疼痛基礎(chǔ)研究方法

疼痛的動物模型與研究方法

AnimalModelsandMethods

InPainResearch常用的動物模型神經(jīng)病理性痛模型神經(jīng)損傷：神經(jīng)瘤、慢性壓迫性損傷、部分神經(jīng)損傷、背根節(jié)慢性壓迫、低溫神經(jīng)損傷中樞神經(jīng)痛模型炎癥痛模型癌癥痛模型甩尾反射模型熱輻射或熱水甩尾機械刺激甩尾熱(冷)板反應(yīng)模型內(nèi)臟痛模型化學(xué)誘導(dǎo)的軀體扭動模型膨脹結(jié)腸模型常用的動物模型外周炎性痛模型皮膚炎性痛模型：Formalintest,BeeVenom致炎劑模型：白陶土-鹿角菜膠炎癥模型紫外線致炎扭體模型關(guān)節(jié)炎模型單關(guān)節(jié)炎模型多關(guān)節(jié)炎模型實驗型肌炎模型手術(shù)創(chuàng)傷模型常用的動物模型炎癥痛模型外周炎性痛模型皮膚炎性痛模型：Formalintest,BeeVenom致炎劑模型：角叉菜膠模型紫外線致炎關(guān)節(jié)炎模型單關(guān)節(jié)炎模型多關(guān)節(jié)炎模型實驗型肌炎模型常用的動物模型神經(jīng)病理性痛模型神經(jīng)損傷：神經(jīng)瘤、慢性壓迫性損傷、部分神經(jīng)損傷、背根節(jié)慢性壓迫、低溫神經(jīng)損傷中樞神經(jīng)痛模型內(nèi)臟痛模型化學(xué)誘導(dǎo)的軀體扭動模型膨脹結(jié)腸模型癌癥痛模型大鼠脛骨乳腺癌痛模型AnimalmodelsofpainAcutestimulus-evokedpainThetail-flicktestThehot-platetestTheformalintestThepawflicktestImmersiontestforthermalhypersensitivityCold-allodyniatestThepin-pricktestformechano-hyperalgesiavonfreyHairtestformechano-allodyniaThewrithingtestTheDistensionofahollowviscusMusclepainAnimalmodelsofpainModelsofchronicinflammatorypainAdjuvant-inducedarthritisUnilateralarthritisInflammationofahollowviscusUreteralcalculosis扭體模型可采用小鼠或大鼠有多種刺激物都可誘發(fā)動物扭體(writhing)行為最常見的刺激物是醋酸(aceticacid)。將1克阿拉伯膠(arabicgum)加入9ml濃度為1%的醋酸溶液中，再注入實驗動物體內(nèi)，觀察注射后90分鐘期間每15分鐘內(nèi)出現(xiàn)典型扭體癥狀的次數(shù)該模型可以模擬腹腔炎癥引起的腹痛癥狀TheAbdominalConstriction(Writhing)Test

