帕金森病的6-羟基多巴胺大鼠模型
Summary
6-羟基多巴胺（6-OHDA）模型已经使用了几十年，以推进对帕金森病的理解。在该协议中，我们展示了如何通过在内侧前脑束中注射6-OHDA来执行大鼠的单侧黑质纹状体病变，评估运动缺陷并使用步进测试预测病变。
Abstract
帕金森病 （PD） 的运动症状 – 运动迟缓、运动障碍和休息时震颤 – 是黑质 （SNc） 中多巴胺能神经元神经变性和多巴胺能纹状体缺陷的结果。动物模型已被广泛用于在实验室中模拟人类病理学。啮齿动物是PD最常用的动物模型，因为它们易于处理和维护。此外，PD的解剖学和分子，细胞和药理学机制在啮齿动物和人类中是相似的。将神经毒素6-羟基多巴胺（6-OHDA）输注到大鼠的内侧前脑束（MFB）中再现多巴胺能神经元的严重破坏并模拟PD症状。该协议演示了如何在PD的大鼠模型中对MFB中6-OHDA进行单侧显微注射，并通过步进测试显示了6-OHDA诱导的运动缺陷和预测的多巴胺能病变。6-OHDA导致对侧前肢进行的步数显着受损。
Introduction
PD 的主要神经病理学特征是黑质 （SNc） 中多巴胺能神经元的慢性进行性神经变性以及存在含有α-突触核蛋白的路易体1。当 SNc 多巴胺能神经元通过纹状体通路将其轴突投射到纹状体中时，SNc 中神经元的神经变性导致纹状体中多巴胺能缺陷2。纹状体中多巴胺的缺失导致直接和间接运动控制途径的活动失衡，这是PD的主要运动症状的原因：运动障碍（运动缓慢），运动迟缓（开始运动困难），肌肉僵硬和休息时的震颤3，4，5。
由于参与PD发作的分子和生理机制仍未完全了解，目前可用的主要治疗方法旨在通过药物治疗，深部脑刺激6，7，遗传疗法8和细胞移植来缓解运动症状9。因此，临床前研究对于阐明PD发病的机制以及发现早期诊断的新方法和新疗法以防止或阻止受PD10影响的神经元变性至关重要。
动物模型已被广泛用于在实验室中模拟人类病理学，为医学和科学的进步做出了贡献11，12，13，14。然而，必须强调的是，动物模型的正确选择是研究成功的基础。因此，动物模型必须在三个主要方面进行验证：i）面部有效性，其中动物模型必须具有人类病理学的特征;ii）建设性有效性，其中动物模型必须具有坚实的理论基础;iii）预测有效性，其中动物模型必须以与临床治疗类似的方式对治疗做出反应。
目前，几种动物被用作PD的动物模型。主要群体包括哺乳动物，如啮齿动物，灵长类动物，迷你猪，狗和猫，以及其他群体，如果蝇和斑马鱼。啮齿动物是PD最经典的动物模型，由于其易于处理和维护而使用最多。此外，PD的解剖学和分子，细胞和药理学机制在啮齿动物和人类中是相似的15。
Kin及其同事在2019年发表的一篇综述分析了2000年代用于PD的主要动物模型方法，发现最常用的动物模型涉及神经毒素，如6-羟基多巴胺（6-OHDA）和1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）。这两种神经毒素都会导致黑纹状体通路中多巴胺能神经元的线粒体失调，导致细胞死亡16。另一种广泛使用的模型涉及通过参与PD发作的特定基因的突变进行遗传操作，从而导致线粒体失调17。神经毒素模型通常用于评估和比较治疗方法，而遗传模型用于研究预防性疗法和特发性PD15的发展。
神经毒素MPTP在20世纪80年代中期被发现会导致帕金森综合征，此前有七名患者使用该物质并表现出严重的PD症状。除了症状之外，患者对L-DOPA的治疗也有反应，这使得研究人员将分子直接与PD联系起来。该病例于1986年发表后，一些研究人员开始在临床前PD研究中使用MPTP18。研究人员发现，作为一种亲脂性分子，MPTP可以穿过血脑屏障（BBB）并转化为MPP + 19。这种有毒物质积聚在神经元内，对线粒体呼吸链的复合物1造成损害，导致多巴胺能神经元死亡20。
6-OHDA神经毒素模型于196821年首次用于诱导黑纹状体通路单胺神经元的变性。6-OHDA模型通常用于引起黑纹状体通路中的神经变性，因为它是一种多巴胺类似物，对含儿茶酚胺的细胞有毒。6-OHDA进入大脑后，它可能被多巴胺能神经元中的多巴胺转运蛋白（DAT）吸收，导致黑纹状体通路变性22。由于6-OHDA不穿透BBB，因此必须直接通过脑内立体定向注射给药23。去甲肾上腺素再摄取抑制剂通常与 6-OHDA 显微注射相结合，以保留去甲肾上腺素能纤维，并提供更具选择性的多巴胺能神经元变性24。
DAT吸收6-OHDA后，它会积聚在神经元的细胞质基质中，产生活性氧（ROS）并导致细胞死亡15。经常使用6-OHDA的三种不同病变模型：i）SNc25，26的病变;ii）纹状体病变27，28;iii）MFB29，30的病变。纹状体引起的病变导致SNpc中多巴胺能神经元的缓慢和逆行变性。相反，在SNpc和MFB中引起的病变导致神经元的快速和完全变性，导致更晚期的帕金森症状31。
单侧或双侧注射 6-OHDA 可导致多巴胺能神经元的神经变性。6-OHDA并不总是对神经元造成严重损害;有时，注射会导致部分损坏，这也用于模拟PD32的早期阶段。单侧注射更常用，因为该模型能够评估动物的运动缺陷，并通过安非他明/阿扑吗啡诱导的旋转和步进测试等测试预测细胞损失29。双侧注射最常用于评估空间记忆和识别33。
苯丙胺/阿扑吗啡诱导的旋转试验是一种行为试验，通常用于预测黑纹状体通路中的细胞损失。它被定义为重复施用多巴胺激动剂导致6-OHDA病变动物旋转行为加剧的过程34。旋转行为包括量化单侧病变啮齿动物中苯丙胺诱导的同侧旋转或阿扑吗啡诱导的对侧旋转。药物诱导的旋转行为受到批评，因为旋转与人类的PD症状不对应，并且可能受到耐受性，致敏和“启动”等变量的影响35。
启动是这些行为测试中最关键的因素之一。据报道，一些病例中，单剂量的左旋多巴导致旋转行为失败36。此外，与苯丙胺诱导试验和阿扑吗啡诱导试验联合应用并行使用相关的另一个关键因素是，由于作用机制不同，它们测量不同的终点，反映了不同信号传导机制和途径的失活。此外，苯丙胺诱导的试验更准确地测量高于50-60%的黑质纹状体病变，而阿扑吗啡诱导的试验对于80%以上的病变更准确37。
