疼痛基础研究方法

1、万有北京大学基础医学院神经生物学系1/16/2022Neuroscience Research Institute, Peking University1常用的动物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1/16/2022Neuroscience Research Institute, Peking University2常用的动物模型常用的动物模型外周炎性痛模型皮肤炎性痛模型：Formal

2、in test, Bee Venom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1/16/2022Neuroscience Research Institute, Peking University3常用的动物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：Formalin test, Bee Venom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1/16/2022Neuroscience Research Institute, Peking University4常用的动物模

3、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1/16/2022Neuroscience Research Institute, Peking University5Animal models of painAcute stimulus-evoked painThe tail-flick testThe hot-plate testThe formalin testThe paw flick testImmersion test for ther

4、mal hypersensitivityCold-allodynia testThe pin-prick test for mechano-hyperalgesiavon frey Hair test for mechano-allodyniaThe writhing testThe Distension of a hollow viscusMuscle pain1/16/2022Neuroscience Research Institute, Peking University6Animal models of painModels of chronic inflammatory painA

5、djuvant-induced arthritisUnilateral arthritisInflammation of a hollow viscusUreteral calculosis1/16/2022Neuroscience Research Institute, Peking University7扭体模型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(acetic acid)。将1克阿拉伯胶(arabic gum)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

6、模拟腹腔炎症引起的腹痛症状1/16/2022Neuroscience Research Institute, Peking University8The Abdominal Constriction (Writhing) Test tonic inflammatory pain spinally mediated visceral/subcutaneous0.9% Acetic Acid(10 ml/kg; intraperitoneal)1/16/2022Neuroscience Research Institute, Peking University9白陶土白陶土-鹿角菜胶炎症模型鹿角菜

7、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1/1

8、6/2022Neuroscience Research Institute, Peking University10福尔马林致痛模型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 1/16/2022Neuroscience Researc

9、h Institute, Peking University11福尔马林致痛模型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1/16/2022Neuroscience Research Institute, Peking University12慢性病理性疼痛慢性病理性疼痛慢性病理痛炎症性痛(inflammatory pain)神经病理性痛(neuropathic pain)癌症痛(cancer pain)病理性痛时，共同存在：痛觉过敏(hyperalges

10、ia): 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(ongoing pain or spontaneous pain).1/16/2022Neuroscience Research Institute, Peking University13炎症痛模型炎症痛模型 inflammatory pain model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

11、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1/16/2022Neuroscience Research Institute, Peking University14Ankle joint:intra-articular injection of CFAWeek 1: acute periodWeek 2-3: subacute periodWeek 4-9: chronic periodChronic Inflammatory Pain Model - Monoarthritis1/16/2022Ne

12、uroscience Research Institute, Peking University15012345690246810#/*Scores of extension pain test#/Time (weeks after injection of CFA)IFA: Incomplete Freunds AdjuvantCFA: Complete Freunds Adjuvantn=10 / group*p0.05, *p0.01, *p0.001 compared with IFA group#p0.05, #p0.01, #p0.001 compared with left an

13、kleChronic Inflammatory Pain Model-MonoarthritisIFA leftCFA left IFA rightCFA right1/16/2022Neuroscience Research Institute, Peking University16Animal models of painNeuropathic pain modelsExperimental anesthesia dolorosaExperimental models of painful peripheral neuropathy due to traumatic, partial n

14、erve damageChronic constriction injuryPartial nerve transection injurySpinal nerve transection injuryExperimental models of painful diabetic neuropathyChemotherapy-evoked painful peripheral neuropathy1/16/2022Neuroscience Research Institute, Peking University17Neuropathic pain from nerve inflammatio

15、nEliav and his colleagues have developed an en experimental model of a neuritis. The rat aciatic nerve is exposed and loosely wrapped with oxidized cellulose that is saturated with CFA. Within 24 and 48 h the animals develop heat-hyperalgesia, mechano-hyperalgesia, mechano-allodynia, and (to a lesse

16、r degree) cold-evoked pains last until 5 to 6 days after treatment, after which responses all return to normal. (Eliav, E. et al. Neuropathic pain from an experimental neuritis of the rat sciatic nerve. Pain 1999; 83:169)1/16/2022Neuroscience Research Institute, Peking University18L2L3L4L5L6L2L3L4L5

17、ChungsCCISeltzer1/16/2022Neuroscience Research Institute, Peking University191/16/2022Neuroscience Research Institute, Peking University20Allodynia in rats infected with varicella zoster virusa small animalmodel for post-herpetic neuralgiaFollowing VZV infection of the left footpad rats develop a ch

