左旋多巴诱导的帕金森病大鼠模型中运动障碍的诱导与评估
Summary
本文描述了在帕金森病的大鼠模型中诱导和评估左旋多巴诱导的运动障碍的方法。该协议提供了有关一系列运动障碍行为（包括肌张力障碍和运动过度）的强度和频率的详细信息，为测试针对这种未满足的医疗需求的治疗提供了可靠的工具。
Abstract
左旋多巴（L-DOPA）仍然是用于治疗帕金森病（PD）运动症状的金标准疗法。然而，长期使用这种多巴胺前体会发展为称为 L-DOPA 诱导的运动障碍 （LID） 的不需要的不自主运动。据估计，在治疗后 10-15 年内，LID 的发病率上升到约 90% 的 PD 患者。了解这种疾病的机制并开发新颖有效的抗运动障碍治疗需要为治疗干预的临床前测试提供一致和准确的建模。本文介绍了在PD大鼠模型中6-OHDA诱导的黑粒病变后对LID进行可靠诱导和全面评估的详细方法。大鼠中可靠的LID评估提供了一种强大的工具，可以很容易地在实验室中用于测试新兴疗法，重点是减少或消除PD患者的这种常见治疗负担。
Introduction
虽然左旋多巴（L-DOPA）首次作为PD1，2患者的治疗方法已经超过50年，但它仍然是帕金森病运动症状最有效的治疗方法。与PD相关的临床运动症状源于黑质（SN）中多巴胺（DA）神经元的丧失，导致纹状体中可用多巴胺的显着减少。左旋多巴可有效恢复纹状体 DA 水平，从而在疾病早期带来运动益处3，4.不合时宜地，通过长期治疗，大多数PD患者将发展为L-DOPA诱导的运动障碍（LID），包括舞蹈病，肌张力障碍和手足徐动症，这些通常会显着影响日常生活活动5，6，7。
虽然存在几种啮齿动物LID行为模型，但LID的建模和行为评估的差异使人们对实验室之间结果的可重复性以及这些实验工具在临床前PD研究中的可靠性提出了质疑。与临床运动障碍专家8联合开发，目前的方案是LID诱导和评级的直接方法，适用于利用6-羟基多巴胺（6-OHDA）诱导的单侧黑粒病变的大鼠PD模型9，10。此处提供的LID评分量表包括对身体各个部位运动障碍行为的强度和频率的评分。还提供了有关实验工作流程优化以及帕金森病和运动障碍动物的适当护理和处理的相关信息。
Protocol
这里介绍的动物按照机构指南进行了维护和处理。所有动物程序均由密歇根州立大学机构动物护理和使用委员会（IACUC）批准，符合联邦和州法规。 1. 无药物确认 6-OHDA 病变状态 姿势尾挂试验11，12，13注意：在实验受试者（例如，雄性或雌性，成年Sprague Dawley或Fisher 344大鼠）的6-OHDA?…
Representative Results
帕金森病大鼠的LID可以表现为一系列异常不自主运动（AIM），包括肌张力障碍，运动过度和刻板行为。此处介绍了此类行为的LID评级标准，包括强度（表1）和频率（表2）。这为每只大鼠提供了总体LID严重程度评分，反映了在每个评级时间点参与这些行为的质量（强度）和时间（频率）的数量。最终的LID严重程度评分是通过将强度评分乘以每个行为成分的频率评分来计算?…
Discussion
这里介绍的是黑纹体DA系统单侧6-OHDA病变后帕金森病大鼠模型中LID的可重复诱导和评级的详细信息。虽然曾经认为啮齿动物没有发展LID并且旋转不对称可能是大鼠31中LID的类似物，但在过去二十年中已经对大鼠和小鼠模型进行了表征，并且是LID研究的公认工具15，32，33，34。这里给出?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要承认所有帕金森病患者的挣扎以及他们每天表现出的力量和韧性，特别是KSC心爱的父亲Mark Steece。这里代表的工作得到了国家神经疾病和中风研究所（NS090107，NS110398）和帕金森病基金会国际研究资助计划（现为帕金森基金会）的支持。我们还要感谢Molly VanderWerp出色的编辑协助。
Materials
| 100 Minutes Digital Timer | Staples | 1111764 | |
| Compass CX Compact Scale | Ohaus | 30428202 | |
| 5-(2-aminoethyl)-1,2,4-benzenetriol, monohydrobromide | Cayman Chemicals | 25330 | 6-OHDA is a catecholaminergic neurotoxin that is used to induce dopaminergic lesions and parkinsonian symptoms in rodents. |
| Allentown cages | Allentown, LLC | Rat900 | Allentown cages provide the ability to view the rats from all sides. |
| BD Allergist Trays with Permanently Attached Needle | BD | BD 305540 | For subcutaneous L-DOPA injections |
| Benserazide hydrochloride | Sigma-Aldrich | B7283 | Benserazide is a peripheral decarboxylase inhibitor used with L-DOPA to to induce dyskinesia in rodent models of PD. |
| Glass amber scintillation vials | Thermo Scientific | B7921 | Used for storage of L-DOPA/benserazide at -20 °C until mixed with sterile saline. |
| L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride | Sigma-Aldrich | D1507 | L-3,4-Dihydroxyphenylalanine methyl ester is a precursor to L-DOPA that crosses the blood-brain barrierand use to treat parkinsonian symptoms in rodents. |
| Paper Mate Sharpwriter Mechanical Pencils | Staples | 107250 | |
| Rodent nutritionally complete enrichment treats | Bio-Serv | F05478 | |
| Round Ice Bucket with Lid, 2.5 L | Corning | 432129 | |
| Standard Plastic Clipboard | Staples | 1227770 | |
| Steel wired 6' long movable shelving units | Uline | H9488 | Width/Height can be adjusted to need/number of rats per experiment |
| Sterile Saline 0.9% | Covidien/Argyle | 1020 | For mixing with L-DOPA/benserazide prior to subcutaneous injections. |
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Cite This Article
Caulfield, M. E., Stancati, J. A., Steece-Collier, K. Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson’s Disease. J. Vis. Exp. (176), e62970, doi:10.3791/62970 (2021).