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

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