18、ronic mechanical allodynia, which is present for longer than 60 days post-infection and which resolves by 100 days post-infection. The model is robust and reproducible with animals consistently developing allodynia by 3 days post-infection and continuing to present with symptoms for at least 30 days

19、. The reproducible nature of the induction and course of the allodynia allows the use of this model to determine the effect of various compounds on, and to investigate the pathogenic mechanisms underlying the development of VZV-induced allodynia. Comparative studies using HSV-1 show that the inducti

20、on of the chronic allodynia is VZV-specific and is not a result is of virus replication-induced tissue damage or accompanying inflammation.1/16/2022Neuroscience Research Institute, Peking University21Fig. 1. Duration of VZV-induced allodynia1/16/2022Neuroscience Research Institute, Peking University

21、22Fig. 2. Reproducibility of the modelThe mean withdrawal thresholds observed in four individual VZV studies (n=24) are presented individually (, ,). The data from the controls (n=24) from these four studieswere pooled and are plotted as a single line ().1/16/2022Neuroscience Research Institute, Pek

22、ing University23Fig. 3. Specificity of the modelAnimals (n=20) were infected with 107 pfu of HSV-1 in 50 l PBS. Control animals (n=6) received heat-inactivated HSV-1. Allodynia was assessed using an electronic von Frey hair daily up to day 6 post-infection. One group (n=10) of infected animals was t

23、reated with valaciclovir (50 mg/kg twice daily by oral gavage) from day 0 to day 6 post-infection. The mean withdrawal thresholds measured in grams for were determined ipsilateral paws and plotted against time post-infection in days for each group and SEM shown. HSV-1 (), HSV plus valaciclovir (), c

24、ontrol (). (B) Animals were injected in the left hindpaw on day 0 with either 48106 VZV-infected CV-1 cells (VZV, n=12) or uninfected CV-1 cells (control, n=6). One group (n=6) of infected animals were treated with valaciclovir (50 mg/kg twice daily by oral gavage) from day 0 to day 10 post-infectio

25、n. The mean withdrawal thresholds measured in grams were determined for ipsilateral paws and plotted against time post-infection in days for each group and SEM shown. VZV (), VZV plus valaciclovir (), control (). The line above the graphs indicates the duration of administration of valaciclovir.1/16

26、/2022Neuroscience Research Institute, Peking University24Animal models of painVisceral pain modelsColonic-rectal distension (CRD)Small bowel distensionArtificial kidney stonesUrinary bladder distensionUrinary bladder irritantsIschemic stimuli (coronary artery occlusion)1/16/2022Neuroscience Research

27、 Institute, Peking University25Chemotherapy-evoked painful peripheral neuropathy (1)Painful peripheral neuropathy is a common, although seldom acknowledged, side effect of cancer chemotherapy.Chemotherapy-evoked neuropathic pain has been made using vincristine and paclitaxel. The use of dose that ar

28、e considerably lower than those used previously. Aley et al injected vincristine 5 days per week for 2 weeks. They found that doses of 50 and 75 g/kg produced a significant mechano-hyperalgesia beginning around the time of the last injection on day 10 and continuing for at least 12 days after dosing

29、 ceased. Both doses produced a significantly increased threshold to heat-evoked pain. (Aley KO, et al. Vincristine hyperalgesia in the rat: a model of painful cincristine neuropathy in humans, Neuroscience 1996; 73: 259)1/16/2022Neuroscience Research Institute, Peking University26Chemotherapy-evoked

30、 painful peripheral neuropathy (2)Polomano et al described a paclitaxel-evoked painful peripheral neuropathy in the rat that is not associated with any evidence of injury to sensory or motor axons and that is not accompanied by significant effects on the animals general health. Rats were treated wit

31、h paclitaxel via 4 i.p. injections given on alternate days with doses of 0.5, 1.0, or 2.0 mg/kg. All three doses produced heat-hyperalgesia, mechano-hyperalgesia, mechano-allodynia, and cold-allodynia. The abnormal pain sensations began within several days of the initiation of treatment and lasted f

32、or at least several weeks afterward. (Polomano RC, et al. A painful peripheral neuropathy in the rat produced by the chemotherapeutic drug, paclitaxel. Pain 2001; 94: 293-304)1/16/2022Neuroscience Research Institute, Peking University27Colonic-Rectal DistensionIn rats, a flexible latex balloon fixed