tonicinflammatorypainspinallymediatedvisceral/subcutaneous0.9%AceticAcid(10ml/kg;intraperitoneal)白陶土-鹿角菜膠炎癥模型白陶土(Kaolin)是一種細顆粒狀物質(zhì)，成分為氧化鋁，起機械刺激作用；鹿角菜膠(carrageenan)是由水生植物鹿角菜中提取的膠體物質(zhì)，具有過敏刺激作用。鹿角菜膠單獨實驗即可誘發(fā)炎癥，若與白陶土合并使用，則炎癥更為強烈可采用家兔或大鼠麻醉動物，由一側(cè)后肢足底注入4%白陶土混懸液0.1ml，并按摩5分鐘使之在組織中分散。在注射后1小時，再注入2%鹿角菜膠溶液0.05ml并按摩5分鐘。炎癥過程一般在第一次注射后2小時內(nèi)開始。動物后足紅腫，皮溫升高，PWT值降低等類似痛敏的癥狀一般能持續(xù)12小時以上，24小時后基本復(fù)原。因而本模型屬于亞急性炎癥痛模型范圍本模型亦可采用關(guān)節(jié)腔注射福爾馬林致痛模型模擬組織急性炎癥損傷所致的持續(xù)性疼痛大鼠或小鼠足底福爾馬林致痛模型：在動物一肢足底皮下注射稀釋的福爾馬林(formalin)溶液，動物的行為改變，如安靜時的屈腿、運動時的跛行以及舔足等。這些行為的程度(如舔足時間)與福爾馬林濃度成正比面部福爾馬林致痛模型：把不同濃度的福爾馬林溶液(0.2~10%)皮下注射到大鼠的右上唇，記錄注射后每3分鐘時間內(nèi)動物用同側(cè)前肢或后肢摩擦注射部位的秒數(shù)作為痛分數(shù)福爾馬林致痛模型各種癥狀普遍分為兩個時相：急性相或第一相：前5分鐘。之后有5-10分鐘的間歇持續(xù)相或第二相：15~60分鐘兩相均可用于實驗，但以第二相為常用。兩個時相的發(fā)生機制并不相同慢性病理性疼痛慢性病理痛炎癥性痛(inflammatorypain)神經(jīng)病理性痛(neuropathicpain)癌癥痛(cancerpain)病理性痛時，共同存在：痛覺過敏(hyperalgesia):對傷害性刺激敏感性增強和反應(yīng)閾值降低；觸誘發(fā)痛(allodynia):非痛刺激誘發(fā)持續(xù)性痛和自發(fā)痛(ongoingpainorspontaneouspain).炎癥痛模型inflammatorypainmodel多發(fā)性佐劑關(guān)節(jié)炎模型含高濃度結(jié)核桿菌的福氏佐劑，向大鼠尾根部或足底作皮內(nèi)注射，一側(cè)或雙側(cè)后肢通常首先出現(xiàn)多個關(guān)節(jié)的炎癥單發(fā)性佐劑關(guān)節(jié)周圍炎模型完全福氏佐劑注射到動物后肢足底，造成單個關(guān)節(jié)周圍局部組織的炎癥反應(yīng)單發(fā)性佐劑關(guān)節(jié)腔炎模型將高濃度的福氏佐劑直接注射到大鼠后肢踝關(guān)節(jié)腔中，引起一個具有急性、慢性兩相的高度局限的關(guān)節(jié)炎癥福氏佐劑關(guān)節(jié)炎模型福氏佐劑足底炎癥模型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大鼠脛骨乳腺癌痛模型小鼠足底癌痛模型癌痛實驗進展情況培養(yǎng)腫瘤細胞，建立癌癥痛模型行為學(xué)指標

痛覺過敏、痛覺超敏、自發(fā)性疼痛病理學(xué)指標

腫瘤形態(tài)大小、腫瘤病理切片、骨病理小鼠腳掌皮膚癌痛模型動物：C57BL6,Male,6weeksoldB16-BL6(黑色素瘤細胞)模型組：右側(cè)腳掌皮下接種：B16-BL6105/20ul

左側(cè)：0.1MPBS20ul對照組：右側(cè):B16-BL6105/20ul（heatkilled)

