大鼠行为学实验评价汇总.docx

大鼠行为学实验评价汇总大鼠行为学评定方法比较大鼠进行行为学评定(behaviortest)的十分重要，对其评定的方法也颇多，但究竟那种方法更适用，目前未有人进行过比较。为了合理选择MCAO后的评定方法，如下对目前常用的几种行为学评定方法进行比较。行为学检查方法:由3位参加试验的人员分别以单盲法对试验的大鼠进行打分和记录.然后将3组的记分结果进行平均后的得分进行统计计算。(国内)由一个对试验实施过程不了解的观察者对大鼠进行行为学评测。评测续贯进行。如果大鼠在一次评测中出现恰当的行为，而以后却未出现,按前者记分。(国外)1. 神经行为学检查LOnga评分法2. BerderSon评分法姿势反射测验(POStUraIrefIeXteSt)3. 攀绳实验4. 网屏测验(screentest)5. 肢体放置测验(limb-placementtest)ElicitedForelimbPlacing6. 开野试验(OPen-Field法)测定行为7. MNSS8. 转动杆测验(rotatingpoletest)9. Rotation10. 肢体对称试验评分法11. rotarodtest12. adhesive-removalsomatosensorytest13. SpontaneousActivity14. SymmetryintheMovementofFourLimbs15. ForepawOutstretching16. Climbing17. BodyProprioception18. ResponsetoVibrissaeTouch改善记忆作用1 .跳台试验2 .避暗试验3 .穿梭箱试验4 .水迷宫试验Motorbehavior(1) observationofspontaneousipsilateralcircling,(2) contralateralhindlimbretraction,(3) beamwalkingability,平衡木测验(balancebeamtest)(4) bilateralforepawgrasp,Skilledforelimbfunction(I)Staircasefeedingapparatusnociception(1)plantartest1.神经行为学检查：1.onga评分法神经学检查分5个等级：0分：正常，无神经功能缺损；1分：左侧前爪不能完全伸展，轻度神经功能缺损；2分：行走时，大鼠向左侧（瘫痪侧）转圈，中度神经功能缺损；3分：行走时，大鼠身体向左侧（瘫痪侧）倾倒。重度神经功能缺损;4分：不能自发行走，有意识丧失。2.BerderSOn评分法姿势反射测验（POStUraIreflextest）提尾悬空试验大鼠提尾悬空一米,观察前肢屈曲情况。正常大鼠向地面伸展双上肢。可伸展双上肢向地面并无其它神经缺陷大鼠得0分。梗死后大鼠持续屈曲损伤半球对测患肢；各种不同姿势，从轻度腕屈曲、肩内收、肘伸展到严重姿势，完全腕、肘屈曲，肩内旋转并内收。有任何持续前肢屈曲和其它不正常得1分。大鼠被放于大的软垫子上，表面有大鼠可用爪子牢牢抓住的塑料压膜纸。用手抓其尾巴，缓慢在鼠肩后给予侧压直至前肢滑动儿英寸。此手法在每个方向重复几次。正常或轻度功能损伤大鼠两侧抵抗滑动力相同。严重功能损伤大鼠瘫痪侧始终抵抗力下降，得2分。然后让大鼠自由活动，观察转圈行为。大鼠持续向瘫痪侧转圈得3分。前肢屈曲带有侧推抵抗力下降经常见到；双上肢屈曲并侧推抵抗力下降大鼠经常有转圈行为。神经检查在3-5分钟内完成。正常O分未见行为缺陷中度1分前肢屈曲（即提尾悬空实验阳性）重度2分侧推抵抗力下降（即侧向推力实验阳性）.伴前肢屈曲.无转圈行为3分同2级行为.伴自发性旋转。提起鼠尾.使其离地0.33m高.观察前肢情况。正常大鼠两前肢对称向前伸开;如果有肩部内旋、前肢内收现象发生者.根据其严重程度.最高评分为4分.正常评分为0分。将动物置于平滑地板上.分别推其左、右肩向对侧移动，此时检查动物抵抗推动时的阻力，正常大鼠两侧推动的阻力明显对称。如一侧阻力下降者.可根据下降程度评13分.正常评分为0分。观察两前肢肌力。将动物两前肢置于一金属网上.然后轻提起大鼠.检查两前肢肌力。正常左右对称.如有一侧前肢肌力下降者.根据下降程度评为13分.正常评分为0分。根据上述行为检查的情况.积分值为010分。积分越高说明动物行为障碍越严重。此测验对皮层和纹状体的损伤较敏感。3攀绳实验：本实验主要检查大鼠双前肢和双后肢的协调配合能力。取直径1.5cm的麻绳从高1m、长和宽分别为15、50cm的平台上垂下，手术前Id训练每只大鼠.使之能够爬到绳子的上l3o每个测试日测试3次。评分的标准如下:0分.不需任何刺激在IOS内爬上平台;1分.不需任何刺激在15s内爬上平台;2分.刺激次数小于5次.在30s内爬上平台;3分.刺激次数小于5次.在60s内爬上平台;4分喇激次数大于5次,在60s内爬上平台或是刺激小于5次.但爬上平台时间超过60s;5分.大鼠不能爬上平台。4 .网屏测验(screentest)网屏训练:网屏为50cmX40cm网带.网眼为IcmXICm.网板的左右和上方都用25Cm高的木板框边.网屏距地面高度为80cm.下方铺以12cm厚的海绵。先将网屏水平放置.将老鼠放在其上.然后缓缓地将其一端抬高.在2s内将此屏风变成垂直位.保持5s.观察大鼠是否会从网屏上下来或用前爪抓握住网屏，从而评价前爪抓握能力及肌力情况。网屏实验:评分标准分为4个等级。0分:前爪握住网屏达5s之久，不会掉下来;1分:暂时握住网屏，滑落一段距离，但没有掉下来;2分:在5s内掉下来;3分:网屏转动时，鼠即刻掉下来。5 .肢体放置测验(Iilnb-PIaCeinenttest)ElicitedForelimbPlacing肢体放置于三种独立刺激(视觉、触觉、本体感觉)以评测运动感觉完整性。