大鼠关节内注射单碘乙酸酯诱导的骨关节炎疼痛模型
Summary
本研究描述了大鼠关节内注射单碘乙酸酯的方法，并讨论了由此产生的疼痛相关行为和组织病理学变化，为今后的应用提供参考。
Abstract
目前的骨关节炎（OA）动物模型可分为自发模型和诱导模型，两者都旨在模拟人类OA的病理生理变化。但是，疼痛作为OA晚期的主要症状，影响患者的日常生活，可用的模型并不多。单碘乙酸（MIA）诱导模型是应用最广泛的OA疼痛模型，主要用于啮齿动物。MIA是甘油醛-3-磷酸脱氢酶的抑制剂，可导致软骨细胞死亡，软骨变性，骨赘和动物行为的可测量变化。此外，在MIA诱导的模型中可以检测到基质金属蛋白酶（MMP）和促炎细胞因子（IL1 β和TNF α）的表达变化。这些变化与人类OA病理生理状况一致，表明MIA可以诱导可测量和成功的OA疼痛模型。本研究旨在描述大鼠关节内注射MIA的方法，并讨论由此产生的疼痛相关行为和组织病理学变化。
Introduction
骨关节炎（OA）是世界上最常见的关节疾病，估计影响成人10-12%的人口1。最普遍受累的关节是膝关节，OA在老年人中发病率较高，尤其是女性2。作为一种慢性疾病，OA在几十年内逐渐发展为关节衰竭，症状包括软骨丢失，滑膜炎症，骨癣菌病，功能下降和慢性疼痛3。根据世界卫生组织（WHO）的数据，OA是女性中第四大最普遍的疾病，也是男性中第八大最流行的疾病。到2020年，OA可能成为人类第四大致残性疾病4。然而，目前可用的OA疗法仅解决症状，并延长关节置换手术的时间5。
人类患者的自发性OA往往需要很长时间才能产生关节相关疼痛等临床症状6。在OA的早期阶段，疼痛通常是间歇性的，并且随着疾病的进展变得更加频繁和严重，使其成为患者的主要主诉7。因此，在过去的半个世纪中，已经开发了广泛的OA疼痛动物模型来促进疼痛缓解疗法。OA模型通常分为自发模型和诱导模型。自发模型包括自然发生的模型和转基因模型，它们可以更密切地模拟人类原发性OA的过程8。诱导模型一般可分为两类：1）手术或其他创伤诱导的创伤后OA;或2）关节内注射软骨毒性或促炎物质3。这些模型为OA的病理生理学研究奠定了基础，并为开发减轻疼痛和增加功能的药物做出了巨大贡献。
最近，OA建模最广泛使用的诱导剂是单碘乙酸酯（MIA）。MIA是甘油醛-3-磷酸脱氢酶的抑制剂，可引起软骨基质的变化、降解、软骨流失、滑膜炎等变化，与人类骨关节炎的病理变化相似9.已经注意到，关节内注射MIA在MIA给药后28天引起持续疼痛，表明MIA模型可能有助于研究慢性伤害性疼痛10，11，12。在这项研究中，雄性Sprague-Dawley大鼠接受关节内注射，膝关节中含有0.5、1.5或3mg的MIA。通过注射后1、7、14、21、28和35天的机械和热敏感性评估来测量MIA诱导的关节疼痛的严重程度。在此基础上，选择1.5mg的MIA作为最终浓度，以评估注射后28天的步态模式和组织学变化。
Protocol
涉及动物受试者的程序已获得浙江中医大学医学规范和伦理委员会的批准，并符合中国关于实验动物使用和护理的立法。 1.膝关节内注射单碘乙酸酯 适应一周后，将40只体重180-200g（4-5周龄）的雄性Sprague-Dawley大鼠随机平均分为四组（n = 10只大鼠/组）。注意：对照组的大鼠将关节内注射50μL盐水，而实验组中的大鼠将分别用溶解在50μL盐水中的0.5，1.5或3mgMIA处理。</li…
Representative Results
通过这种方法，我们在大鼠中建立了OA疼痛模型并检测了由此产生的变化。MWT和TWL分别反映机械性异常性疼痛和热痛觉过敏。如图 1所示，MIA诱导的机械性异常性疼痛和热痛觉过敏以剂量依赖性方式存在。值得注意的是，MWT的下降从21 d达到峰值至28 d，然后反弹，表明现阶段可能发生关节修复，但3 mg MIA组的MWT仍处于较低水平。TWL的变化与MWT大致一致（图2</stron…
Discussion
MIA诱导的OA大鼠模型是一种成熟且广泛使用的模型。关节内注射MIA最初引起严重和急性炎症，这导致OA的更长和退行性阶段17，18。在这项研究中，我们通过MWT和TWL测量了伤害敏感性，并使用成像系统评估了步态改变。先前的报告发现，注射MIA可以提高传入膝关节纤维的敏感性，导致伤害感受，这反映在热痛觉过敏和降低的机械阈值19<sup…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
本研究由中国浙江省自然科学基金（批准号：LY17H270016），中国国家自然科学基金（批准号：81774331，81873049和81673997）和中国浙江省中医药科学技术项目（批准号：2013ZQ007和2016ZZ011）资助。
Materials
| Anti-Collagen II antibody | Abcam(UK) | 34712 | Primary antibody for immunohistochemistry (IHC) |
| Anti-Collagen X (Col10) antibody | Abcam(UK) | 49945 | Primary antibody for IHC |
| DigiGait Imaging System | Mouse Specifics (Boston, MA, USA) | Equipment for gait patterns analyses | |
| Eosin | Sigma-Aldrich | 861006 | The dye for HE staining |
| Fast Green FCF | Sigma-Aldrich | F7252 | The dye for SO staining |
| Goat anti-mouse antibody | ZSGQ-BIO (Beijing, China) | PV-9002 | Secondary antibody for IHC |
| Goat anti-rabbit antibody | ZSGQ-BIO (Beijing, China) | PV-9001 | Secondary antibody for IHC |
| Hematoxylin | Sigma-Aldrich | H3163 | The dye for HE staining |
| MIA | Sigma-Aldrich | I4386-10G | powder |
| MMP13 | Cell Signaling Technology, Inc. (Danvers, MA, USA) | 69926 | Primary antibody for IHC |
| Modular tissue embedding center | Thermo Fisher Scientific (USA) | EC 350 | Produce paraffin blocks. |
| Plantar Test apparatus | UgoBasile (Italy) | 37370 | Equipment for TWL assay |
| PrimeScript RT reagent Kit (Perfect Real Time) | TaKaRa Biotechnology Co. Ltd. (Dalian, China) | RR037A | Extracte total RNA from cultured cells |
| Rotary and Sliding Microtomes | Thermo Fisher Scientific (USA) | HM325 | Precise paraffin sections. |
| Safranin-O | Sigma-Aldrich | S2255 | The dye for SO staining |
| Tissue-Tek VIP 5 Jr | Sakura (Japan) | Vacuum Infiltration Processor |
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Citar este artículo
Xu, J., Yan, L., Yan, B., Zhou, L., Tong, P., Shan, L. Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats. J. Vis. Exp. (159), e60649, doi:10.3791/60649 (2020).