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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,常用的动物模型,外周炎性痛模型 皮肤炎性痛模型：Formalin test, Bee Venom 致炎剂模型：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：Formalin test, Bee Ven
2、om 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,Animal models of pain,Acute stimulus-evoked pain The tail-flick test The hot-plate test The formalin test The paw flick test Immersion test for
3、thermal hypersensitivity Cold-allodynia test The pin-prick test for mechano-hyperalgesia von frey Hair test for mechano-allodynia The writhing test The Distension of a hollow viscus Muscle pain,Animal models of pain,Models of chronic inflammatory pain Adjuvant-induced arthritis Unilateral arthritis
4、Inflammation of a hollow viscus Ureteral calculosis,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 该模型可以模拟腹腔炎症引起的腹痛症状,The Abdominal Constriction (Writhing) Test,tonic inflammatory pain spinally mediated visc
5、eral/subcutaneous,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
6、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(0.210%)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,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
7、 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(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(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(ongoing pain or spontaneous pain).,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
8、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,Ankle joint: intra-articular injection of CFA,Week 1: acute period Week 2-3: subacute period Week 4-9: chronic period,Chronic Inflammatory Pain Mode
9、l - Monoarthritis,Animal models of pain,Neuropathic pain models Experimental anesthesia dolorosa Experimental models of painful peripheral neuropathy due to traumatic, partial nerve damage Chronic constriction injury Partial nerve transection injury Spinal nerve transection injury Experimental model
10、s of painful diabetic neuropathy Chemotherapy-evoked painful peripheral neuropathy,Neuropathic pain from nerve inflammation,Eliav 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 w
11、ith CFA. Within 24 and 48 h the animals develop heat-hyperalgesia, mechano-hyperalgesia, mechano-allodynia, and (to a lesser 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
12、of the rat sciatic nerve. Pain 1999; 83:169),Allodynia in rats infected with varicella zoster virusa small animal model for post-herpetic neuralgia,Following VZV infection of the left footpad rats develop a chronic mechanical allodynia, which is present for longer than 60 days post-infection and whi
13、ch 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. The reproducible nature of the induction and course of the allodynia allows the use of th
14、is 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 induction of the chronic allodynia is VZV-specific and is not a result is of virus replication-ind
15、uced tissue damage or accompanying inflammation.,Fig. 1. Duration of VZV-induced allodynia,Fig. 2. Reproducibility of the model,The mean withdrawal thresholds observed in four individual VZV studies (n=24) are presented individually (, ,). The data from the controls (n=24) from these four studieswer
16、e pooled and are plotted as a single line ().,Fig. 3. Specificity of the model,Animals (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 (
17、n=10) of infected animals was treated 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
18、(), HSV plus valaciclovir (), control (). (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) fro
19、m day 0 to day 10 post-infection. 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 admi
20、nistration of valaciclovir.,Animal models of pain,Visceral pain models Colonic-rectal distension (CRD) Small bowel distension Artificial kidney stones Urinary bladder distension Urinary bladder irritants Ischemic stimuli (coronary artery occlusion),Chemotherapy-evoked painful peripheral neuropathy (
21、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 are considerably lower than those used previously. Aley et al injected vincristine
22、 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 ceased. Both doses produced a significantly increased threshold to heat-evoked
23、pain. (Aley KO, et al. Vincristine hyperalgesia in the rat: a model of painful cincristine neuropathy in humans, Neuroscience 1996; 73: 259),Chemotherapy-evoked painful peripheral neuropathy (2),Polomano et al described a paclitaxel-evoked painful peripheral neuropathy in the rat that is not associa
24、ted 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 with 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
25、, mechano-hyperalgesia, mechano-allodynia, and cold-allodynia. The abnormal pain sensations began within several days of the initiation of treatment and lasted for at least several weeks afterward. (Polomano RC, et al. A painful peripheral neuropathy in the rat produced by the chemotherapeutic drug,
26、 paclitaxel. Pain 2001; 94: 293-304),Colonic-Rectal Distension,In rats, a flexible latex balloon fixed 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 b
27、e used as the balloon. The catheter in rats is Tygon flexible tubing . For a 7 to 8-cm long balloon, 6 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 v
