作者感谢里斯凡Asselberghs和安凡Santvoort的出色技术援助。研究由内陆水运-VLAANDEREN（IWT SBO / 80020），该FWO VLAANDEREN（G.0768.10）资助，由EC-FP6项目"迪米"（LSHB-CT-2005-512146）中，FP7项目RTD MEFOPA（HEALTH -2009-241791），在FP7计划"INMiND"（健康F2-2011-278850），在鲁汶（IOF-KP / 07/001，OT / 08 / 052A，IMIR PF / 10/017），和MJFox基金会（目标验证2010）。 A.范德Perren和C Casteels是科学研究的弗拉芒基金的博士后。 K.范Laere是科学研究的弗拉芒基金的高级研究员临床。

所有的动物实验是由比利时鲁汶大学生物伦理委员会（比利时）按照1986年11月24日（86/609 / EEC）的欧洲共同体委员会指令进行批准。 1.重组腺相关病毒生产和纯化注意:rAAV载体生产和纯化由鲁汶病毒载体核（LVVC）如先前所述17进行。 <ol><li>简言之，转染汇合低（&lt;50）通道使用25KD线性聚乙烯亚胺150纳米的NaCl转染溶液和三种不同的质粒以1:1的比例粘附的HEK 293T细胞:1:1的DMEM培养基2％胎牛血清。后在37℃下在5％CO 2温育24小时，替换以新鲜的DMEM培养基2％胎牛血清的培养基。 注意:质粒包括用于AAV7血清型的构建体中，CMVIE的控制下，α-SYN编码人A53T突变的AAV转移质粒增强SYNAPSIN1子和pAdvDeltaF6腺病毒辅助质粒17。 </li><li>收获中5天瞬时转染后使用切向流过滤17集中。 </li><li>净化从浓缩介质使用碘克沙醇步骤梯度17 rAAV载体颗粒。 </li><li>使用实时PCR基因组副本（GC）测定的标准技术。在这个协议中，3.0 E11 GC的向量滴度/ ml的用于开发基于α-SYN大鼠模型PD 17。 </li></ol> 2.重组腺相关病毒α-SYN的立体定向注射载体在大鼠的SN（图2） <ol><li>众议院8周龄雌性Wistar大鼠可免费使用颗粒状的食物和自来水一个正常的12小时光/暗周期下重约200-250克</li><li>提交老鼠腹腔内（IP）含有氯胺酮（60毫克/千克），美托咪定（0.4毫克/公斤）混合物麻醉。一旦大鼠麻醉，并没有反应挤压不同爪子时，管理对大鼠的用微型发射机应答器植入背面为进一步识别微发射机应答皮下。检查，如果微发射机应答器被正确定位，并且可以通过读取装置读出。 </li><li>切头皮上的顶部的毛发。在头皮和耳都应用局部麻醉剂。使用无菌技术在层流下进行手术操作的其余部分。 </li><li>将使用两个耳朵酒吧，嘴和鼻子吧的立体定向头架的老鼠。覆盖纸毛毯的大鼠的体以避免在体温下降。应用眼部润滑剂，以防止眼睛变干。 </li><li>消毒在异丙醇70％与jodium 1％头皮和作一小切口在头皮的中线。轻轻刮掉头骨膜和用生理盐水冲洗。让头骨干几分钟。观察颅缝和两个参考点:前囟门和Lambda。 </LI&gt; <li>注入rAAV载体进入SN，定义坐标对前囟门（正位5.3毫米;内外侧斜:2.0毫米，背腹:从硬脑膜计算7.2毫米）。 注意:为每个感兴趣的区域的三维坐标可以使用大鼠大脑的立体图集进行计算，施加前囟如解剖参考点。 </li><li>填有10微升注射针筒（30计20毫米），腺相关病毒载体，并将其放置在与机动微量注射泵连接在立体定位仪。通过释放载体一滴控制音量和消除多元清洁剂pH值为9（ 如 RBS）。 </li><li>目视检查头直在头部框架固定和评估的左右轴。仔细直观定义前囟门和Lambda前后及中侧坐标和立体定位框架的背腹臂用30计20毫针刺测量自己的身高。 <ol><li>允许时在囟门和Lambda之间的高度0.3毫米差aximum。放置针回到前囟门和通过移动的前后和立体定位框架的中侧臂施加的前后和中侧坐标。 </li></ol></li><li>在注射位置，测量头骨的高度，并确保它不会从囟的高度相差大于0.3毫米。钻在头骨的孔的直径为约2毫米。测量硬脑膜的高度，这将作为应用背腹坐标参考。可替代地减去对颅骨（0.9毫米）的固定厚度。 </li><li>使用26号针头穿透硬脑膜，用无菌组织吸收血液。等到所有出血已在继续之前停止。 </li><li>慢慢插入10微升注射注射器预加载了载体溶液进入脑到预先确定的深度（背腹坐标）。等待1分钟，以代替针。注射3微升使用机动微量注射泵，吞吐量0.25微升（的rAAV2 / 7α-SYN或绿色荧光蛋白控制向量的3.0 E11基因组拷贝/ ml（中等矢量剂量）或1.0 E12 GC /毫升（高矢量剂量））向量解/分钟。 </li><li>注射后，保持针到位再过5分钟，慢慢地，再取出。使用拼接编织涂层聚酯3.0头皮，消毒用1％jodium在70％异丙醇和轻轻地从立体定向仪取出的动物。首先松开鼻子和嘴棒，那么这两个耳酒吧。 </li><li>扭转麻醉，用0.5毫克/千克腹膜内阿替美唑注入大鼠和放置老鼠在一个干净的笼子上的38℃的加热板，直到其被唤醒。覆盖纸毛毯大鼠防止体温下降。 </li><li>提供容易获得食物和水的第一个小时。监测的最初几天的老鼠。如果需要应用镇痛。 注意:没有必要去除从t线圈他的头骨。 1-2周后颅骨完全修复和拆线松动。 </li></ol> 3.的rAAV2评估/ 7α-SYN注射的大鼠使用非侵入性的PET显像，行为测试和免疫组化分析<ol><li>跟进黑质纹状体多巴胺神经变性的动力学非侵入过在动物个体时，量化多巴胺转运蛋白（DAT），使用小动物正电子发射断层扫描（PET）和DA转运如的示踪剂结合[18 F] -FECT 16 。 </li><li>审查多巴胺能神经变性的程度是否足以诱发在大鼠运动障碍，若使大鼠在气缸试验，评价自发前肢使用。 <ol><li>放置老鼠在期间沿后部后壁和到达垂直运动宽透明玻璃筒和录像带的行为20厘米分数由每个前爪做触点数量共计20联系人。对的评分标准的详细描述，参见Schallert 等人 18快速的受损前肢触点（ 例如左前爪），为总前肢接点的百分比（左加右前爪）的数目。 注意:在使用两个爪同样非损伤对照大鼠应在此测试得分50％左右。 </li></ol></li><li>进行免疫组织化学（IHC）分析来评估转基因表达和多巴胺能细胞损失的水平。 <ol><li>在不同的最终阶段，处死大鼠用戊巴比妥钠（60毫克/千克，IP）的过量，并用冷盐水随后在PBS 19 4％多聚甲醛执行心脏内灌注。固定的大脑在4℃过夜，并用振动切片机切成50μm厚冠状脑切片。 </li><li>使用抗α-SYN和酪氨酸羟化酶的抗体来分析α-SYN表达水平和乐自由浮动的部分进行IHC染色神经退行性疾病16德维尔。 </li></ol></li></ol>