左側(cè):0.1MPBS20ulReference:SasamuraTetal.EurJPharmacol,2002小鼠腳掌腫瘤生長情況疼痛的常見癥狀人類的“疼痛”與動物的“傷害性感受”常見癥狀：主要包括ongoingpainandstimulus-evokedpain自發(fā)痛(ongoingpain)誘發(fā)痛(stimulus-evokedpain)，包括痛覺過敏hyperalgesia和痛覺超敏（觸誘發(fā)痛allodynia）更為復(fù)雜的幻肢痛、鏡像痛、動物的自噬等動物模型上研究的策略是，通過觀察動物的行為，實驗者來推測動物是否發(fā)生了“疼痛”慢性痛的常見癥狀自發(fā)痛spontaneouspain持續(xù)存在的通感覺痛覺過敏hyperalgesia弱的痛刺激引起強的痛感覺痛覺超敏allodynia，或稱觸誘發(fā)痛非痛刺激引起痛感覺痛敏的種類與機制痛敏的種類(typesofhyperalgesia)痛敏包括痛覺過敏(hyperalgesia)與痛覺超敏(allodynia，也稱觸痛)原發(fā)性(primary)和繼發(fā)性(secondary)痛敏(hyperalgesia)繼發(fā)性痛敏：病區(qū)周圍非炎癥區(qū)也發(fā)生痛敏⊙軸—軸反射末梢釋放SP+EAAPrimaryhyperalgesia原發(fā)性痛敏Secondaryhyperalgesia繼發(fā)性痛敏Allodynia痛覺超敏(觸痛)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單通道電流的記錄Neher&Sakmann(1976,1981)微玻管去神經(jīng)肌膜1991獲諾貝爾獎EricR.Kandel,etal.PrinciplesofNeuroscience4thedition.Fig.11-8.PatchClamp技術(shù)影響傷害性感受測量的因素動物種類、品系、性別的選擇傷害性敏感度的晝夜變化身體不同部位的傷害性感受閾值得差異刺激區(qū)域的大小和連續(xù)刺激的間隔對閾值和反應(yīng)的影響皮膚基礎(chǔ)溫度對傷害性熱刺激閾值得影響Strainandsexdifferencesinbasalthresholdinmice測量疼痛的兩類方法第一類：測量產(chǎn)生傷害性反應(yīng)所需的刺激的閾值。即設(shè)定一個標準反應(yīng)，當(dāng)發(fā)生了傷害性反應(yīng)時，測定刺激的強度和時程。第二類：測量產(chǎn)生傷害性反應(yīng)所需的刺激強度和時程。即刺激是標準化的。與第一類不同，它測定的不是閾值，而是反應(yīng)的大小。常用的痛刺激方法熱刺激冷刺激機械刺激化學(xué)刺激電刺激缺血實驗動物的疼痛評價方法較理想的行為學(xué)評價方法應(yīng)該具備能區(qū)分動物對傷害性和非傷害性刺激的不同反應(yīng)痛刺激引起的行為反應(yīng)隨刺激強度從痛閾到耐痛閾間出現(xiàn)相應(yīng)改變測得的行為改變可以反映動物的痛感受動物的行為反應(yīng)對鎮(zhèn)痛藥物的處理敏感能將非感覺性變化，如注意力、活動能力等與感覺性變化區(qū)分開反復(fù)刺激不引起或只引起極小的組織損傷實驗動物的疼痛評價方法簡單的反射行為甩尾實驗(tailflicktest)鉀離子測痛法(Potassiumiontophoreticdolorimetry)缺血實驗：尾部束縛缺血后，動物搖頭、前肢回縮非訓(xùn)練學(xué)會的組合行為熱板測痛法(hotplatetest)冷板測痛法(coldplatetest)翻滾實驗(wrotjomgresponse)發(fā)聲反應(yīng)(colcalization)訓(xùn)練學(xué)會的或自發(fā)反應(yīng)逃跑或躲避反應(yīng)(escapeandavoidancebehaviors)動機性選擇(motivationalchoiceparadign)動物疼痛的行為學(xué)研究方法外周神經(jīng)損傷后的機械痛敏OXOXOXCold-inducedOngoingPain

AfterPeripheralNerveInjury5

C輻射熱甩尾測定痛閾（電針）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現(xiàn)代神經(jīng)科學(xué)研究的一部分從傳統(tǒng)的行為學(xué)、藥理學(xué)、臨床觀察，到電生理學(xué)、神經(jīng)化學(xué)，以及到現(xiàn)代的細胞學(xué)、組織學(xué)、分子生物學(xué)、影像學(xué)、蛋白質(zhì)組的方法臨床研究遵循隨機、對照、多中心，以及志愿、雙盲等基本原則基礎(chǔ)研究的多學(xué)科方法行為學(xué)藥理學(xué)（包括腦內(nèi)核團立體定位注射、蛛網(wǎng)膜下腔注射等）生理學(xué)（包括電生理學(xué)）細胞學(xué)解剖學(xué)（如神經(jīng)示蹤）與組織學(xué)（包括一般組織學(xué)與免疫組織化學(xué)）生物化學(xué)和分子生物學(xué)基因組學(xué)和蛋白質(zhì)組學(xué)……脊髓丘腦束神經(jīng)元中樞敏化痛敏，通覺超敏（allodynia)遞質(zhì)釋放配體/受體反應(yīng)跨膜信號轉(zhuǎn)導(dǎo)基因轉(zhuǎn)錄因子基因激活SPEAANK1RNMDARNO/CGMPNO/PKGPKCPKAMARPKCREBFOS通道、受體基因表達痛敏傷害性刺激短時程中時程長時程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(‘na?¨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