疼痛基础研究方法

疼痛的动物模型与研究方法

AnimalModelsandMethods

InPainResearch常用的动物模型神经病理性痛模型神经损伤：神经瘤、慢性压迫性损伤、部分神经损伤、背根节慢性压迫、低温神经损伤中枢神经痛模型炎症痛模型癌症痛模型甩尾反射模型热辐射或热水甩尾机械刺激甩尾热(冷)板反应模型内脏痛模型化学诱导的躯体扭动模型膨胀结肠模型常用的动物模型外周炎性痛模型皮肤炎性痛模型：Formalintest,BeeVenom致炎剂模型：白陶土-鹿角菜胶炎症模型紫外线致炎扭体模型关节炎模型单关节炎模型多关节炎模型实验型肌炎模型手术创伤模型常用的动物模型炎症痛模型外周炎性痛模型皮肤炎性痛模型：Formalintest,BeeVenom致炎剂模型：角叉菜胶模型紫外线致炎关节炎模型单关节炎模型多关节炎模型实验型肌炎模型常用的动物模型神经病理性痛模型神经损伤：神经瘤、慢性压迫性损伤、部分神经损伤、背根节慢性压迫、低温神经损伤中枢神经痛模型内脏痛模型化学诱导的躯体扭动模型膨胀结肠模型癌症痛模型大鼠胫骨乳腺癌痛模型AnimalmodelsofpainAcutestimulus-evokedpainThetail-flicktestThehot-platetestTheformalintestThepawflicktestImmersiontestforthermalhypersensitivityCold-allodyniatestThepin-pricktestformechano-hyperalgesiavonfreyHairtestformechano-allodyniaThewrithingtestTheDistensionofahollowviscusMusclepainAnimalmodelsofpainModelsofchronicinflammatorypainAdjuvant-inducedarthritisUnilateralarthritisInflammationofahollowviscusUreteralcalculosis扭体模型可采用小鼠或大鼠有多种刺激物都可诱发动物扭体(writhing)行为最常见的刺激物是醋酸(aceticacid)。将1克阿拉伯胶(arabicgum)加入9ml浓度为1%的醋酸溶液中，再注入实验动物体内，观察注射后90分钟期间每15分钟内出现典型扭体症状的次数该模型可以模拟腹腔炎症引起的腹痛症状TheAbdominalConstriction(Writhing)Test

