疼痛基礎(chǔ)研究方法[共87頁(yè)]

1、1 萬(wàn)有 北京大學(xué)基礎(chǔ)醫(yī)學(xué)院神經(jīng)生物學(xué)系 2 常用的動(dòng)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(jī)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(dòng)模型 膨脹結(jié)腸模型 3 常用的動(dòng)物模型 外周炎性痛模型 皮膚炎性痛模型：Formalin test, Bee Venom 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(shí)驗(yàn)型肌炎模型 手術(shù)創(chuàng)傷模型 4 常用的動(dòng)物模型 炎癥痛模型 外周炎性痛模型 皮

2、膚炎性痛模型：Formalin test, Bee Venom 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(shí)驗(yàn)型肌炎模型 5 常用的動(dòng)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(dòng)模型 膨脹結(jié)腸模型 癌癥痛模型 大鼠脛骨乳腺癌痛模型 6 Animal models of pain Acute stimulus-evoked pain The tail-flick test The hot-plate test The formalin test The

3、paw flick test Immersion test for 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 7 Animal models of pain Models of chronic inflammatory pain Adjuvant-ind

4、uced arthritis Unilateral arthritis Inflammation of a hollow viscus Ureteral calculosis 8 扭體模型 可采用小鼠或大鼠 有多種刺激物都可誘發(fā)動(dòng)物扭體(writhing)行為 最常見的刺激物是醋酸(acetic acid)。將1克阿拉 伯膠(arabic gum)加入9ml濃度為1%的醋酸溶液中， 再注入實(shí)驗(yàn)動(dòng)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 9 The Abdominal Constriction (Writhing) Test ton

5、ic inflammatory pain spinally mediated visceral/subcutaneous 0.9% Acetic Acid (10 ml/kg; intraperitoneal) 10 白陶土-鹿角菜膠炎癥模型 白陶土(Kaolin)是一種細(xì)顆粒狀物質(zhì)，成分為氧化鋁， 起機(jī)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(guò)敏刺激作用。鹿 角菜膠單獨(dú)實(shí)驗(yàn)即可誘發(fā)炎癥，若與白陶土合并使用， 則炎癥更為強(qiáng)烈 可采用家兔或大鼠 麻醉動(dòng)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(shí)，

6、再注入2% 鹿角菜膠溶液0.05ml并按摩5分鐘。炎癥過(guò)程一般在第一次 注射后2小時(shí)內(nèi)開始。動(dòng)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(shí)以上，24小時(shí)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 11 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(dòng)物一肢足底皮下注 射稀釋的福爾馬林(formalin)溶液，動(dòng)物的行為改 變，如安靜時(shí)的屈腿、運(yùn)動(dòng)時(shí)的跛行以及舔足等。 這些行為的程度(如舔足時(shí)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(0.210%)皮下注射

7、到大鼠的右上唇，記錄 注射后每3分鐘時(shí)間內(nèi)動(dòng)物用同側(cè)前肢或后肢摩 擦注射部位的秒數(shù)作為痛分?jǐn)?shù) 12 福爾馬林致痛模型 各種癥狀普遍分為兩個(gè)時(shí)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：1560分鐘 兩相均可用于實(shí)驗(yàn)，但以第二相為常用。 兩個(gè)時(shí)相的發(fā)生機(jī)制并不相同 13 慢性病理性疼痛 慢性病理痛 炎癥性痛(inflammatory pain) 神經(jīng)病理性痛(neuropathic pain) 癌癥痛(cancer pain) 病理性痛時(shí)，共同存在： 痛覺過(guò)敏(hyperalgesia): 對(duì)傷害性刺激敏感性增強(qiáng)和 反應(yīng)閾值降低； 觸誘發(fā)痛(allodynia):

8、 非痛刺激誘發(fā) 持續(xù)性痛和自發(fā)痛(ongoing pain or spontaneous pain). 14 炎癥痛模型 inflammatory pain model 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(gè)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(dòng)物后肢足底，造成單個(gè)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(gè) 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 15 Ankle joint: intra-a

9、rticular injection of CFA Week 1: acute period Week 2-3: subacute period Week 4-9: chronic period Chronic Inflammatory Pain Model - Monoarthritis 16 01234569 0 2 4 6 8 10 # # / * * * * Scores of extension pain test # # # # # # # / / / / Time (weeks after injection of CFA) IFA: Incomplete Freunds Adj