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作者宣称，有没有利益实际或潜在的冲突。

有该协议中的几个关键步骤。载体滴度以及载体纯度直接影响该模型的表型结果。过度载体滴度或充分纯化的载体批次，可能会导致非特异性毒性。因此，使用高品质的矢量批次，适当的控制载体是必不可少的。此外，鼠的头在立体框架的精确定位和坐标的精确确定是在指定目标的黑质是必不可少的。在注射部位钻在头骨的孔后，它在针插入直入老鼠的大脑不接触任何边距是重要的。针应注射病毒载体后慢慢取出，以防止载体的泄漏。最后，缝合后，头皮应与在70％的异丙醇1％jodium消毒由其他动物，以避免拆线的咬入。或者其他防腐试剂可以使用。 所描述的方法还可以用于开发用于帕金森氏病24的rAAV2 / 7α-SYN基于小鼠模型。在小鼠中，我们注入2微升rAAV载体的SN中的体积。相比大鼠，小鼠的DN似乎有点到α-SYN过表达，导致神经变性的延迟表现不太敏感。此外，在脑的其他区域（ 例如纹状体，海马，皮质， 等 ）可被靶向。对于不同脑区的坐标可以在立体图谱中找到。的坐标的最优化可以通过中国墨或通过使用编码报道基因的病毒载体（ 例如 ，eGFP的）来完成。不同载体系统（腺相关病毒，LV 等 ）可根据应用使用。 这种技术具有每个动物有单独注入的限制。因此，一个训练有素的人，应执行注射，以闵不同的动物之间imize变化。另一个限制是，该方法是耗时（当由一个受过训练的人执行它需要每只动物约45分钟）。只有八到十个动物能在一天之内注射。 病毒载体介导的基因递送允许大脑区域的特异性靶向。使用病毒载体，高转基因表达水平可以实现的，这是至关重要的，因为疾病发作和严重程度取决于α-SYN表达水平。此外，不同的剂量可以应用，这将导致在动物模型中显示神经变性的慢或更快的动力学。最后，该技术可以被用来创建使用相同的载体制备不同的动物物种和菌株的模型。 此过程可用于递送病毒载体以及毒素（ 如 6-OHDA）到由载体编码的brain.The转基因的不同区域可以是一个报告蛋白，治疗性protein对于基因治疗的应用8-10或用于疾病建模11-14疾病相关的蛋白质。这种技术可以用于开发新颖的动物模型其允许临床前药物测试，并且可以在研究帕金森氏病，以及许多其它神经变性疾病的分子机制是有利的。

研究PD的病理生理学和开发新的治疗策略，对于动物模型可以非常类似于人PD的神经病理学，生理和运动症状的迫切需要。较高的预测值，我们就能更好地转化动物模型的新疗法给患者。 α-突触核蛋白的发现（α-SYN）在1997年第一PARK基因导致了第一个基因PD模型的发展。许多表达人野生型（WT）或突变（A30P，A53T）的转基因小鼠α-SYN已在过去十年中产生的。 α-SYN过表达的水平已被证明是在病理学的发展是至关重要的。另外，小鼠品系，内源性α-SYN的和是否全长或截短形式表示存在或不存在，播放（由洋红色和Chesselet 1详细审查）的作用。 WT和人类的几个临床突变体的过表达＃945; -SYN在转基因小鼠诱导α-SYN病理累积和神经元功能障碍2-6。然而，直到现在大多数转基因α-SYN的小鼠模型未能显示效果清晰多巴胺细胞损失和多巴胺相关的行为缺陷。 这种障碍是由黑质（SN）用病毒载体过表达α-SYN的直接针对克服。病毒载体是从病毒，可以很容易感染细胞，引入遗传物质引入其宿主基因组中，并迫使宿主细胞中复制的病毒基因组中，以产生新的病毒颗粒衍生的。病毒可以被工程化以保留其进入细胞并引入基因的能力的非复制的病毒载体。通过删除的病毒基因组的某些部分与由感兴趣的基因替换它们，载体的应用将导致在单轮感染而不会在宿主细胞中，通常被指定为"转导"复制。病毒载体Ç一个同时用于过表达和基因沉默。所表达的转基因可以是一个报告蛋白（ 例如绿色荧光蛋白或萤火虫萤光素酶）7，治疗性蛋白质的基因治疗应用8-10，或如我们将集中于在本文中，用于疾病建模11的疾病相关蛋白质-14。 病毒载体介导的基因递送提供了另一种方式来表达在CNS转基因与几个优点。利用当地的转基因传送，特定的大脑区域可以有针对性。此外，转基因的表达可以减少成年后的补偿机制发展过程中的风险过程中引起的。此外，可以在不同的物种和株被创建的模型。最后，不同的转基因可以很容易地进行组合。使用病毒载体，高转基因表达水平可以实现，因为该疾病发病和严重程度常常取决于曝光过度的水平，这可能是重要的ression。 根据不同的病毒数载体系统已经开发出来。载体系统的选择依赖于感兴趣的基因，基因表达，靶细胞和生物安全问题的需要的持续时间的大小。在大脑中，慢病毒（LV）的稳定的基因转移和重组腺相关病毒（腺相关病毒）载体现在被认为是选择的载体系统，因为它们导致在啮齿动物脑有效和长期的基因表达。对于SN的多巴胺能神经元（DN）特异性靶向，腺相关病毒载体已逐渐outcompeted由于其较高的效价和DN的转染效率的LV载体。 目前可用的最佳α-SYN基于啮齿动物模型已经从使用较新的AAV血清型的组合的方法显影（腺相关病毒1，5，6，7，8）和优化的载体构建体，滴度和纯度15,16。载体滴度以及载体纯度直接影响该模型的表型结果。过度载体滴度或充分纯化的载体批次，可能会导致非特异性毒性。因此，适当的控制向量是不可缺少的。在病毒载体生产，像素提升和纯化过程相当长的时间也投资已经证明必不可少获得可重复性和高品质的矢量批次。