tonicinflammatorypainspinallymediatedvisceral/subcutaneous0.9%AceticAcid(10ml/kg;intraperitoneal)白陶土-鹿角菜胶炎症模型白陶土(Kaolin)是一种细颗粒状物质，成分为氧化铝，起机械刺激作用；鹿角菜胶(carrageenan)是由水生植物鹿角菜中提取的胶体物质，具有过敏刺激作用。鹿角菜胶单独实验即可诱发炎症，若与白陶土合并使用，则炎症更为强烈可采用家兔或大鼠麻醉动物，由一侧后肢足底注入4%白陶土混悬液0.1ml，并按摩5分钟使之在组织中分散。在注射后1小时，再注入2%鹿角菜胶溶液0.05ml并按摩5分钟。炎症过程一般在第一次注射后2小时内开始。动物后足红肿，皮温升高，PWT值降低等类似痛敏的症状一般能持续12小时以上，24小时后基本复原。因而本模型属于亚急性炎症痛模型范围本模型亦可采用关节腔注射福尔马林致痛模型模拟组织急性炎症损伤所致的持续性疼痛大鼠或小鼠足底福尔马林致痛模型：在动物一肢足底皮下注射稀释的福尔马林(formalin)溶液，动物的行为改变，如安静时的屈腿、运动时的跛行以及舔足等。这些行为的程度(如舔足时间)与福尔马林浓度成正比面部福尔马林致痛模型：把不同浓度的福尔马林溶液(0.2~10%)皮下注射到大鼠的右上唇，记录注射后每3分钟时间内动物用同侧前肢或后肢摩擦注射部位的秒数作为痛分数福尔马林致痛模型各种症状普遍分为两个时相：急性相或第一相：前5分钟。之后有5-10分钟的间歇持续相或第二相：15~60分钟两相均可用于实验，但以第二相为常用。两个时相的发生机制并不相同慢性病理性疼痛慢性病理痛炎症性痛(inflammatorypain)神经病理性痛(neuropathicpain)癌症痛(cancerpain)病理性痛时，共同存在：痛觉过敏(hyperalgesia):对伤害性刺激敏感性增强和反应阈值降低；触诱发痛(allodynia):非痛刺激诱发持续性痛和自发痛(ongoingpainorspontaneouspain).炎症痛模型inflammatorypainmodel多发性佐剂关节炎模型含高浓度结核杆菌的福氏佐剂，向大鼠尾根部或足底作皮内注射，一侧或双侧后肢通常首先出现多个关节的炎症单发性佐剂关节周围炎模型完全福氏佐剂注射到动物后肢足底，造成单个关节周围局部组织的炎症反应单发性佐剂关节腔炎模型将高浓度的福氏佐剂直接注射到大鼠后肢踝关节腔中，引起一个具有急性、慢性两相的高度局限的关节炎症福氏佐剂关节炎模型福氏佐剂足底炎症模型Anklejoint:intra-articularinjectionofCFAWeek1:acuteperiodWeek2-3:subacuteperiodWeek4-9:chronicperiodChronicInflammatoryPainModel-Monoarthritis012345690246810###//**********Scoresofextensionpaintest##############////////Time(weeksafterinjectionofCFA)IFA:IncompleteFreund'sAdjuvantCFA:CompleteFreund'sAdjuvantn=10/group*p<0.05,**p<0.01,***p<0.001comparedwithIFAgroup#p<0.05,##p<0.01,###p<0.001comparedwithleftankleChronicInflammatoryPainModel-MonoarthritisIFAleftCFAleftIFArightCFArightAnimalmodelsofpainNeuropathicpainmodelsExperimentalanesthesiadolorosaExperimentalmodelsofpainfulperipheralneuropathyduetotraumatic,partialnervedamageChronicconstrictioninjuryPartialnervetransectioninjurySpinalnervetransectioninjuryExperimentalmodelsofpainfuldiabeticneuropathyChemotherapy-evokedpainfulperipheralneuropathyNeuropathicpainfromnerveinflammationEliavandhiscolleagueshavedevelopedanenexperimentalmodelofaneuritis.TherataciaticnerveisexposedandlooselywrappedwithoxidizedcellulosethatissaturatedwithCFA.Within24and48htheanimalsdevelopheat-hyperalgesia,mechano-hyperalgesia,mechano-allodynia,and(toalesserdegree)cold-evokedpainslastuntil5to6daysaftertreatment,afterwhichresponsesallreturntonormal.(Eliav,E.etal.Neuropathicpainfromanexperimentalneuritisoftheratsciaticnerve.Pain1999;83:169)L2L3L4L5L6L2L3L4L5Chung’sCCISeltzerAllodyniainratsinfectedwithvaricellazostervirus—asmallanimal

modelforpost-herpeticneuralgiaFollowingVZVinfectionoftheleftfootpadratsdevelopachronicmechanicalallodynia,whichispresentforlongerthan60dayspost-infectionandwhichresolvesby100dayspost-infection.Themodelisrobustandreproduciblewithanimalsconsistentlydevelopingallodyniaby3dayspost-infectionandcontinuingtopresentwithsymptomsforatleast30days.Thereproduciblenatureoftheinductionandcourseoftheallodyniaallowstheuseofthismodeltodeterminetheeffectofvariouscompoundson,andtoinvestigatethepathogenicmechanismsunderlyingthedevelopmentofVZV-inducedallodynia.ComparativestudiesusingHSV-1showthattheinductionofthechronicallodyniaisVZV-specificandisnotaresultisofvirusreplication-inducedtissuedamageoraccompanyinginflammation.Fig.1.DurationofVZV-inducedallodyniaFig.2.ReproducibilityofthemodelThemeanwithdrawalthresholdsobservedinfourindividualVZVstudies(n=24)arepresentedindividually(,,,

).Thedatafromthecontrols(n=24)fromthesefourstudieswerepooledandareplottedasasingleline(

).Fig.3.SpecificityofthemodelAnimals(n=20)wereinfectedwith107pfuofHSV-1in50

lPBS.Controlanimals(n=6)receivedheat-inactivatedHSV-1.AllodyniawasassessedusinganelectronicvonFreyhairdailyuptoday6post-infection.Onegroup(n=10)ofinfectedanimalswastreatedwithvalaciclovir(50mg/kgtwicedailybyoralgavage)fromday0today6post-infection.Themeanwithdrawalthresholdsmeasuredingramsforweredeterminedipsilateralpawsandplottedagainsttimepost-infectionindaysforeachgroupandSEMshown.HSV-1(