10、uvant CFA: Complete Freunds Adjuvant n=10 / group *p0.05, *p0.01, *p0.001 compared with IFA group #p0.05, #p0.01, #p0.001 compared with left ankle Chronic Inflammatory Pain Model-Monoarthritis IFA leftCFA left IFA rightCFA right 17 Animal models of pain Neuropathic pain models Experimental anesthesi

11、a 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 models of painful diabetic neuropathy Chemotherapy-evoked painful peripheral neuropathy 18

12、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 with CFA. Within 24 and 48 h the animals develop heat-hyperalgesia, mechano- hyperal

13、gesia, 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 of the rat sciatic nerve. Pain 1999; 83:169) 19 L2 L3 L4 L5 L6 L2 L3 L4 L5 Chungs

14、CCI Seltzer 20 21 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 which resolves by 100 days pos

15、t- 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 this model to determine the

16、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-induced tissue damage or acco

17、mpanying inflammation. 22 Fig. 1. Duration of VZV-induced allodynia 23 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 studieswere pooled and are plo

18、tted as a single line (). 24 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 (n=10) of infecte

19、d 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 (), HSV plus val

20、aciclovir (), 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) from day 0 to day 1

21、0 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 administration of va

22、laciclovir. 25 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) 26 Chemotherapy-evoked painful peripheral neuropathy (1) Painful

23、 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 5 days pe

24、r 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 pain. (Ale

25、y KO, et al. Vincristine hyperalgesia in the rat: a model of painful cincristine neuropathy in humans, Neuroscience 1996; 73: 259) 27 Chemotherapy-evoked painful peripheral neuropathy (2) Polomano et al described a paclitaxel-evoked painful peripheral neuropathy in the rat that is not associated wit

26、h 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, mecha

27、no-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, paclit

28、axel. Pain 2001; 94: 293- 304) 28 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 be u

29、sed 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 visc

30、eral pain, in Methods in Gastrointestinal pharmacology, Gaginella, TS Ed, CRC Press, Boca Ratom 1996, 359) 29 Animal models of pain Models of cancer pain 大鼠脛骨乳腺癌痛模型 小鼠足底癌痛模型 30 癌痛實(shí)驗(yàn)進(jìn)展情況 培養(yǎng)腫瘤細(xì)胞，建立癌癥痛模型培養(yǎng)腫瘤細(xì)胞，建立癌癥痛模型 ?行為學(xué)指標(biāo)行為學(xué)指標(biāo) 痛覺過(guò)敏、痛覺超敏、自發(fā)性疼痛 ?病理學(xué)指標(biāo)病理學(xué)指標(biāo) 腫瘤形態(tài)大小、腫瘤病理切片、骨病理 31 小鼠腳掌皮膚癌痛模型 動(dòng)物：C57BL6, Ma

31、le, 6 weeks old B16-BL6 (黑色素瘤細(xì)胞) 模型組：右側(cè)腳掌皮下接種：B16-BL6 105/20ul 左側(cè)： 0.1M PBS 20ul 對(duì)照組：右側(cè): B16-BL6 105/20ul（heat killed) 左側(cè): 0.1M PBS 20ul Reference: Sasamura T et al. Eur J Pharmacol, 2002 32 小鼠腳掌腫瘤生長(zhǎng)情況 33 疼痛的常見癥狀 人類的“疼痛”與動(dòng)物的“傷害性感受” 常見癥狀：主要包括ongoing pain and stimulus- evoked pain 自發(fā)痛(ongoing pain ) 誘

32、發(fā)痛(stimulus-evoked pain)，包括痛覺過(guò)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(dòng)物的自噬等 動(dòng)物模型上研究的策略是，通過(guò)觀察動(dòng)物的行為， 實(shí)驗(yàn)者來(lái)推測(cè)動(dòng)物是否發(fā)生了“疼痛” 34 慢性痛的常見癥狀 自發(fā)痛spontaneous pain 持續(xù)存在的通感覺 痛覺過(guò)敏hyperalgesia 弱的痛刺激引起強(qiáng)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 35 痛敏的種類與機(jī)制 痛敏的種類(types of hyperalgesia) 痛敏包括痛覺過(guò)敏(hyperalgesia)與痛覺超敏(all

33、odynia，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 36 軸軸反射 末梢釋放 SP+EAA Primary hyperalgesia 原發(fā)性痛敏 Secondary hyperalgesia 繼發(fā)性痛敏 Allodynia 痛覺超敏 (觸痛) 37 Philosophy of Measuring Pain The human subject can report his sensations to us. He does so with an act, some sort of behavio

34、r- 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 private subjective experience (a “sharper” image) is the same as what he would experience under the same circumstances. 38 Phi

