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Biology

实验性自身免疫性脑脊髓炎的诱导及多样评价指标

Published: September 09, 2022
doi:
10.3791/63866
, , , , , , , , , , ,
1Putuo People’s Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology,Tongji University, 2National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics,Chinese Academy of Sciences, 3Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital,Fudan University

Summary

本协议描述了使用髓鞘少突胶质细胞糖蛋白在小鼠模型中诱导实验性自身免疫性脑脊髓炎，并使用临床评分系统监测疾病过程。采用小鼠股骨显微计算机断层扫描分析和开放现场试验分析实验性自身免疫性脑脊髓炎相关症状，全面评估疾病过程。

Abstract

多发性硬化症 （MS） 是一种典型的中枢神经系统 （CNS） 自身免疫性疾病，其特征是炎症浸润、脱髓鞘和轴索损伤。目前，没有完全治愈MS的措施，但有多种疾病改善疗法（DMT）可用于控制和减轻疾病进展。实验性自身免疫性脑脊髓炎（EAE）和MS患者的CNS病理特征有显着相似之处。EAE已被广泛用作确定MS药物疗效和探索MS疾病新疗法开发的代表性模型。在小鼠中主动诱导EAE具有稳定且可重复的作用，特别适用于研究药物或基因对自身免疫性神经炎症的影响。主要分享用髓磷脂少突胶质细胞糖蛋白（MOG35-55）免疫C57BL / 6J小鼠的方法和使用临床评分系统的疾病症状的日常评估。鉴于MS病因复杂，临床表现多样，现有的临床评分体系无法满足疾病治疗的评估。为了避免单一干预的缺点，创建了基于MS患者焦虑样情绪和骨质疏松症的临床表现来评估EAE的新指标，以提供更全面的MS治疗评估。

Introduction

自身免疫性疾病是由免疫系统对其自身抗原的免疫反应引起的一系列疾病，导致组织损伤或功能障碍1。多发性硬化症（MS）是中枢神经系统（CNS）多发性神经病的慢性自身免疫性疾病，其特征是炎症浸润，脱髓鞘和神经元轴突变性2，3。目前，MS已影响全球多达250万人，其中大多数是20-40岁的年轻人和中年人，他们往往是家庭和社会的支柱。这对家庭和社会造成了相当大的影响和伤害2，4。

MS是一种多因素疾病，临床表现多样且复杂。除了以炎症浸润和脱髓鞘为特征的经典神经系统疾病外，MS还经常表现出视力障碍，肢体运动障碍以及认知和情绪障碍5，6，7。如果MS患者没有得到适当和正确的治疗，其中一半将在20年后生活在轮椅上，其中近一半会出现抑郁和焦虑症状，导致自杀意念水平比一般人群高得多8，9。

尽管研究时间较长，但MS的病因仍然难以捉摸，MS的发病机制尚未阐明。尽管啮齿动物和人类免疫系统之间存在显着差异，但MS的动物模型允许作为探索疾病发展和新治疗方法的测试工具，同时共享一些基本原理。实验性自身免疫性脑脊髓炎（EAE）是目前研究MS的理想动物模型，利用髓鞘蛋白自身抗原免疫诱导易感小鼠对CNS成分的自身免疫，并加入完全弗氏佐剂（CFA）和百日咳毒素（PTX）增强体液免疫应答。根据遗传背景和免疫抗原，获得不同的疾病过程，包括急性、复发缓解或慢性，以模拟各种临床形式的 MS 10，11，12。构建EAE模型常用的相关免疫原来自自身CNS蛋白，如髓鞘碱性蛋白（MBP）、蛋白脂蛋白（PLP）或髓鞘少突胶质细胞糖蛋白（MOG）。MBP或PLP免疫的SJL / L小鼠发展复发 – 缓解过程，MOG触发C57BL / 6小鼠的慢性进行性EAE11，12，13。