参照KaWamata等采用的动物行为测试评分方法。具体实验有:前肢放置实验,包括视觉亚实验,即前方刺激实验者将动物握于手中,使其前爪悬空,自桌面上方10cm处向桌面缓慢斜线靠近（此时桌子位于大鼠前方）,大鼠正常反应为前肢即刻抓向桌面,损伤大鼠则表现为肢体反应延迟。O分动物肢体放置反应正常;1分反应延迟但不超过2s;2分反应延迟且超过2sO侧方刺激,此时桌子位于动物侧方,其余实验方法及评分标准同前方刺激。（2）触觉亚实验,将动物置于桶状凹陷实验器内,使其前爪悬空,此时大鼠应该既看不见,也不能用胡须触及桌面,用其前爪背侧轻触桌面,刺激深度仅达皮肤和毛发,动物反应及评分同视觉亚实验,触觉刺激同样分前方及侧方刺激。（3）本体觉亚实验,操作及评分同触觉亚实验,仅刺激深度不同,本体觉亚实验给予前爪较大压力,刺激深达肌肉及关节。该亚实验只有前方刺激。动物前肢放置实验总分范围为O10分,功能损伤越重,得分越高。6 .开野试验（OPenField法）测定行为方法：本实验所用敞箱为正方形,长、宽各80cm,高40cm,底面由面积相等边长16Cm的正方形25块组成,以动物穿越底面的块数为水平活动（CrOSSing）得分,以直立次数为垂直活动（rearing）得分海只动物共进行7次测定,每次3minOpen2Field法是测定抑郁症大鼠行为学的经典方法,分水平运动和垂直运动2个观察面,以观察得分为评分计算单位，记各组大鼠的水平运动评分及垂直运动评分。Elevatedopenfieldactivityischaracteristicofhippocampallydamagedanimals19202122andhasbeenobservedinanimalswithstriatal/corticaldamageaswell.23Ratsareplacedinanovelenvironment(3x3ft)with3-inch-squaregridmarkingsonthebase.Thenumberoftimesaratcrossesagridlineisrecordedfora5-minuteperiod.7 .肢体对称试验评分法将大鼠置于网中，网眼大小为2.3x2.3cm,当大鼠在网络中行走时，计数在2分钟内其前爪漏到网眼中的次数。计算公式为：(脑病变对侧前爪的错步数一病变同侧前爪的错步数)/总步数。其分值如为正数表明脑病变对侧功能缺损；如为负数表明脑病变同侧功能缺损。8 .mNSSmNSS体系的设计原理,评分越低表示功能越健全，0分表示功能完全健全的大鼠。modifiedNeurologicalSeverityScores见附件19 .转动杆测验(rotatingpoletest)转棒上行走测评:评分标准分为4个等级。0分:转动过程中.鼠可在棒上行走;1分:转动过程中.60s以上时间.鼠不会掉下来;2分:转动开始后.鼠从棒上掉下来;3分:转动开始前.鼠从棒上掉下来。10 .Thedetectionwasoperatedat1weekaftersurgerybyinjecting0.5mg/kgofapomorphineintoabdominalcavityforobservationofthechangesinThebehavioursforonceaweekandcontinuousdetectedfor6weeks.Ifconstantright-siderotationoccurredandThenumbersofThecirclesrotatedweremorethan210rper30minutes,thoseratswereconsideredassuccessfulmodels:ifnorotationoccurredorThedirectionofrotationwasnotconstantorconstantright-sideorlell-siderotationbutThespeedwasslowerthan210rper30minutes,thoseanimalswereconsideredasunsuccessfulmodels.Inadditionaltorotation,othercompanionedabnormalbehavioursalsoshouldbeobserved,suchas,tremor,sluggish,grab,andsmellexploring,etc.11 .rotarodtestratswereplacedonanacceleratingrotarodcylinder,andthetimetheanimalsremainedontherotarodwasmeasured.Thespeedwasslowlyincreasedfrom4to40rpmwithin5minutes.Atrialendediftheanimalfellofftherungsorgrippedthedeviceandspunaroundfor2consecutiverevolutionswithoutattemptingtowalkontherungs.Theanimalsweretrained3daysbeforeMCAO.Themeanduration(inseconds)onthedevicewasrecordedwith3rotarodmeasurements1daybeforesurgery.Motortestdataarepresentedaspercentageofmeanduration(3trials)ontherotarodcomparedwiththeinternalbaselinecontrol(beforesurgery).12.adhesive-removalsomatosensorytestsomatosensorydeficitwasmeasuredbothbeforeandaftersurgery.Allratswerefamiliarizedwiththetestingenvironment.Intheinitialtest,2smallpiecesofadhesive-backedpaperdots(ofequalsize,113.1mm2)wereusedasbilateraltactilestimulioccupyingthedistal-radialregiononthewristofeachforelimb.Theratwasthenreturnedtoitscage.Thetimetoremoveeachstimulusfromforelimbswasrecordedon5trialsperday.Individualtrialswereseparatedbyatleast5minutes.Beforesurgery,theanimalsweretrainedfor3days.Oncetheratswereabletoremovethedotswithin10seconds,theyweresubjectedtoMCAO.13. SpontaneousActivityTheanimalwasobservedfor5minutesinitsnormalenvironment(cage).Therat'sactivitywasassessedbyitsabilitytoapproachallfourwallsofthecage.Scoresindicatethefollowing:3,ratmovedaround,exploredtheenvironment,andapproachedatleastthreewallsofthecage;2,slightlyaffectedratmovedaboutinthecagebutdidnotapproachallsidesandhesitatedtomove,althoughiteventuallyreachedatleastoneupperrimofthecage;1,severelyaffectedratdidnotriseupatallandbarelymovedinthecage;andO,ratdidnotmoveatall.14. SymmetryintheMovementofFourLimbsTheratwasheldintheairbythetailtoobservesymmetryinthemovementofthefourlimbs.Scoresindicatethefollowing:3,allfourlimbsextendedsymmetrically;2,limbsonleftsideextendedlessormoreslowlythanthoseontheright;1,limbsonleftsideshowedminimalmovement;andO,forelimbonleftsidedidnotmoveatall.JulioH.Garcia,MD;SimoneWagner,MD;Kai-FengLiu,MD;Xiao-jiangHu,MD,PhD。NeurologicalDeficitandExtentofNeuronalNecrosisAttributabletoMiddleCerebralArteryOcclusioninRatSoStroke.1995;26:627-635o15. ForepawOutstretchingTheratwasbroughtuptotheedgeofthetableandmadetowalkonforelimbswhilebeingheldbythetail.Symmetryintheoutstretchingofbothforelimbswasobservedwhiletheratreachedthetableandthehindlimbswerekeptintheair.Scoresindicatethefollowing:3,bothfrelimbswereoutstretched,andtheratwalkedsymmetricallyonforepaws;2,leftsideoutstretchedlessthantheright,andforepawwalkingwasimpaired;1,leftfbrelimbmovedminimally;andO,leftforelimbdidnotmove.JulioH.Garcia,MD;SimoneWagner,MD;Kai-FengLiu,MD;Xiao-jiangHu,MD,PhDoNeurologicalDeficitandExtentofNeuronalNecrosisAttributabletoMiddleCerebralArteryOcclusioninRatSoStroke.1995;26:627-635o16.ClimbingTheratwasplacedonthewallofawirecage.Normallytheratusesallfourlimbstoclimbupthewall.Whentheratwasremovedfromthewirecagebypullingitoffbythetail,thestrengthofattachmentwasnoted.Scoresindicatethefollowing:3,ratclimbedeasilyandgrippedtightlytothewire;2,leftsidewasimpairedwhileclimbingordidnotgripashardastherightside;and1,ratfailedtoclimbortendedtocircleinsteadofclimbing17. BodyProprioceptionTheratwastouchedwithabluntstickoneachsideofthebody,andthereactiontothestimuluswasobserved.Scoresindicatethefollowing:3,ratreactedbyturningheadandwasequallystartledbythestimulusonbothsides;2,ratreactedslowlytostimulusonleftside;and1,ratdidnotrespondtothestimulusplacedontheleftside.18. ResponsetoVibrissaeTouchAbluntstickwasbrushedagainstthevibrissaeoneachside;thestickwasmovedtowardthewhiskersfromtherearoftheanimaltoavoidenteringthevisualfields.Scoresindicatethefollowing:3,ratreactedbyturningheadorwasequallystartledbythestimulusonbothsides;2,ratreactedslowlytostimulusonleftside;and1,ratdidnotrespondtostimulusontheleftside.改善记忆作用1 .跳台试验跳台装置为IOcmX15CmX60Cm的被动条件反射箱，四周用黑色塑料板、底面铺可通电的铜栅，每间内放置一高4.5cm,直径4.5Cm的橡皮垫作为动物回避电击的安全区，先将动物放在反应箱内的橡皮台上适应3min,然后立即通以40V交流电，动物受到电击后逃避反应为跳上橡皮台。记录5min内大鼠受到电击次数，称为错误次数。24h后在底部铜栅通电情况下，直接将动物置于平台上，记录第1次跳下的潜伏期和5min内的错误次数。2 .避暗实验Avoidanceresponse根据大鼠有嗜暗习性分设明暗两室。明室上方20Cm处悬一个60W日光灯，在暗室的中后部分底部铜栅加40V电压，两室之间设有一直径为3cm大小的圆洞。实验时将大鼠背对洞口放入明室，同时启动计时表，动物自放入明室至进入暗室遭电击所需时间为潜伏期，鼠受电击后可自行逃出暗室。记录潜伏期及5min内动物受电击次数，24h后复测。3 .穿梭箱试验4 .水迷宫试验(Morris)4.1 Morris水迷宫为圆形水池.直径100Cm.深50Cm.水深30Cm.水温(24士2)°C.水而覆一层塑料泡沫屑.池壁上4个等距离点分水池为4个象限.在第三象限中央放置平台.平台无色透明.直径6cm.高28cm.平台没于水而下2cm.水池周围参照物保持不变。试验包括:定位航行试验(PlaCenavigationtest):实验历时7do将大鼠从入水面向池壁置入水槽中.记录大鼠从入水到爬上平台所需的时间.即逃避潜伏期。每只大鼠每人从4个不同象限入水点入水进行训练各1次.取其平均值为其每人逃避潜伏期。训练中.若大鼠在60s内找到平台.让其于平台上站立IOs;若未找到用棒将其引上平台.并让其站立10s.潜伏期限记为60So将大鼠从平台上拿卜来休息60s之后.再按序由下一入水点入水进行下一次实验。空间探索试验(SPatiaIprobetest)第8天撤去平台.将大鼠从第二象限入水点放入水槽.记录60s内其在平台象限的滞留时间。4.2 水迷宫试验主要用于测试大鼠对空间位置觉和方位觉的学习记忆能力5'水迷宫试验测试的一个重要指标是大鼠的平均逃避潜伏期”平均逃避潜伏期指的是大鼠在放入迷宫后学会在5中内找到平台所需要的平均时间,主要与海马功能有关,与大脑皮层也有一定的关系。MorrisWaterMazeThisisataskthatmeasureslearningandmemoryinanaqueousenvironment.Briefly,theratwasplacedina4-ft-diameterwatertankthatisvisuallyseparatedintofourquadrants.Inthecenterofonequadrant,thegoalquadrant,aplatformishidden1inchbelowthewaterline.Theratwasplacedinthetankatoneoffourstartlocations(north,south,east,west)andswamforupto60secondsuntilitfoundtheplatform.Oncetheanimallocatedtheplatform,itremainedonitfor20seconds.Intheeventthatananimaldidnotlocatetheplatform,theobserverpositionedtheanimalontheplatformattheendofthe60-secondswimperiod.Duringa2-to4-minuteintertrialinterval,theanimalswerekeptwarmbyinfraredheatinglamps.Thetemperatureofthewatermazewas20÷0.5oC.Thelatencyandpathlengthoftheratweremeasuredbyavideotrackingsysteminterfacedtoacomputer.NancyE.Alexis,MS;W.DaltonDietrich,PhD;EdwardJ.Green,PhD;RicardoPrado,MD;BrantD.Watson,PhD.NonocclusiveCommonCarotidArteryThrombosisintheRatResultsinReversibleSensorimotorandCognitiveBehavioralDeficits.Stroke.1995;26:2338-2346.4.3 Y迷宫学习记忆训练及测试:Y型电迷宫臂长30cm.