35、losophy of Measuring Pain We assume that other people see 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

36、 experience we are discussing. 39 Philosophy of Measuring Pain 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 damag

37、e. It is reasonable to argue that this relationship has obvious 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 th

38、e similarity between pain in man and other mammals: the rank order of the potency of opioids is the same as in human beings and rats. 40 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

39、 that lasts 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 model But pain from different causes and from different tissues may be dissimilar in important ways. Abdominal pain may be uniquely modulated by drugs t

40、hat block a opioid-like receptors. 41 Methods in Pain Research Behavioral: 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

41、, molecular, and genetic Morphological: Histochemical, immunohistochemical, fluorescent Electrophysiological: Extracellular, multi-channel recording patch clamp Evoked potential Non-invasive: PET, fMRI Combination of methods at different levels, integration of above methods 42 單通道電流的記錄 Neher intrape

42、ritoneal) 63 64 65 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(xì)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(jī)、對(duì)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)行為學(xué) 藥理學(xué)（包括腦內(nèi)核團(tuán)立藥理學(xué)（包括腦內(nèi)核團(tuán)立 體定位注射、蛛網(wǎng)膜下腔體定位注射、蛛網(wǎng)膜下腔 注射等）注射等） 生理學(xué)（包括電生理學(xué)）生理學(xué)（包括電生理學(xué)） 細(xì)胞學(xué)細(xì)胞學(xué) 解剖學(xué)（如神經(jīng)示蹤）與解剖學(xué)（如神經(jīng)示蹤）與 組織學(xué)（包括一般組織學(xué)組織學(xué)（包括一般組織學(xué) 與免疫組織化學(xué)）與

43、免疫組織化學(xué)） 生物化學(xué)和分子生物學(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)基因組學(xué)和蛋白質(zhì)組學(xué) 66 脊髓丘腦束神經(jīng)元中樞敏化脊髓丘腦束神經(jīng)元中樞敏化痛敏，通覺超敏痛敏，通覺超敏 （allodynia) SP EAA NK1R NMDAR NO/CGMP NO/PKG PKC PKA MARPK CREB FOS 通道、受體 基因表達(dá) 痛敏 傷害性 刺激 67 68 Vanilloid receptor type 1 (VR1) Distribution in DRG in Normal Rats IB4VR1merged Scale bar = 40 m 69 Change of VR1

44、 Expression in DRG after CFA Injection Ctrl137142128 0 10 20 30 * * * * * Days after CFA injection Average VR1 immunoreactivity Average VR1-ir = average gray value(mean density)-background n = 3, * p 0.05 * p 200 (5) 125 (3) 188 (4) 460 (10) 560 (9) Postoperative time Proportion of filaments with ED

45、 (%) 0-4 h 4-8 h 8-12 h 12-16 h 16-20 h 20-24 h d 2 d 3 d 5 d 7 d 14 0 5 10 15 20 25 30 B 27 121 10279 76 83 129 109 106 112 Postoperative time Average frequency (Hz) Changes of ectopic discharges after SNL 74 0-4 h4-8 h8-12 h12-16 h 16-20 h 20-24 h 0 4 8 12 16 proportion of ED(%) 50% PWT 0 20 40 60

46、 80 100 A Postoperative time 50% PWT (g) Proportion of ED(%) 0255075100 0 4 8 12 16 B r = -0.958, p 0.003 proportion of ED (%) 50% PWT (g) 0-4 h4-8 h8-12 h12-16 h 16-20 h 20-24 h 0 4 8 12 16 Frequency of ED50% PWT 0 10 20 30 C Postoperative time 50% PWT (g) Frequency of ED 0510152025 0 4 8 12 16 D r

47、 = -0.955, p 0.003 Frequency of ED 50% PWT (g) Correlation analysis between tactile allodynia and ectopic activity in the first 24 hours after SNL 75 1235714 0 4 8 12 16 Proportion of ED(%)50% PWT 0 25 50 75 100 A Postoperative time (day) 50% PWT (g) Proportion of ED(%) 255075100 0 1 2 3 B r = -0.00

48、7, p = 0.989 Proportion of ED(%) 50% PWT (g) 1235714 0 4 8 12 16 Frequency of ED50% PWT 0 10 20 30 C Postoperative time (day) 50% PWT (g) Frequency of ED 0102030 0 1 2 3 D r = 0.177, p = 0.738 Frequency of ED 50% PWT (g) Correlation analysis between tactile allodynia and ectopic activity in the days

49、 1-14 after SNL 76 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 spontaneous 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