步进测试已成为一种行为测试，表明与多巴胺能神经元变性和治疗效果相关的缺陷。它能够分析由多巴胺能神经元中的6-OHDA病变引起的运动障碍，而无需药物诱导的程序。此外，自1995年以来，该测试已经得到很好的建立和广泛使用，当时Olsson等人首次对其进行了描述。1999年，Chang等人还分析 并比较了大鼠在步进试验中的表现与6-OHDA引起的变性水平，发现在步进试验中表现较差的动物也具有更显着的多巴胺能神经元变性。
步进试验是预测6-OHDA病变大鼠严重多巴胺能黑质纹状体损伤的极好方法。有证据表明，当 SNc 中的多巴胺能损失程度为 >90%39 时，在步进试验期间，6-OHDA 输注的对侧前肢会出现运动功能障碍。本文介绍了用于进行立体定向手术的方案，方法和材料，用于将6-OHDA单侧输注到大鼠的MFB中，以及如何通过步进测试预测由毒素引起的多巴胺能病变。
Protocol
Representative Results
Discussion
本文描述了一种在MFB中对6-OHDA进行单侧微输注手术的方案，能够在黑纹状体通路的神经元中引起强大的病变并在动物中产生运动障碍。还描述了用于执行步进测试的方案，这是一种易于应用且无创的测试，可用于证明病变的成功并评估前肢运动障碍。正如代表性结果所示，接受6-OHDA的动物显示出与损伤对侧调整步骤的次数减少，这意味着6-OHDA受伤的动物在输液手术后2周内表现出强烈的运动障碍。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了圣保罗研究基金会（FAPESP，拨款2017/00003-0）的支持。我们感谢提高高等教育人员的协调（CAPES）。我们感谢Anthony R. West博士，Heinz Steiner博士和Kuei Y. Tseng博士的支持和指导。
Materials
| 6-OHDA | Sigma Aldrich | H4381 | https://www.sigmaaldrich.com/catalog/product/sigma/h4381?lang=pt®ion=BR&cm_sp=Insite-_-caSrpResults_srpRecs_srpModel _6-ohda-_-srpRecs3-1 |
| 70% Alcohol | |||
| Ascorbic acid | Sigma Aldrich | 795437 | https://www.sigmaaldrich.com/catalog/product/sial/795437?lang=pt®ion=BR&gclid= Cj0KCQjw4cOEBhDMARIsAA3XD RipyOnxOxkKAm3J1PxvIsvw09 _kfaS2jYcD9E5OyuHYr4n89kO 6yicaAot6EALw_wcB |
| Cotton | |||
| Drill or tap | |||
| Gauze | |||
| Hamilton syringe 50 uL | Hamilton | 80539 | https://www.hamiltoncompany.com/laboratory-products/syringes/80539 |
| Imipramine | Alfa Aeser | J63723 | https://www.alfa.com/pt/catalog/J63723/ |
| Infusion pump | Insight | EFF-311 | https://insightltda.com.br/produto/eff-311-bomba-de-infusao-2-seringas/ |
| Ketamine (Dopalen) | Ceva | https://www.ceva.com.br/Produtos/Lista-de-Produtos/DOPALEN | |
| Machine for trichotomy | |||
| Meloxicam (Maxicam 2% Ourofino) | Ourofino | https://terrazoo.com.br/produto/maxicam-injetavel-2-50ml-ouro-fino/ | |
| Metal Disposal | |||
| Paper towels | |||
| Pentabiotic | Zoetis | https://www.zoetis.com.br/pentabiotico-veterinario.aspx | |
| Plastic waste garbage can | |||
| Poly-antibiotic | Pentabiotic (Wealth) | ||
| Povidone-iodine | |||
| Scalpel and blades | |||
| Scissors | |||
| Scraper | |||
| Stereotaxic apparatus | Insight | EFF-331 | https://insightltda.com.br/produto/eff-331-estereotaxico-1-torre/ |
| Sterile saline solution | |||
| Swabs | |||
| Temperature probe | |||
| Timer | |||
| Tweezers | |||
| Xylazine (Anasedan) | Ceva | https://www.ceva.com.br/Produtos/Lista-de-Produtos/ANASEDAN |
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