疼痛基础研究方法ppt课件

疼痛的动物模型与研究方法AnimalModelsandMethodsInPainResearch,1,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,2,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,3,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,4,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,5,Animalmodelsofpain,Acutestimulus-evokedpainThetail-flicktestThehot-platetestTheformalintestThepawflicktestImmersiontestforthermalhypersensitivityCold-allodyniatestThepin-pricktestformechano-hyperalgesiavonfreyHairtestformechano-allodyniaThewrithingtestTheDistensionofahollowviscusMusclepain,6,Animalmodelsofpain,ModelsofchronicinflammatorypainAdjuvant-inducedarthritisUnilateralarthritisInflammationofahollowviscusUreteralcalculosis,7,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,8,TheAbdominalConstriction(Writhing)Test,tonicinflammatorypainspinallymediatedvisceral/subcutaneous,9,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,10,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(0.210%)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,11,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：1560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,12,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).,13,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,14,Anklejoint:intra-articularinjectionofCFA,Week1:acuteperiodWeek2-3:subacuteperiodWeek4-9:chronicperiod,ChronicInflammatoryPainModel-Monoarthritis,15,16,Animalmodelsofpain,NeuropathicpainmodelsExperimentalanesthesiadolorosaExperimentalmodelsofpainfulperipheralneuropathyduetotraumatic,partialnervedamageChronicconstrictioninjuryPartialnervetransectioninjurySpinalnervetransectioninjuryExperimentalmodelsofpainfuldiabeticneuropathyChemotherapy-evokedpainfulperipheralneuropathy,17,Neuropathicpainfromnerveinflammation,Eliavandhiscolleagueshavedevelopedanenexperimentalmodelofaneuritis.TherataciaticnerveisexposedandlooselywrappedwithoxidizedcellulosethatissaturatedwithCFA.Within24and48htheanimalsdevelopheat-hyperalgesia,mechano-hyperalgesia,mechano-allodynia,and(toalesserdegree)cold-evokedpainslastuntil5to6daysaftertreatment,afterwhichresponsesallreturntonormal.(Eliav,E.etal.Neuropathicpainfromanexperimentalneuritisoftheratsciaticnerve.Pain1999;83:169),18,19,20,Allodyniainratsinfectedwithvaricellazostervirusasmallanimalmodelforpost-herpeticneuralgia,FollowingVZVinfectionoftheleftfootpadratsdevelopachronicmechanicalallodynia,whichispresentforlongerthan60dayspost-infectionandwhichresolvesby100dayspost-infection.Themodelisrobustandreproduciblewithanimalsconsistentlydevelopingallodyniaby3dayspost-infectionandcontinuingtopresentwithsymptomsforatleast30days.Thereproduciblenatureoftheinductionandcourseoftheallodyniaallowstheuseofthismodeltodeterminetheeffectofvariouscompoundson,andtoinvestigatethepathogenicmechanismsunderlyingthedevelopmentofVZV-inducedallodynia.ComparativestudiesusingHSV-1showthattheinductionofthechronicallodyniaisVZV-specificandisnotaresultisofvirusreplication-inducedtissuedamageoraccompanyinginflammation.,21,Fig.1.DurationofVZV-inducedallodynia,22,Fig.2.Reproducibilityofthemodel,ThemeanwithdrawalthresholdsobservedinfourindividualVZVstudies(n=24)arepresentedindividually(,).Thedatafromthecontrols(n=24)fromthesefourstudieswerepooledandareplottedasasingleline().,23,Fig.3.Specificityofthemodel,Animals(n=20)wereinfectedwith107pfuofHSV-1in50lPBS.Controlanimals(n=6)receivedheat-inactivatedHSV-1.AllodyniawasassessedusinganelectronicvonFreyhairdailyuptoday6post-infection.Onegroup(n=10)ofinfectedanimalswastreatedwithvalaciclovir(50mg/kgtwicedailybyoralgavage)fromday0today6post-infection.Themeanwithdrawalthresholdsmeasuredingramsforweredeterminedipsilateralpawsandplottedagainsttimepost-infectionindaysforeachgroupandSEMshown.HSV-1(),HSVplusvalaciclovir(),control().