),HSVplusvalaciclovir(

),control(

).(B)Animalswereinjectedinthelefthindpawonday0witheither4–8

106VZV-infectedCV-1cells(VZV,n=12)oruninfectedCV-1cells(control,n=6).Onegroup(n=6)ofinfectedanimalsweretreatedwithvalaciclovir(50mg/kgtwicedailybyoralgavage)fromday0today10post-infection.Themeanwithdrawalthresholdsmeasuredingramsweredeterminedforipsilateralpawsandplottedagainsttimepost-infectionindaysforeachgroupandSEMshown.VZV(

),VZVplusvalaciclovir(

),control(

).Thelineabovethegraphsindicatesthedurationofadministrationofvalaciclovir.AnimalmodelsofpainVisceralpainmodelsColonic-rectaldistension(CRD)SmallboweldistensionArtificialkidneystonesUrinarybladderdistensionUrinarybladderirritantsIschemicstimuli(coronaryarteryocclusion)Chemotherapy-evokedpainfulperipheralneuropathy(1)Painfulperipheralneuropathyisacommon,althoughseldomacknowledged,sideeffectofcancerchemotherapy.Chemotherapy-evokedneuropathicpainhasbeenmadeusingvincristineandpaclitaxel.Theuseofdosethatareconsiderablylowerthanthoseusedpreviously.Aleyetalinjectedvincristine5daysperweekfor2weeks.Theyfoundthatdosesof50and75g/kgproducedasignificantmechano-hyperalgesiabeginningaroundthetimeofthelastinjectiononday10andcontinuingforatleast12daysafterdosingceased.Bothdosesproducedasignificantlyincreasedthresholdtoheat-evokedpain.(AleyKO,etal.Vincristinehyperalgesiaintherat:amodelofpainfulcincristineneuropathyinhumans,Neuroscience1996;73:259)Chemotherapy-evokedpainfulperipheralneuropathy(2)Polomanoetaldescribedapaclitaxel-evokedpainfulperipheralneuropathyintheratthatisnotassociatedwithanyevidenceofinjurytosensoryormotoraxonsandthatisnotaccompaniedbysignificanteffectsontheanimals’generalhealth.Ratsweretreatedwithpaclitaxelvia4i.p.injectionsgivenonalternatedayswithdosesof0.5,1.0,or2.0mg/kg.Allthreedosesproducedheat-hyperalgesia,mechano-hyperalgesia,mechano-allodynia,andcold-allodynia.Theabnormalpainsensationsbeganwithinseveraldaysoftheinitiationoftreatmentandlastedforatleastseveralweeksafterward.(PolomanoRC,etal.Apainfulperipheralneuropathyintheratproducedbythechemotherapeuticdrug,paclitaxel.Pain2001;94:293-304)Colonic-RectalDistensionInrats,aflexiblelatexballoonfixedtoapliablecatheterispalcedintothedescendingcolonand/orrectumtransanally,securingthecathetertothetailwithtape.Briefly,eitheralatexcondomorafingerfromalatexglovemaybeusedastheballoon.ThecatheterinratsisTygon®flexibletubing.Fora7to8-cmlongballoon,6cmofoneendoftheflexibletubingisrepeatedlyperforatedwitha#35holepunch(20to25holes),insertedintheballoon,andtiedtightlywithsilksuture.(GebhartGF,etal.evaluationofvisceralpain,inMethodsinGastrointestinalpharmacology,Gaginella,TSEd,CRCPress,BocaRatom1996,359)AnimalmodelsofpainModelsofcancerpain大鼠胫骨乳腺癌痛模型小鼠足底癌痛模型癌痛实验进展情况培养肿瘤细胞，建立癌症痛模型行为学指标

痛觉过敏、痛觉超敏、自发性疼痛病理学指标

肿瘤形态大小、肿瘤病理切片、骨病理小鼠脚掌皮肤癌痛模型动物：C57BL6,Male,6weeksoldB16-BL6(黑色素瘤细胞)模型组：右侧脚掌皮下接种：B16-BL6105/20ul

左侧：0.1MPBS20ul对照组：右侧:B16-BL6105/20ul（heatkilled)