33、 to a pliable catheter is palced into the descending colon and/or rectum transanally, securing the catheter to the tail with tape. Briefly, either a latex condom or a finger from a latex glove may be used as the balloon. The catheter in rats is Tygon flexible tubing . For a 7 to 8-cm long balloon, 6

34、 cm of one end of the flexible tubing is repeatedly perforated with a #35 hole punch (20 to 25 holes), inserted in the balloon, and tied tightly with silk suture. (Gebhart GF, et al. evaluation of visceral pain, in Methods in Gastrointestinal pharmacology, Gaginella, TS Ed, CRC Press, Boca Ratom 199

35、6, 359)1/16/2022Neuroscience Research Institute, Peking University28Animal models of painModels of cancer pain大鼠胫骨乳腺癌痛模型小鼠足底癌痛模型1/16/2022Neuroscience Research Institute, Peking University29癌痛实验进展情况癌痛实验进展情况 培养肿瘤细胞，建立癌症痛模型培养肿瘤细胞，建立癌症痛模型?行为学指标行为学指标 痛觉过敏、痛觉超敏、自发性疼痛?病理学指标病理学指标 肿瘤形态大小、肿瘤病理切片、骨病理1/16/2022N

36、euroscience Research Institute, Peking University30小鼠脚掌皮肤癌痛模型小鼠脚掌皮肤癌痛模型动物：C57BL6, Male, 6 weeks oldB16-BL6 (黑色素瘤细胞)模型组：右侧脚掌皮下接种：B16-BL6 105/20ul 左侧： 0.1M PBS 20ul对照组：右侧: B16-BL6 105/20ul（heat killed) 左侧: 0.1M PBS 20ulReference: Sasamura T et al. Eur J Pharmacol, 20021/16/2022Neuroscience Research In

37、stitute, Peking University31小鼠脚掌肿瘤生长情况小鼠脚掌肿瘤生长情况1/16/2022Neuroscience Research Institute, Peking University32疼痛的常见症状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ongoing pain and stimulus-evoked pain自发痛(ongoing pain )诱发痛(stimulus-evoked pain)，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，

38、通过观察动物的行为，实验者来推测动物是否发生了“疼痛”1/16/2022Neuroscience Research Institute, Peking University33慢性痛的常见症状慢性痛的常见症状自发痛spontaneous pain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1/16/2022Neuroscience Research Institute, Peking University34痛敏的种类与机制痛敏的种类与机制痛敏的种类(types of hyperalgesia)痛敏包括痛觉过

39、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1/16/2022Neuroscience Research Institute, Peking University35轴轴反射末梢释放 SP+EAAPrimary hyperalgesia原发性痛敏Secondary hyperalgesia继发性痛敏Allodynia痛觉超敏 (触痛)1/16/2022Neuroscience Research Institute, Peking Univers

40、ity36Philosophy of Measuring PainThe human subject can report his sensations to us. He does so with an act, some sort of behavior- the spoken word, a pencil mark on a ruled line, etc.What then of measuring sensation in an animal? The optometrists procedure is based on the implicit assumption that my

41、 private subjective experience (a “sharper” image) is the same as what he would experience under the same circumstances. 1/16/2022Neuroscience Research Institute, Peking University37Philosophy of Measuring PainWe assume that other people see like us because they look like us. Rats do not look like u

42、s. Can we make the assumption that a rats private and subjective experience is Iike ours? In its broadest sense, the question is difficult to answer and depends on exactly what kind of experience we are discussing.1/16/2022Neuroscience Research Institute, Peking University38Philosophy of Measuring P

43、ainWe find that the average rat heat-pain threshold is about 45C. It is also true for a human being.The threshold for denaturation of many proteins is 45CUnder normal circumstances, the sensation of pain is tightly related to tissue damage. It is reasonable to argue that this relationship has obviou

44、s evolutionary value. It is also an obviously primitive relationship that is likely to be highly conserved in man, rat, other mammals, and probably in all animals with a nervous system.There is pharmacological evidence that argues for the similarity between pain in man and other mammals: the rank or

45、der of the potency of opioids is the same as in human beings and rats.1/16/2022Neuroscience Research Institute, Peking University39Measuring pain in animalsAcute and chronic painThe distinction is arbitrary“acute” refers to pain that lasts for seconds to about a day“chronic” refers to pain that last

46、s for at least several days.In theory, on could produce any sort of injury to any body part in the anial and declare that one had a pain modelBut pain from different causes and from different tissues may be dissimilar in important ways.Abdominal pain may be uniquely modulated by drugs that block a o

