冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响 精灵论文.docx
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冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响 精灵论文.docx
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冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响精灵论文
冬眠对达乌尔黄鼠Spermophilusdauricus趾长伸肌形态结构及肌纤维类型的影响精灵论文
Hibernationeffectsonmusclemorphologicalcharacteristics
andfibertypecompositionindauriagroundsquirrels
(Spermophilusdauricus)12111GAOYunfang,WANGJun,WANGHuiping,FENGBan,DANGKai,13WANGQi,HelmutG.Hinghofer-Szalkay
(1.KeyLaboratoryofResourceBiologyandBiotechnologyinWesternChina(Northwest
University),MinistryofEducation,Xi’an710069,China;
2.DepartmentofObstetricsandGynecology,TangduHospital,ForthMilitaryMedicalUniversity,
Xi'an710038,China;
3.InstituteofAdaptiveandSpaceflightPhysiology,Wormgasse9,A-8010Graz,Austria)Abstract:
Aim:
Westudiedeffectsofhibernationonextensordigitorumlongus(EDL)weight,fibercross-sectionalarea(CSA),fibertypedistribution,andmyosinATPaseactivityindauriagroundsquirrels(Spermophilusdauricus).Methods:
Musclemass,fiberCSA(videoanalysis),fibertype,andmyosinATPaseactivity(Ca2+-ATPase)weremeasuredbeforeandafter1and2monthsofhibernation,and2-4daysafterarousalindifferentgroupsofgroundsquirrels.Results:
Comparedwithpre-hibernation,bodyweightdecreasedbyabout10%perhibernationmonthandmusclewetweightwasnotaltered.However,muscle-to-bodyweightratioincreasedsignificantly.BothtypeIandtypeIIfiberCSAincreasedbyabout8%duringhibernation,withnoobviousinfluenceofhibernationduration.
HibernationcausedaslightbutsignificantdecreaseinbothATPaseactivityandthepercentageoftypeIIfibers.2-4daysafterarousal,bothfiberCSAandfibertypedistributionreturnedtopre-hibernationlevels.NotypeIIfibersub-typecanbeidentifiedindauriagroundsquirrelEDLsamples.Conclusion:
WereportfirstdataonEDLfibertypedistributionandhibernationeffectsindauriagroundsquirrels.EDLdidnotshowanysignofatrophyoverthe3-monthwinterdormancy.Theseresultssuggestthatapotentialmechanismexistsinhibernatorsthatwouldallowthemtopreventmuscleatrophyfortheprolongeddisuse.
Keywords:
Dauriagroundsquirrels;extensordigitorumlongus;myosinATPase;hibernation
0Introduction
Decreasedskeletalmuscleactivationaswithinactivity,bedrest,orspaceflightleadstodisuseatrophy.Astronautmuscularperformancediminishesbothin-andpostflight,andrehabilitationfromlongtimebedrestisdelayedinpatients.Innon-hibernatinganimals,thisatrophyisassociatedwithreducedcross-sectionalmusclearea(CSA)anddecreasedoxidativecapacity.Ashiftfromslow-tofast-twitchfiberisseenafter?
7daysdisuseinantigravitymusclesthatarerich
inslowtwitchfibers,andafter?
2weeksdisuseinmuscleswithahighpercentageoffasttwitch
fiber(1,7,12,23).Ifsuchchangesoccurredinhibernatinganimals,bothlocomotorandthermogenicfunctionwouldbecompromised.Thiswouldbedangerousorevendeadlyforhibernatinganimalsbecausetherecoverywouldbeslowlydespitethequickdemandasanemergencyappeared.(3,6).Asanimalsbeinghibernating,itsskeletalmuscleisundoubtedinalongtimedisusedstatus.Now,availableevidenceonmusclephysiologyinhibernationislimitedandpartlycontroversial(2,9,16).Somehaveobservedatrophyeffectsassociatedwithhibernation(5,18,22),somedidnot(8,10,11,19).
Westudiedchangesofmuscleweight,fibertypedistribution,cross-sectionalarea(CSA),andmyosinATPaseactivityindauriagroundsquirrelextensordigitorumlongus(EDL)samplesafter
variousdurationofhibernation,andcompareddatafrombeforeandduringhibernation,andafter
Foundations:
NationalNatureScienceFoundationofChina(No.30770273),KeyLaboratoryfoundationofResourceBiologyandBiotechnologyinWesternChina(KH09026),andScienceFoundationofNorthwestUniversity(No.NH0918).
Briefauthorintroduction:
GAOYunfang(1958-),female,Prof.PhD,physiology.E-mail:
gaoyunf@
arousal.WereportanunexpectedincreaseinfiberCSAandbelievetobefirsttopresentdetailsontheseadaptivechangesingroundsquirrelmuscletissueduringhibernation.
1Proceduresandmethods
1.1Animalsandexperimentalprocedures
32dauriagroundsquirrelsfromtheWeinanregion,ShaanxiprovinceofChina,ofeithersexwerematchedforbodymassandrandomlyassignedtofourgroupequally:
1.Pre-hibernationgroup(Pre-H)
2.After1and2monthsofhibernationgroup,respectively.(H-1monandH-2mon)
3.Post-hibernationgroup(Pos-H,i.e.2-4daysafterarousingfrom>3monthshibernation.)
Theanimalswereraisedindividuallyinconventionalplasticcagesandprovidedwithstandardlaboratoryratchowandwateradlibitum,andwerehousedinanenvironmentwithnaturallight,followinginternationalguidelinesforthecareanduseoflaboratoryanimals.TheLaboratoryAnimalCareCommitteeoftheP.R.ChinaMinistryofHealthapprovedallprocedures.Thegroundsquirrelswerebroughttoadark,hibernationroom(4-6?