<table><tbody><tr><td>Female 8 weeks old Wistar rats</td><td>Janvier</td><td>/</td><td>200-250 g</td></tr><tr><td>Ketamine (Nimatek)</td><td>Eurovet animal health</td><td>804132</td><td></td></tr><tr><td>Medetomidine (Dormitor)</td><td>Orion-Pharma/ Janssen Animal Health</td><td>1070-499</td><td></td></tr><tr><td>&amp;nbsp;Local anesthetic for scalp and ears: Xyloca&amp;iuml;ne 2% gel</td><td>Astrazeneca</td><td>0137-547</td><td></td></tr><tr><td>Terramycine</td><td>Pfizer</td><td>0132-472</td><td></td></tr><tr><td>Bupr&amp;eacute;norphine (Vetergesic)</td><td>Ecuphar</td><td>2623-627</td><td></td></tr><tr><td>Jodium 1% isopropanol</td><td>VWR</td><td>0484-0100</td><td></td></tr><tr><td>stereotactic head frame</td><td>Stoeling</td><td>/</td><td></td></tr><tr><td>Hamilton Syringe (30 gauge -20 mm -pst 2)</td><td>Hamilton/ Filter Service</td><td>7803-07</td><td></td></tr><tr><td>atipamezole (Antisedan)</td><td>Orion-Pharma/Elanco</td><td>1300-185</td><td></td></tr><tr><td>rAAV A53T &amp;alpha;-SYN vector</td><td>LVVC, KU Leuven</td><td>/</td><td>https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular-virology-and-gene-therapy/lvvc/</td></tr><tr><td>sodium pentobarbital (Nembutal)</td><td>Ceva Sant&amp;eacute;</td><td>0059-444</td><td></td></tr><tr><td>microtome</td><td>Microm</td><td>HM650</td><td></td></tr><tr><td>rabbit polyclonal synuclein Ab</td><td>Chemicon</td><td>5038</td><td>1:5,000</td></tr><tr><td>rabbit polyclonal TH Ab</td><td>Chemicon</td><td>152</td><td>1:1,000</td></tr><tr><td>Lutetium oxyorthosilicate detector-based FOCUS 220 tomograph</td><td>Siemens/ Concorde Microsystems</td><td>/</td><td></td></tr><tr><td>radioligand: 18F-FECT</td><td>In house</td><td>/</td><td></td></tr><tr><td>L-dopa: Prolopa 125</td><td>Roche</td><td></td><td>6 mg/kg i.p.</td></tr><tr><td>DMEM, Glutamax</td><td>Life Technologies</td><td>N&amp;deg; 31331-093</td><td></td></tr><tr><td>Foetal bovine serum</td><td>Life Technologies</td><td>N&amp;deg; 10270-106</td><td></td></tr><tr><td>25 kD linear polyethylenimine (PEI)</td><td>Polysciences</td><td>/</td><td></td></tr><tr><td>OptiPrep Density Gradient Medium: Iodixanol</td><td>Sigma</td><td>D1556-250ML</td><td></td></tr><tr><td>Optimen</td><td>Life Technologies</td><td>N&amp;deg; 51985-026</td><td></td></tr><tr><td>Paxinos 1 watston steretactic atlas, fourth Edition</td><td>Elsevier</td><td>/</td><td></td></tr></tbody></table>

development of an alpha-synuclein based rat model for parkinson's disease via stereotactic injection of a recombinant adeno-associated viral vector

