelatestageosteoblasticboneformation[53,54]andtheabsenceofSPreducestherateof
boneformation[30].Gra¨sselbelievesthatduringendochondralossification,sensoryneuro-
peptideSPpromotesproliferationofstemcellsandgrowthplatechondrocytes[55].Inthis
study,aftersciaticnerveinjury,tibialSPexpressionwassignificantlyhigherinthetreadmill
treatmentgroupandhighestinthegroupthatreceivedbothtreadmillandJiaji-EAtherapy.
BMDandbonestrengthwerepositivelycorrelatedwithSPexpression.Thebonehealthofthe
juvenilerabbits’tibiaswasimprovedbytreadmillrunning.AddingJiaji-EAtothetherapypro-
ducedincreasedbenefits.
Limitations
ThecurrentfindingspointtobetterrecoveryintheETgroup,however,theseresultsarelim-
itedbyhavingnowaytodetermineifthecurrentresultswereduespecificallytostimulationof
theJiaji-EAortheeletrostimularionofthegeneralregion.Furtherstudieswillbeneededto
determinethis.Anotherlimitationistheabsenceofdirectevidenceregardingthepathby
whichtreadmillcombinedwithJiaj-EAincreasedSPexpressionandimprovedbonemass
afterperipheralnerveinjury.Furtherstudieswillberequiredtoidentifythemechanismby
whichtreadmillcombinedwithJiaji-EAreversesdenervationbonelossviasignalingpathways.
PLOSONE|DOI:10.1371/journal.pone.0164652 November23,2016 8/12