C)atatimeinyearwhenanimalsusuallystarttohibernate.
1.2SamplePreparation
Animalswereanaesthetizedwith90-mg/kgsodiumpentobarbitali.p.,oneextensordigitorumlongus(EDL)musclewasremovedandweighedto0.1mgaccuracy(Sartorius,BS210S,Germany).5mmsamplesfromtheEDLmid-bellywerepreparedandputintocold30%sucrosesolution,embeddedverticallywithanOptimalCuttingTemperaturegum(SakuraFinetekUSAInc.,Torrance,CA,USA),and10µmslicescutinacryostat(Leica,Wetzlar,CM1850,Germany)at-25ºC.Tissuesectionswereputonmicroscopeslides,whichwerepre-treatedwithPoly-L-Lysine(10g/L).Attheendofthesurgicalintervention,theanimalsweresacrificedbyoverdoseinjectionofsodiumpentobarbital.
1.3HistochemicalAnalyses
After5minutesacidpreincubationatpH4.6and30secondsalkalinepreincubationatpH10.4(0.02Msodiumbarbital,0.036Mcalciumchloride),musclesampleswereincubatedfor45minutesinanATPasereactionsolution(0.02Msodiumbarbital,0.018Mcalciumchloride,0.3%Adenosine-5`-triphosphoricaciddisodiumsalt-Genview,Houston,Texas,USA,0.06%2,4-dinitrophenol,atpH9.4and37?
C).
WefollowedDubowitz-Brookefiberclassification-slowfiber(typeI),fastoxidativeglycolytic(typeIIA)andfastglycolytic(typeIIB)(4).ItisgenerallyassumedthatpreincubationatpH4.4inducesdarkstainingoftypeI,lightornegativestainingoftypeIIB,and"intermediate"stainingoftypeIIA.TypeIIfibersinratEDLcanbeclassifiedintotypeIIAandtypeIIBfiberswithacidpre-incubatedm-ATPasestainingmethods(17,20).Inthisstudy,wemadeadifferentiationintoslowfiber(typeI)andfastfiber(typeII)only,sincenotypeIIsubtypescanbeidentifiedindauriagroundsquirrelEDL.However,therewasacleardistinctionbetweenthedarkstainedTypeIandlighterstainedTypeIIfibers.
Thefibertypedistributionisexpressedasaratiobetweenthenumberoffibersofeachtypeandthetotalnumberoffibers.Measurementsweremadeforallfibersineachsection.TheCSAofnolessthan100fibersofeachtypewasmeasuredwithaQuantimet-570(Leica,Cambridge,UK)videoimageanalyzerandcolordigitalvideoenhancement.
1.4StatisticalAnalysis
SPSS10.0wasusedforallstatisticalanalyses.DataareindicatedasMean?
SD.Aone-wayANOVAwasusedtodetermineoveralldifferences;Fisher’sLSDposthoctestwasusedto
determinegroupdifferences.ANOVA-Dunnett’sT3methodwasusedwhennohomogeneitywas
detected.Paired-samplesTtestwasappliedtotestforbodyweightloss.Significancewasassumedwithalphaerrorp<0.05.
2Results
2.1BodyandEDLmuscleweight
Bodyweight(BW)decreasedupto36%andEDLmusclewetweight(MWW)decreased13%afterhibernationmorethan3months.Meanwhile,themuscle-to-bodyweightratiopresentedanobviousincreaseduringhibernationof1,2andpost-hibernation(>3months),respectively(Table1).
Tab1Bodyweight,EDLmusclewetweightandmuscle-to-bodyweightratiobefore,duringandafterhibernation(Mean?
SD,n=8)EDLMWWatEDLMWW/BWatBWlossBWbeforeBWatexperimentGroupexperimenttimeexperimenttimehibernation(g)time(g)(%)(mg)(mg/g)Pre-H345.38?
17.96345.38?
17.960.00132.48?
12.610.385?
0.045
H-1mon362.50?
35.66314.88?
30.10***13.05?
3.31127.33?
11.150.406?
0.043###H-2mon332.75?
30.54253.88?
22.29***23.46?
6.13122.31?
13.180.483?
0.042
####Pos-H349.38?
32.89219.50?
18.99***36.54?
9.40115.85?
14.790.529?
0.061
Cross-sectionalfiberarea(CSA)
CSAoftypeIandIIfibersinextensordigitorumlongus(EDL)samplesbothincreasedduringhibernationcomparedwiththatofpre-hibernation.TypeIfiberCSAincreased11.3%and9.6%for1and2monthshibernation(0.05
2800TypeITypeII2400)2?
?
2000
1600
1200
800
EDLmusclefiberscrosssectionalarea(mµ400
0Pre-HH-1monH-2monPost-H
Group
Figure1.EDLmusclefibercrosssectionalareabefore,duringandafterhibernation.
CSA=crosssectionalarea.?
0.05
2.2MyosinATPaseactivityandfibertypedistribution
Comparedwithpre-hibernationgroup,weobservedashiftoffibertypefromtypeIItotypeIduringhibernation(Figure2).
Figure2.EDLmusclefibermyosinATPaseactivitybefore,duringandafterhibernationI=typeIfiber,II=typeIIfiber.Magnificationx200.PanelA:
Pre-hibernation.
2.3PanelB:
Duringhibernation.PanelC:
2daysafterarousal.
ThepercentageoftypeIfiberincreasedsignificantlyby42.49%and47.28%after1and2monthshibernation,respectively.Thepercentageofty
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