左侧:0.1MPBS20ulReference:SasamuraTetal.EurJPharmacol,2002小鼠脚掌肿瘤生长情况疼痛的常见症状人类的“疼痛”与动物的“伤害性感受”常见症状：主要包括ongoingpainandstimulus-evokedpain自发痛(ongoingpain)诱发痛(stimulus-evokedpain)，包括痛觉过敏hyperalgesia和痛觉超敏（触诱发痛allodynia）更为复杂的幻肢痛、镜像痛、动物的自噬等动物模型上研究的策略是，通过观察动物的行为，实验者来推测动物是否发生了“疼痛”慢性痛的常见症状自发痛spontaneouspain持续存在的通感觉痛觉过敏hyperalgesia弱的痛刺激引起强的痛感觉痛觉超敏allodynia，或称触诱发痛非痛刺激引起痛感觉痛敏的种类与机制痛敏的种类(typesofhyperalgesia)痛敏包括痛觉过敏(hyperalgesia)与痛觉超敏(allodynia，也称触痛)原发性(primary)和继发性(secondary)痛敏(hyperalgesia)继发性痛敏：病区周围非炎症区也发生痛敏⊙轴—轴反射末梢释放SP+EAAPrimaryhyperalgesia原发性痛敏Secondaryhyperalgesia继发性痛敏Allodynia痛觉超敏(触痛)PhilosophyofMeasuringPainThehumansubjectcanreporthissensationstous.Hedoessowithanact,somesortofbehavior-thespokenword,apencilmarkonaruledline,etc.Whatthenofmeasuringsensationinananimal?Theoptometrist’sprocedureisbasedontheimplicitassumptionthatmyprivatesubjectiveexperience(a“sharper”image)isthesameaswhathewouldexperienceunderthesamecircumstances.PhilosophyofMeasuringPainWeassumethatotherpeopleseelikeusbecausetheylooklikeus.Ratsdonotlooklikeus.Canwemaketheassumptionthatarat’sprivateandsubjectiveexperienceisIikeours?Initsbroadestsense,thequestionisdifficulttoansweranddependsonexactlywhatkindofexperiencewearediscussing.PhilosophyofMeasuringPainWefindthattheaverageratheat-painthresholdisabout45°C.Itisalsotrueforahumanbeing.Thethresholdfordenaturationofmanyproteinsis45°CUndernormalcircumstances,thesensationofpainistightlyrelatedtotissuedamage.Itisreasonabletoarguethatthisrelationshiphasobviousevolutionaryvalue.Itisalsoanobviouslyprimitiverelationshipthatislikelytobehighlyconservedinman,rat,othermammals,andprobablyinallanimalswithanervoussystem.Thereispharmacologicalevidencethatarguesforthesimilaritybetweenpaininmanandothermammals:therankorderofthepotencyofopioidsisthesameasinhumanbeingsandrats.MeasuringpaininanimalsAcuteandchronicpainThedistinctionisarbitrary“acute”referstopainthatlastsforsecondstoaboutaday“chronic”referstopainthatlastsforatleastseveraldays.Intheory,oncouldproduceanysortofinjurytoanybodypartintheanialanddeclarethatonehadapainmodelButpainfromdifferentcausesandfromdifferenttissuesmaybedissimilarinimportantways.Abdominalpainmaybeuniquelymodulatedbydrugsthatblockaopioid-likereceptors.MethodsinPainResearchBehavioral:hot(cold)plate,vonFreyhair,painscorePharmacological:antagonist,radioligandbindingassayPsychologicalNeurochemical:neurotransmittercontentmeasurementwithhighperformanceliquidchromatography(HPLC)Cellular,molecular,andgeneticMorphological:Histochemical,immunohistochemical,fluorescentElectrophysiological:Extracellular,multi-channelrecordingpatchclampEvokedpotentialNon-invasive:PET,fMRICombinationofmethodsatdifferentlevels,integrationofabovemethods单通道电流的记录Neher&Sakmann(1976,1981)微玻管去神经肌膜1991获诺贝尔奖EricR.Kandel,etal.PrinciplesofNeuroscience4thedition.Fig.11-8.PatchClamp技术影响伤害性感受测量的因素动物种类、品系、性别的选择伤害性敏感度的昼夜变化身体不同部位的伤害性感受阈值得差异刺激区域的大小和连续刺激的间隔对阈值和反应的影响皮肤基础温度对伤害性热刺激阈值得影响Strainandsexdifferencesinbasalthresholdinmice测量疼痛的两类方法第一类：测量产生伤害性反应所需的刺激的阈值。即设定一个标准反应，当发生了伤害性反应时，测定刺激的强度和时程。第二类：测量产生伤害性反应所需的刺激强度和时程。即刺激是标准化的。与第一类不同，它测定的不是阈值，而是反应的大小。常用的痛刺激方法热刺激冷刺激机械刺激化学刺激电刺激缺血实验动物的疼痛评价方法较理想的行为学评价方法应该具备能区分动物对伤害性和非伤害性刺激的不同反应痛刺激引起的行为反应随刺激强度从痛阈到耐痛阈间出现相应改变测得的行为改变可以反映动物的痛感受动物的行为反应对镇痛药物的处理敏感能将非感觉性变化，如注意力、活动能力等与感觉性变化区分开反复刺激不引起或只引起极小的组织损伤实验动物的疼痛评价方法简单的反射行为甩尾实验(tailflicktest)钾离子测痛法(Potassiumiontophoreticdolorimetry)缺血实验：尾部束缚缺血后，动物摇头、前肢回缩非训练学会的组合行为热板测痛法(hotplatetest)冷板测痛法(coldplatetest)翻滚实验(wrotjomgresponse)发声反应(colcalization)训练学会的或自发反应逃跑或躲避反应(escapeandavoidancebehaviors)动机性选择(motivationalchoiceparadign)动物疼痛的行为学研究方法外周神经损伤后的机械痛敏OXOXOXCold-inducedOngoingPain