疾病修正疗法（DMT）的主要目的是尽量减少疾病症状并改善功能6。临床上有几种药物用于缓解MS，但尚未使用任何药物来完全治愈它，这表明协同治疗的必要性。C57BL/6小鼠是目前最常用于构建转基因小鼠的方法，本工作采用MOG35-55 诱导的5点量表C57BL/6J小鼠EAE模型监测疾病进展。EAE模型还患有焦虑样情绪和骨质流失，以及广为人知的脱髓鞘病变。在这里，还描述了使用开放现场测试和显微计算机断层扫描（Micro-CT）分析从多个角度评估EAE症状的方法。

Protocol

同济大学动物护理委员会批准了目前的工作，并遵循了所有动物护理指南。使用8-12周龄的雄性或雌性C57BL / 6J小鼠进行实验。确保实验组的年龄和性别相同;否则，对该疾病的易感性受到影响。将小鼠饲养在特定的无病原体环境中，在恒定条件下（室温23±1°C，湿度50%±10%）交替进行12小时的光照和黑暗循环，并自由获取小鼠食物和水。 1.MOG35-55 乳液的制备…

Representative Results

小鼠免疫后，每天记录小鼠的体重，并根据上述方案评估其临床症状（步骤4）。在免疫MOG肽的C57BL/6J小鼠中，由于病变的位置主要局限于脊髓，EAE小鼠的发病机制从尾端扩散到头部。在疾病开始时，EAE小鼠表现出尾巴无力和下垂，随后是后肢无力，运动不协调和瘫痪。随着病情的恶化，逐渐发展为前肢无力，瘫痪，严重时导致小鼠移动困难，甚至濒临死亡。如图 1A所示，具?…

Discussion

MS是中枢神经系统的脱髓鞘炎症性疾病，是导致年轻人慢性残疾的最常见神经系统疾病之一，给家庭和社会带来巨大负担3，4。MS一直被归类为器官特异性T细胞介导的自身免疫性疾病，诱导自身免疫系统缓慢侵蚀CNS，这将涉及全身的多个系统27。典型的临床症状包括视力障碍、运动障碍、认知和情绪障碍等6，<s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢中国国家自然科学基金（32070768，31871404，31900658，32270754）和药物研究国家重点实验室的支持。

Materials

1 mL syringe(with 26 G needle) Shanghai Kindly Medical Instruments Co., Ltd 60017031
2 mL microcentrifuge tube HAIKELASI KY-LXG2A
22 G needle Shanghai Kindly Medical Instruments Co., Ltd 60017208
Complete Freund’s Adjuvant Sigma F5881 Stored at 4 °C, 1 mg of heat-inactivated MTB (H37Ra) per mL
Conditioned place preference system Shanghai Jiliang Software Technology Co., Ltd Animal behavior
Ethanol Sinopharm Chemical Reagent Co., Ltd 10009218 Stored at RT
Locomotion activity (open field) video analysis system Shanghai Jiliang Software Technology Co., Ltd DigBehv-002 Animal behavior
MOG35-55 peptide Gill Biochemical Co., Ltd GLS-Y-M-03590 Stored at -20 °C
Mycobacterium tuberculosis H37Ra BD 231141 Stored at 4 °C
Open field reaction chamber Shanghai Jiliang Software Technology Co., Ltd Animal behavior
Pertussis toxin Calbiochem 516560 Stored at 4 °C
Phosphate Buffered Saline Made in our laboratory
Scissor Shanghai Medical Instrument (group) Co., Ltd J21010
Sealing film Heathrow Scientific HS 234526B
Sorvall Legend Micro 21R Microcentrifuge Thermo Scientific 75002447
Steel ball QIAGEN 69975
TissueLyser II QIAGEN 85300
Tweezer Shanghai Medical Instrument (group) Co., Ltd JD1060
μCT 35 desktop microCT scanner Scanco Medical AG, Bassersdorf, Switzerland
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Tags

Experimental Autoimmune EncephalomyelitisMultiple SclerosisMyelin Oligodendrocyte Glycoprotein PeptideFreud’s AdjuvantPertussis ToxinOpen Field Reaction ChamberMouse Locomotion
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Wang, C., Lv, J., Zhuang, W., Xie, L., Liu, G., Saimaier, K., Han, S., Shi, C., Hua, Q., Zhang, R., Shi, G., Du, C. Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis. J. Vis. Exp. (187), e63866, doi:10.3791/63866 (2022).
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