为了研究帕金森氏病（PD）的分子途径，并制定新的治疗策略，科学调查依靠动物模型。的PD-相关基因的鉴定已经导致遗传的PD模型的发展。大多数转基因α-SYN的小鼠模型开发逐渐α-SYN病理，但未能显示效果清晰多巴胺细胞损失和多巴胺相关的行为缺陷。这一障碍，通过用病毒载体过量表达的PD-相关基因的黑质的直接靶向克服。使用病毒载体本地基因递送提供了一个有吸引力的方式来表达在中枢神经系统中的转基因。特定的大脑区域可以靶向（ 例如黑质），表达可在成人设置被诱导，并且可以达到高表达水平。此外，可以使用基于各种病毒不同载体系统。的协议概括了所有关键步骤来执行的病毒载体注射的大鼠的黑质开发用于帕金森氏病的病毒的基于矢量的α-突触核蛋白的动物模型。

实验的总体方案在图1中所描绘 A53Tα-SYN的重组腺相关病毒2月7日-介导的过度表达导致多巴胺相关运动障碍。 为了检验α-SYN过表达水平是否足以诱发在大鼠运动障碍，我们进行了大鼠的气缸试验，评价自发前肢使用（ 图3A）。 3周至注射后，一个显著运动功能障碍主要出现在接受剂量3.0 E11 GC / ml的A53Tα-SYN的rAAV2 / 7载体的作用。在注射4周后没有观察到自发对侧（左）前爪使用降低50％，而对照的eGFP的rAAV2 / 7注射的动物显示在前爪使用（ 图3B）无不对称。这获得了较高的αA53T-SYN的rAAV2 / 7载体剂量大鼠表现出更为明显的impairm在29天前爪使用（70％）的耳鼻喉科注射后（ 图3C）。为了证明所观察到的运动功能障碍是多巴胺依赖性，我们给予L-DOPA（6毫克/公斤的ip）的以高矢量剂量注射的大鼠的单剂量。当我们重复的L-DOPA治疗，前爪使用在A53T完全康复α-SYN的rAAV2 / 7注射的动物中观察到（ 图3C）后汽缸测试45分钟。 PET成像可以α-SYN逐步诱导的神经退行性疾病无创成像。 跟进黑质纹状体多巴胺神经变性的动力学非侵入过在动物个体时，我们量化多巴胺转运蛋白（DAT），使用小动物正电子发射断层扫描（PET）与[18 F] -FECT作为放射性配体结合。 DAT在A53T同侧尾状核结合显著减少α-SYN的rAAV2 / 7 injec特德大鼠随着时间的推移，但在绿色荧光蛋白对照动物保持稳定（ 图4A - 4B）。的A53Tα-SYN的rAAV2 / 7注射的动物的DAT结合定量分析表明注射后7天和21之间的黑质纹状体多巴胺变性的最大速率。 32天后，观察到（ 图4C）中的DAT减少高达85％的结合。作为阳性对照，神经毒素6-羟基多巴胺的SN的注射诱导的DAT的90％的损失于7天（ 图4B - 4C）结合。 在大鼠的SN的rAAV2 / 7 A53Tα-SYN的立体定向注射诱导黑质多巴胺细胞死亡和形成不溶性的α-SYN阳性聚集物。 分析α-SYN过表达和多巴胺能细胞损失的程度，我们处死动物在不同时间点。 IHC于使用抗体AG自由浮动切片上进行ainstα突触核蛋白（兔多克隆1:5000）。这种抗体可以检测人和大鼠α突触核蛋白，但大鼠α突触核蛋白的内源性水平低于黑质细胞的胞体中的检出限，在突触膜由于其主要定位。以评估细胞丧失我们用来对付TH抗体（兔多克隆1:1000）。 大鼠- rAAV载体注射后四天，α-SYN或eGFP的表达在SN（5C 图5A）进行检测。的DN的大部分（&gt; 90％）得到了有效转导和转基因两种蛋白质在细胞机构和轴突本地化。在29天的黑质致密部，观察注射后（PI）α-SYN表达大幅度减少，而它仍然在围绕SN（ 图5B - 5C）的区域检测到。下一步，我们分析了黑质细胞损失的水平。一种快速而逐渐丧失Ø超过29天后，检测到与注射的大鼠TH阳性神经元F的高达80％A53Tα-SYN的rAAV2 / 7（ 图5D - 5H）。值得注意的是，野生型而不是A53Tα-SYN导致类似的多巴胺能神经变性的表达（数据未显示）。的DN SN的损失是由TH阳性神经终端在纹状体（STR）（ 图5F）健壮减少并联。为了排除特定载体批量的影响，不同的α-SYN载体制剂具有相似的结果SN进行了测试。在SN中观察到TH染色没有减少或eGFP的的rAAV2 / 7的STR注射的对照动物（ 图5E-5H）。旁边多巴胺能神经变性，α-共核蛋白病的存在是PD的第二个重要的特点。尽管我们的模型（四周）的短时间过程中，我们在STR（ 图5I观察到SN两个α-SYN阳性细胞质聚合和变性神经突</str<html>翁&gt;）。泛素免疫反应是路易体病理的人的大脑20-22一个显着特点。我们观察到α-SYN表达黑质神经元（ 图5J）的一小部分（±20％）α-SYN的共定位和泛在29天圆周率。 α-SYN聚集体的纤维性质是由硫磺素S（硫代S）染色23进行评价。硫S IN从17天起（ 图5J）的SN检测阳性细胞。 <img alt="图1" src="/files/ftp_upload/53670/53670fig1.jpg" /> 图1:在渐进的神经变性的rAAV2 / 7α-SYN矢量结果立体定向注射于大鼠的SN的rAAV2 / 7α-SYN载体的立体定向注射诱导经由行为分析（圆筒试验），非侵入性的PET测定多巴胺能神经变性成像和分析IHC。