28、isceral pain, in Methods in Gastrointestinal pharmacology, Gaginella, TS Ed, CRC Press, Boca Ratom 1996, 359),Animal models of pain,Models of cancer pain 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, 6 weeks old B16-BL6 (黑色素瘤细胞)
29、 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 20ul,Reference: Sasamura T et al. Eur J Pharmacol, 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ongoing pain and stimulus-evoked pain 自发痛(ongoing pain ) 诱发痛(stimulus-evoked pain)，包括痛觉过敏hyperalg
30、esia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,痛敏的种类与机制,痛敏的种类(types of hyperalgesia) 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(hype
31、ralgesia) 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+EAA,Primary hyperalgesia 原发性痛敏,Secondary hyperalgesia 继发性痛敏,Allodynia 痛觉超敏 (触痛),Philosophy of Measuring Pain,The 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, et
32、c. What then of measuring sensation in an animal? The optometrists procedure is based on the implicit assumption that my private subjective experience (a “sharper” image) is the same as what he would experience under the same circumstances.,Philosophy of Measuring Pain,We assume that other people se
33、e like us because they look like us. Rats do not look like us. 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.,Philosophy of Measuring P
34、ain,We 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 45C Under normal circumstances, the sensation of pain is tightly related to tissue damage. It is reasonable to argue that this relationship has obv
35、ious 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 ran
36、k order of the potency of opioids is the same as in human beings and rats.,Measuring pain in animals,Acute and chronic pain The distinction is arbitrary “acute” refers to pain that lasts for seconds to about a day “chronic” refers to pain that lasts for at least several days. In theory, on could pro
37、duce any sort of injury to any body part in the anial and declare that one had a pain model But 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 opioid-like receptors.,Methods in Pain Research,Be
38、havioral: hot (cold) plate, von Frey hair, pain score Pharmacological: antagonist, radio ligand binding assay Psychological Neurochemical: neurotransmitter content measurement with high performance liquid chromatography (HPLC) Cellular, molecular, and genetic Morphological: Histochemical, immunohist
39、ochemical, fluorescent Electrophysiological: Extracellular, multi-channel recording patch clamp Evoked potential Non-invasive: PET, fMRI Combination of methods at different levels, integration of above methods,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. Principl
40、es of Neuroscience 4th edition. Fig. 11-8.,Patch Clamp 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,Strain and sex differences in basal threshold in mice,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
41、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(tail flick test) 钾离子测痛法(Potassium iontoph
42、oretic 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(wrotjomg 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),动物疼痛的行为学研究方法,辐射热甩尾测定痛阈（电针）,The Abdominal Constriction (Writhing) Test,
43、tonic inflammatory pain spinally mediated visceral/subcutaneous,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
44、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,SP EAA,NK1R NMDAR,NO/CGMP NO/PKG PKC PKA,MARPK CREB FOS,通道、受体 基因表达,痛敏,伤害性 刺激,短时程,中时程,长时程,Vanilloid receptor type 1 (VR1) Distribution in DRG in Normal Rats,IB4,VR1,merged,Scale bar = 40 m,Change of VR1 Expression in DRG after CFA Injection,Aver
45、age VR1-ir = average gray value(mean density)-background,n = 3, * p 0.05 * p 0.01,background,Correlation between Hyperalgesia and VR1 Protein Level,PAIN,Three different typical patterns of ectopic discharges,Changes of ectopic discharges after SNL,Correlation analysis between tactile allodynia and e
46、ctopic activity in the first 24 hours after SNL,Correlation analysis between tactile allodynia and ectopic activity in the days 1-14 after SNL,Neuropathic pain: Early spontaneous afferent activity is the trigger,After CCI nerve injury, both A-fibers (c) and C-fibers (f) displayed high levels of spon
47、taneous activity in the CCI-rats that were untreated (nave) or perfused with saline, but treatment with either bupivacaine or TTX during the first 47 days post-injury inhibited this elevation in spontaneous activity. Wenrui Xie, Judith A. Strong, Johanna T.A. Meij, Jun-Ming Zhang, Lei Yu. Pain xx (2
48、005) 114,Allodynia,C.N.S.,Central Sensitization,(Pain),(Touch = Pain),Wind-up of a WDR neuron,Induction of LTP of C-fiber-evoked potentials in the spinal dorsal horn in sham-operated and neuropathic pain rats,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. Principle
49、s of Neuroscience 4th edition. Fig. 11-8.,Patch Clamp 技术,Schematic drawing of microarrays（微电极芯片）,Microarrays may be one-, two- or three-dimentional Microwires are grouped together to occupy a target area,Donald D. Price. Science 2000; 288: 1769-1772,Psychological and neural mechanisms of the affective dimension of pain.,Human Models for Neuropathic Pain,Diabetic neuropathy (DN) and post-herpetic neuralgia (PHN) are the most prevalent neuropathic pain disorders Majority of randomized controlled trials