47、pioid-like receptors.1/16/2022Neuroscience Research Institute, Peking University40Methods in Pain ResearchBehavioral: hot (cold) plate, von Frey hair, pain scorePharmacological: antagonist, radio ligand binding assayPsychologicalNeurochemical: neurotransmitter content measurement with high performan

48、ce liquid chromatography (HPLC)Cellular, molecular, and geneticMorphological: Histochemical, immunohistochemical, fluorescentElectrophysiological:Extracellular, multi-channel recordingpatch clampEvoked potentialNon-invasive: PET, fMRICombination of methods at different levels, integration of above m

49、ethods1/16/2022Neuroscience Research Institute, Peking University41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Eric R. Kandel, et al. Principles of Neuroscience4th edition. Fig. 11-8.Patch Clamp 技术1/16/2022Neuroscience Research Institute, Peking University42影响伤害性感受测量的因素影响伤害性感受测量的因素

50、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1/16/2022Neuroscience Research Institute, Peking University43Differences of basal thresholds amonginbred strains of mice129AAKR B6B10 B/c C3H C58 RIIIS SM23456n=46-80StrainsBasal threshold (s)Sex difference of basal t

51、hresholdsamong inbred strains129AAKRB6B10B/cC3HC58RIIISSM23456n=14-51StrainsBasal threshold (s)Strain and sex differences in basal threshold in mice1/16/2022Neuroscience Research Institute, Peking University44测量疼痛的两类方法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

52、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。1/16/2022Neuroscience Research Institute, Peking University45常用的痛刺激方法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1/16/2022Neuroscience Research Institute, Peking University46实验动物的疼痛评价方法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

53、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1/16/2022Neuroscience Research Institute, Peking University47实验动物的疼痛评价方法实验动物的疼痛评价方法简单的反射行为甩尾实验(tail flick test)钾离子测痛法(Potassium iontophoretic dolorimetry)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(hot plate test)冷板测痛法(cold plate test)翻滚实验(wrot

54、jomg response)发声反应(colcalization)训练学会的或自发反应逃跑或躲避反应(escape and avoidance behaviors)动机性选择(motivational choice paradign)1/16/2022Neuroscience Research Institute, Peking University48动物疼痛的行为学研究方法动物疼痛的行为学研究方法1/16/2022Neuroscience Research Institute, Peking University49外周神经损伤后的机械痛敏OXOXOXCold-induced Ongoin

55、g Pain After Peripheral Nerve Injury5 C1/16/2022Neuroscience Research Institute, Peking University51辐射热甩尾测定痛阈（电针）辐射热甩尾测定痛阈（电针）1/16/2022Neuroscience Research Institute, Peking University521/16/2022Neuroscience Research Institute, Peking University531/16/2022Neuroscience Research Institute, Peking Uni

56、versity541/16/2022Neuroscience Research Institute, Peking University55012345690246810#/*Scores of extension pain test#/Time (weeks after injection of CFA)IFA: Incomplete Freunds AdjuvantCFA: Complete Freunds Adjuvantn=10 / group*p0.05, *p0.01, *p0.001 compared with IFA group#p0.05, #p0.01, #p0.001 c

57、ompared with left ankleChronic Inflammatory Pain Model-MonoarthritisIFA leftCFA left IFA rightCFA right1/16/2022Neuroscience Research Institute, Peking University561/16/2022Neuroscience Research Institute, Peking University571/16/2022Neuroscience Research Institute, Peking University581/16/2022Neuro

58、science Research Institute, Peking University591/16/2022Neuroscience Research Institute, Peking University601/16/2022Neuroscience Research Institute, Peking University61The Abdominal Constriction (Writhing) Test tonic inflammatory pain spinally mediated visceral/subcutaneous0.9% Acetic Acid(10 ml/kg

59、; intraperitoneal)1/16/2022Neuroscience Research Institute, Peking University621/16/2022Neuroscience Research Institute, Peking University631/16/2022Neuroscience Research Institute, Peking University64疼痛的研究方法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、

60、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1/16/2022Neuroscience Research Institute, Peking University65脊髓丘脑束神经元中枢敏化脊髓丘脑束神经元中枢敏化痛敏，通觉超敏痛敏，通觉超敏 （allodynia)SPEAANK1RNMDARNO/CGMPNO/PKGPKCPKAMARPKCREBFOS通道、受体基因表达痛敏伤害性刺激1/16/2022N