(B)Animalswereinjectedinthelefthindpawonday0witheither48106VZV-infectedCV-1cells(VZV,n=12)oruninfectedCV-1cells(control,n=6).Onegroup(n=6)ofinfectedanimalsweretreatedwithvalaciclovir(50mg/kgtwicedailybyoralgavage)fromday0today10post-infection.Themeanwithdrawalthresholdsmeasuredingramsweredeterminedforipsilateralpawsandplottedagainsttimepost-infectionindaysforeachgroupandSEMshown.VZV(),VZVplusvalaciclovir(),control().Thelineabovethegraphsindicatesthedurationofadministrationofvalaciclovir.,24,Animalmodelsofpain,VisceralpainmodelsColonic-rectaldistension(CRD)SmallboweldistensionArtificialkidneystonesUrinarybladderdistensionUrinarybladderirritantsIschemicstimuli(coronaryarteryocclusion),25,Chemotherapy-evokedpainfulperipheralneuropathy(1),Painfulperipheralneuropathyisacommon,althoughseldomacknowledged,sideeffectofcancerchemotherapy.Chemotherapy-evokedneuropathicpainhasbeenmadeusingvincristineandpaclitaxel.Theuseofdosethatareconsiderablylowerthanthoseusedpreviously.Aleyetalinjectedvincristine5daysperweekfor2weeks.Theyfoundthatdosesof50and75g/kgproducedasignificantmechano-hyperalgesiabeginningaroundthetimeofthelastinjectiononday10andcontinuingforatleast12daysafterdosingceased.Bothdosesproducedasignificantlyincreasedthresholdtoheat-evokedpain.(AleyKO,etal.Vincristinehyperalgesiaintherat:amodelofpainfulcincristineneuropathyinhumans,Neuroscience1996;73:259),26,Chemotherapy-evokedpainfulperipheralneuropathy(2),Polomanoetaldescribedapaclitaxel-evokedpainfulperipheralneuropathyintheratthatisnotassociatedwithanyevidenceofinjurytosensoryormotoraxonsandthatisnotaccompaniedbysignificanteffectsontheanimalsgeneralhealth.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),27,Colonic-RectalDistension,Inrats,aflexiblelatexballoonfixedtoapliablecatheterispalcedintothedescendingcolonand/orrectumtransanally,securingthecathetertothetailwithtape.Briefly,eitheralatexcondomorafingerfromalatexglovemaybeusedastheballoon.ThecatheterinratsisTygonflexibletubing.Fora7to8-cmlongballoon,6cmofoneendoftheflexibletubingisrepeatedlyperforatedwitha#35holepunch(20to25holes),insertedintheballoon,andtiedtightlywithsilksuture.(GebhartGF,etal.evaluationofvisceralpain,inMethodsinGastrointestinalpharmacology,Gaginella,TSEd,CRCPress,BocaRatom1996,359),28,Animalmodelsofpain,Modelsofcancerpain大鼠胫骨乳腺癌痛模型小鼠足底癌痛模型,29,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,30,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:0.1MPBS20ul,Reference:SasamuraTetal.EurJPharmacol,2002,31,小鼠脚掌肿瘤生长情况,32,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”,33,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,34,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,35,轴轴反射末梢释放SP+EAA,Primaryhyperalgesia原发性痛敏,Secondaryhyperalgesia继发性痛敏,Allodynia痛觉超敏(触痛),36,PhilosophyofMeasuringPain,Thehumansubjectcanreporthissensationstous.Hedoessowithanact,somesortofbehavior-thespokenword,apencilmarkonaruledline,etc.Whatthenofmeasuringsensationinananimal?Theoptometristsprocedureisbasedontheimplicitassumptionthatmyprivatesubjectiveexperience(a“sharper”image)isthesameaswhathewouldexperienceunderthesamecircumstances.,37,PhilosophyofMeasuringPain,Weassumethatotherpeopleseelikeusbecausetheylooklikeus.Ratsdonotlooklikeus.CanwemaketheassumptionthataratsprivateandsubjectiveexperienceisIikeours?Initsbroadestsense,thequestionisdifficulttoansweranddependsonexactlywhatkindofexperiencewearediscussing.,38,PhilosophyofMeasuringPain,Wefindthattheaverageratheat-painthresholdisabout45C.Itisalsotrueforahumanbeing.Thethresholdfordenaturationofmanyproteinsis45CUndernormalcircumstances,thesensationofpainistightlyrelatedtotissuedamage.Itisreasonabletoarguethatthisrelationshiphasobviousevolutionaryvalue.Itisalsoanobviouslyprimitiverelationshipthatislikelytobehighlyconservedinman,rat,othermammals,andprobablyinallanimalswithanervoussystem.Thereispharmacologicalevidencethatarguesforthesimilaritybetweenpaininmanandothermammals:therankorderofthepotencyofopioidsisthesameasinhumanbeingsandrats.,39,Measuringpaininanimals,AcuteandchronicpainThedistinctionisarbitrary“acute”referstopainthatlastsforsecondstoaboutaday“chronic”referstopainthatlastsforatleastseveraldays.Intheory,oncouldproduceanysortofinjurytoanybodypartintheanialanddeclarethatonehadapainmodelButpainfromdifferentcausesandfromdifferenttissuesmaybedissimilarinimportantways.Abdominalpainmaybeuniquelymodulatedbydrugsthatblockaopioid-likereceptors.,40,MethodsinPainResearch,Behavioral:hot(cold)plate,vonFreyhair,painscorePharmacological:antagonist,radioligandbindingassayPsy