D / 53670 / 53670fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53670/53670fig2.jpg" /> 图2:在大鼠的SN的rAAV2 / 7载体编码α-SYN的立体定向注射 。 （A，B，E）对大鼠头骨的颅缝，定义了两个参考点:前囟门和Lambda。 （C）大鼠脑组织呈现喷射的区域即SN的立体图谱。 （D）一个只Wistar定位于使用两个耳朵酒吧，嘴和鼻子吧立体定向头架老鼠。 （F）的30号针填充有载体被放置在黑质的位置。 （G），一个小的整体在注射部位钻出和针被放置在适当的位置。 （H）注射后头皮STItched和消毒。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53670/53670fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53670/53670fig3.jpg" /> 图3:重组腺相关病毒2月7日-介导的A53T的过表达α-SYN诱导多巴胺相关运动障碍 （A，B）气缸的rAAV2 / 7 A53Tα-SYN注射后不同时间点的测试。 （平均值±SD，* P &lt;0.05相对于17天，＃P &lt;通过ANOVA和Tukey事后检验0.05 eGFP的对照中，n = 5）。 （C）气缸的rAAV2 / 7 A53Tα-SYN（高剂量的载体）注射后在不同时间点的测试。 （平均值±SD，* P &lt;0.054天对29天的ANOVA和Tukey事后检验，n = 5）。该试验具有或不左旋多巴（L-DOPA）的施用执行的。 （平均值±标准差，* P &lt;0。05未处理的与处理的动物通过ANOVA和Tukey事后检验，n = 5）。从衰老神经生物学，卷转载。 36，范德Perren 等人 ，一个强大的大鼠模型的基于与腺相关病毒载体，1543年至1558年，（2015），爱思唯尔许可α-突触核蛋白过表达帕金森氏病纵向随访和表征。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53670/53670fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53670/53670fig4.jpg" /> 图4:A53T的非侵入性成像α-SYN使用DAT PET显像 （A - B） 引起的多巴胺的细胞死亡水平和冠状切片描绘平均纹状体DAT结合的系列（A）的rAAV2 / 7 A53Tα-SYN注射的动物在differenT时间点注射（N = 7）和（B）后的rAAV2 / 7 eGFP的注射（n = 1时）或6-OHDA处理的对照动物（n = 1时）注射后79天。彩条表示的DAT绑定潜力。的rAAV2 / 7 A53T的（C）中的DAT的定量结合α-SYN，的rAAV2 / 7的eGFP和6-OHDA注射在不同时间点测得的动物（数据代表平均值±SD）。从衰老神经生物学，卷转载。 36，范德Perren 等人 ，一个强大的大鼠模型的基于与腺相关病毒载体，1543年至1558年，（2015），爱思唯尔许可α-突触核蛋白过表达帕金森氏病纵向随访和表征。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53670/53670fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53670/53670fig5.jpg" /> 图5: </strong&gt;的重组腺相关病毒2月7日-介导的A53T的过表达α-SYN诱导多巴胺的细胞死亡，并生成不溶性的α-SYN阳性聚集的（A - B）IHC染色证实α-SYN表达4天29天的重组腺相关病毒介导的转移后大鼠SN。插入显示所选区域的放大倍数。比例尺= 400微米（概观画面左侧），70微米和200微米（插入右侧）。 （C）IHC染色表明绿色荧光蛋白的过表达4天，在大鼠SN重组腺相关病毒介导的转移后29天。比例尺= 400微米。 （D - G）中的SN注射（E，G）的​​rAAV2 / 7的eGFP的后喷射（D，F）的rAAV2 / 7α-SYN或29天之后IHC染色为TH在SN和STR在不同时间点。比例尺一个中，c = 400微米，B，D = 1000微米。 （H）的麻木的体视量化在SN TH阳性神经元的呃随时间的rAAV2 / 7 A53Tα-SYN注射或的rAAV2 / 7 eGFP的对照载体（平均值±SD后，* P &lt;0.05与8天，＃P &lt;0.05相对于通过ANOVA eGFP的控制和图基事后检验，n = 5）。 （Ⅰ）IHC染色证明α-SYN病理学，包括在SN细胞质聚集体和营养不良和鼓出突起在STR，后intranigral的rAAV2 / 7 A53Tα-SYN注射。 （J）为α-SYN（绿色）和泛素（红色）荧光双immunostainings的代表性共聚焦图像显示在共定位随时间（箭头）的增加。比例尺C = 50微米。 SN的硫黄素S染色的rAAV2 / 7 A53Tα-SYN注射后29天。比例尺D = 30微米。从衰老神经生物学，卷转载。 36，范德Perren 等 ，帕金森氏病的基础上α-突触核蛋白与广告的表达纵向随访和表征一个强大的大鼠模型ENO相关病毒载体，1543年至1558年，（2015），经许可爱思唯尔。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53670/53670fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector at JoVE.co

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

development-an-alpha-synuclein-based-rat-model-for-parkinson-s

Universidad San Sebastian

Research

JoVE Journal

Medicine

13.2K 次观看.  KU Leuven. This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease. 

视频: Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

帕金森氏症小鼠模型的感觉功能评估

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

科研

行为学

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

医学

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

生物学

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

从单体和体内使用的Α-syr核 in 预成型纤维的产生

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...

神经科学

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

将RatWalker系统应用于帕金森病遗传大鼠模型中的步态分析

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

帕金森病的6-羟基多巴胺大鼠模型

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.

分析编码人类α突触核蛋白的腺相关病毒载体诱导的帕金森病小鼠模型

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

Generating the 6-hydroxydopamine Rat Model of Parkinson&#39;s Disease

Source: Guimar&#227;es, R. P., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62923">The 6-hydroxydopamine Rat Model of Parkinson&#39;s Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a procedure to create a rat model of Parkinson&#39;s disease by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the brain. The neurotoxin infusion into the medial forebrain bundle (MFB) specifically targets dopaminergic neurons responsible for motor control, leading to their destruction and replicating Parkinson&#39;s disease symptoms.

生成帕金森病的 6-羟基多巴胺大鼠模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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