高15cm.宽15cmo每臂末端有15W信号灯示安全区.箱底铜栅间隔4mm。电流强度0.7mA.选用电压38V.电击延时2s。设一臂为起步区.按I-II-IIM臂顺序轮流作为安全区。大鼠在起步区静置3min.予以电击致其逃至安全区.灯光持续15s.然后熄灯体急45s.开始下一次操作。学习训练及测试:规定大鼠受电击后从起步区直接逃至安全区为“正确反应”.以达到连续10次电击均为正确反应(即10/10)前所需的电击次数(即尝试次数)表示其学习获得能力(acquisition)。若尝试次数超过30次则不再测试.并以30次为最大值计数。记忆再现(retrieval)测试:选上述达到10/10标准的大鼠.休息24h后再次同法测试.以达10/10标准前的尝试次数表示记忆再现能力。Motorbehavior(1) observationofspontaneousipsilateralcircling,gradedfrom0(nocircling)to3(continuouscircling);(2) contralateralhindlimbretraction,whichmeasuredtheabilityoftheanimaltoreplacethehindlimbafteritwasdisplacedlaterallyby2to3cm,gradedfrom0(immediatereplacement)to3(replacementafterminutesornoreplacement);(3) beamwalkingability,graded0foraratthatreadilytraversesa2.4-cm-wide,80-cm-longbeamto3foraratunabletostayonthebeamfor10seconds;平衡木测验(balancebeamtest)平衡木长80Cm,宽2.5Cm的方木棒，平放在距离地面K)Cm处，让大鼠在其上行走。评分标准为6.个等级。O分:能跳上平衡木，在上面行走不会跌倒;T分:能跳上平衡木，在上面行走跌倒机会小于50%；2分：能跳上平衡木，在上面行走跌倒机会大于50%；3分：在健侧后肢帮助下能跳上平衡木，但受累瘫痪侧后肢不能帮助向前移动；4分：在平衡木上不能行走，但可坐在上面；5分：将大鼠放在平衡木上会掉下来。Beambalanceperformanceofanimalswasassessedona6-pointscale17:0=balanceswithsteadyposture;1=graspssideofbeam;2=hugsbeamand1limb,fallsdownfrombeam;3=hugsbeamand2limbs,fallsdownfrombeam,orspinsonbeam>60seconds;4=attemptstobalanceonbeambutfallsoff>40seconds;5=attemptstobalanceonbeambutfallsoff>20seconds;and6=fallsoff,noattempttobalanceorhangontobeam<20secondsThisisatestofsensorimotorintegrationspecificallyexamininghindlimbfunction.ThetestingapparatusandratingprocedureswereadaptedfromFeeneyandcolleagues.16A1-inch-widebeam,4ftinlength,issuspended3ftabovethefloorinadimlylitroom.Atthefarendofthebeamisadarkenedgoalboxwithanarrowentryway.Atequaldistancesalongthebeam,four3-inchmetalscrewsarepositioned,anglingawayfromthebeam'scenter.Awhitenoisegeneratorandbrightlightsourceatthestartofthebeammotivatetheanimaltotraversethebeamandenterthegoalbox.Onceinsidethegoalbox,thestimuliareterminated.Therat,slatencytoreachthegoalbox(inseconds)andhindlimbperformanceashetraversedthebeam(basedona1to7ratingscale)arerecorded.Ascoreof7indicatesnormalbeamwalkingwithlessthan2footslips,andascoreof1indicatesthattheratisunabletotraversethebeaminlessthan80seconds.Eachratistrainedfor3daysbeforesurgerytoacquirethetaskandtoachievenormalperformance(ascoreof7)onthreeconsecutivetrials.Threebaselinetrialsarecollected24hoursbeforesurgery,andthreetestingtrialsarerecordeddailythereafter.Meanvaluesoflatencyandscoreforeachdaywerecomputed.BeamBalanceBeambalanceissensitivetomotorcorticalinsults.1617ThistaskisusedtoassessgrossVestibulomotorfunctionbyrequiringarattobalancesteadilyonanarrowbeam.Thetestinvolvesthree60-secondtrainingtrials24hoursbeforesurgerytoacquirebaselinedata.Theapparatusconsistsofa3/4-inch-widebeam,10inchesinlength,suspended1ftaboveatabletop.Theratispositionedonthebeamandmustmaintainsteadyposturewithalllimbsontopofthebeamfor60seconds.TheanimasperformancewasratedwiththescaleofCliftonandcolleagues,18whichrangesfrom1to6,withascoreof1beingnormalandascoreof6indicatingthattheanimalwasunabletosupportitselfonthebeam.BeamWalkingThisisatestofsensorimotorintegrationspecificallyexamininghindlimbfunction.ThetestingapparatusandratingprocedureswereadaptedfromFeeneyandcolleagues.16A1-inch-widebeam,4ftinlength,issuspended3ftabovethefloorinadimlylitroom.Atthefarendofthebeamisadarkenedgoalboxwithanarrowentryway.Atequaldistancesalongthebeam,four3-inchmetalscrewsarepositioned,anglingawayfromthebeam'scenter.Awhitenoisegeneratorandbrightlightsourceatthestartofthebeammotivatetheanimaltotraversethebeamandenterthegoalbox.Onceinsidethegoalbox,thestimuliareterminated.Therat,slatencytoreachthegoalbox(inseconds)andhindlimbperformanceashetraversedthebeam(basedona1to7ratingscale)arerecorded.Ascoreof7indicatesnormalbeamwalkingwithlessthan2footslips,andascoreof1indicatesthattheratisunabletotraversethebeaminlessthan80seconds.Eachratistrainedfor3daysbeforesurgerytoacquirethetaskandtoachievenormalperformance(ascoreof7)onthreeconsecutivetrials.Threebaselinetrialsarecollected24hoursbeforesurgery,andthreetestingtrialsarerecordeddailythereafter.Meanvaluesoflatencyandscoreforeachdaywerecomputed.(4) bilateralforepawgrasp,whichmeasurestheabilitytoholdontoa2-mm-diametersteelrod,graded0foraratwithnormalforepawgraspingbehaviorto3foraratunabletograspwiththefbrepaws.Thescoresfromall4tests,whichweredoneoveraperiodofabout15minutesoneachassessmentday,wereaddedtogiveamotordeficitscore(maximumpossiblescore,12).Skilledforelimbfunction(I)StaircasefeedingapparatusThisrequiredpretrainingbeforeinductionofICH.Ratshadfreeaccesstofoodandwaterduringfirst2daysafterarrivalfromthesupplier.Theratswerehousedinpairsinstandardplasticboxeswitha12-hourday/nightcycle.Duringthefollowing7days,theratsweref