AfterPeripheralNerveInjury5

C辐射热甩尾测定痛阈（电针）012345690246810###//**********Scoresofextensionpaintest##############////////Time(weeksafterinjectionofCFA)IFA:IncompleteFreund'sAdjuvantCFA:CompleteFreund'sAdjuvantn=10/group*p<0.05,**p<0.01,***p<0.001comparedwithIFAgroup#p<0.05,##p<0.01,###p<0.001comparedwithleftankleChronicInflammatoryPainModel-MonoarthritisIFAleftCFAleftIFArightCFArightTheAbdominalConstriction(Writhing)Test

tonicinflammatorypainspinallymediatedvisceral/subcutaneous0.9%AceticAcid(10ml/kg;intraperitoneal)疼痛的研究方法疼痛研究是现代神经科学研究的一部分从传统的行为学、药理学、临床观察，到电生理学、神经化学，以及到现代的细胞学、组织学、分子生物学、影像学、蛋白质组的方法临床研究遵循随机、对照、多中心，以及志愿、双盲等基本原则基础研究的多学科方法行为学药理学（包括脑内核团立体定位注射、蛛网膜下腔注射等）生理学（包括电生理学）细胞学解剖学（如神经示踪）与组织学（包括一般组织学与免疫组织化学）生物化学和分子生物学基因组学和蛋白质组学……脊髓丘脑束神经元中枢敏化痛敏，通觉超敏（allodynia)递质释放配体/受体反应跨膜信号转导基因转录因子基因激活SPEAANK1RNMDARNO/CGMPNO/PKGPKCPKAMARPKCREBFOS通道、受体基因表达痛敏伤害性刺激短时程中时程长时程Vanilloidreceptortype1(VR1)Distribution

inDRGinNormalRatsIB4VR1mergedScalebar=40µmChangeofVR1ExpressioninDRGafterCFAInjectionAverageVR1-ir=averagegrayvalue(meandensity)-backgroundn=3,*p<0.05**p<0.01backgroundCorrelationbetweenHyperalgesiaandVR1ProteinLevel

PAINThreedifferenttypicalpatternsofectopicdischargesChangesofectopicdischargesafterSNLCorrelationanalysisbetweentactileallodyniaandectopicactivityinthefirst24hoursafterSNLCorrelationanalysisbetweentactileallodyniaandectopicactivityinthedays1-14afterSNLNeuropathicpain:

EarlyspontaneousafferentactivityisthetriggerAfterCCInerveinjury,bothA-fibers(c)andC-fibers(f)displayedhighlevelsofspontaneousactivityintheCCI-ratsthatwereuntreated(‘naı¨ve’)orperfusedwithsaline,buttreatmentwitheitherbupivacaineorTTXduringthefirst4–7dayspost-injuryinhibitedthiselevationinspontaneousactivity.WenruiXie,JudithA.Strong,JohannaT.A.Meij,Jun-MingZhang,LeiYu.Painxx(2005)1–14AllodyniaOpioids--C.N.S.CentralSensitization+NMDA(Pain)(Touch=Pain)NE5-HTWind-upofaWDRneuronInductionof