作者感谢奥尔加·沙玛，特丽萨·亨特和的DZNE显微镜和动物设施的技术支持人员。

所有的程序，包括动物与北莱茵 - 威斯特伐利亚州环境局（LANUV）的动物保护委员会批准执行。动物饲养和照顾根据具有12小时光照/黑暗周期和食物和水自由进入的标准条件。 1.动物模型<ol><li>相互交叉的Tg半合子（GFAP -luc +/-）小鼠与半合子的Tg（M83 +/-）小鼠来生成半合子bigenic的Tg（M83 +/-:GFAP -luc +/-）小鼠15,16。 </li><li>基因型与实时PCR后代对于转基因编码的人类α-突触核蛋白的，并用标准的PCR存在下萤火虫萤光素酶12,17。 </li><li>在六到八周的年龄接种动物。 </li></ol> 2.接种物制备<ol><li>莫准备nomeric重组人α-突触核蛋白在含有150mM NaCl的20mM的Tris-盐酸（pH7.2）中作为已经Tris缓冲盐水（TBS）缓冲液的A53T突变先前描述的14。 </li><li>通过在800rpm和37℃下5天搅拌单体重组人α-突触核蛋白的3微克/μL在定轨摇床中制备原纤维α-突触核蛋白为接种。 </li><li>稀释在磷酸盐缓冲盐水（PBS）原纤维组件，以达到1微克/微升的最终浓度为腹膜内或2微克/μL为intraglossal接种。 </li><li>片段与杆超声波仪的α-突触核蛋白原纤维进行1分钟（0.5秒的持续时间的40个脉冲，每个脉冲和振幅设定为50％之间的一第二暂停）在冰上。为了避免交叉污染播种。用干净的超声探头准备不同的接种。 </li></ol> 3. Intraglossal注射<ol><li>麻醉动物用intraperitoneal注射100mg / kg的氯胺酮和10mg / kg的甲苯噻嗪。捏动物的脚趾，并确认它不撤回其后肢，以确保适当的麻醉。使用对动物的眼睛兽医药膏，以防止干燥而麻醉下。 </li><li>固定narcotized动物小心地用胶带上与它们对研究者面对头背卧位的加热板。 </li><li>填充超声处理的α-突触核蛋白原纤维或PBS的5μL一个27克的一次性皮下注射器。 </li><li>使用带锯齿的提示一个钝头镊子拇指持有动物的嘴打开，第二小钳子小心地拔出舌头，使舌底侧注射访问。 </li><li>注入注射器的针头插入接近舌的左侧或右侧底端到舌下神经。 5秒后缓缓注入接种。慢慢地缩回针之后5秒以确保接种已经渗透组织，同时缩回针不会丢失。 </li><li>从它的注视释放动物和离开它在持续监测加热板，直到完全恢复。 </li></ol> 4.腹腔注射<ol><li>对于腹膜内注射，通过吸入使用的2升/分钟的流速和蒸发器设定为2％异氟烷/氧不久narcotize动物在麻醉室中。捏动物的脚趾，并确认它不撤回其后肢，以确保适当的麻醉。 </li><li>填充超声处理的α-突触核蛋白原纤维或PBS的50μL一个27克的一次性皮下注射器。 </li><li>直接注射到小鼠的腹膜内，并避免通过与来自研究者背向头保持动物处于仰卧位和向下大约45°穿透小肠或盲肠位于腹壁后面。监控动物，直到他们恢复从麻醉。 </li></ol> 5.生物发光成像<ol><li>图像的Tg（M83 +/-:GFAP -luc +/-）小鼠每2-4周使用生物发光成像系统。 </li><li>在成像之前不久在异氟烷/氧气室以2升/分钟的流速和蒸发器设定为2％麻醉小鼠。捏动物的脚趾，并确认它不撤回其后肢，以确保适当的麻醉。 </li><li>刮脸和用脱毛膏脱毛的老鼠的头。颜色黑耳用无刺激性标记以封闭非特异性的生物发光。 </li><li>稀D-荧光素钾盐，萤光素酶的底物，在PBS中的30mg / mL的储备溶液。在-20℃1个毫升等分试样存储此。保护免于暴露溶解D-荧光素发光。 </li><li>前称量动物注射。计算D-荧光素为150毫克/公斤体重注射的准确体积。腹腔注射Ð-luciferin和动物恢复到麻醉室。 </li><li>启动成像软件。成像系统已经达到后其操作温度通过点击在控制面板的"初始化"按钮初始化系统。 </li><li>选择按钮"发光"和"照片"。曝光时间设置为60秒，分级为"中等"，在F /停止"1"，而EM增益为"关闭"。 </li><li>确认该激发滤光片被设置为"阻止"和发射滤波器，以"打开"，并且对象的高度设定至1.50公分。 </li><li>注射D-萤光素后十分钟，将动物在上成像室的加热板，并确保它们的枪口都正确放置在麻醉出口。关闭从吸入室中的异氟烷/氧气流，并打开用于成像室的流动以0.25 L / min的流速和2％的蒸发。关闭成像室PR的门operly。 </li><li>点击"捕获"按钮来衡量生物发光，这需要60秒。停止异氟醚流和监控动物，直到他们返回他们回到笼子后完全恢复。 </li><li>使用图像处理软件，量化生物发光。在该"工具选项板"选择"ROI工具"。根据"投资回报工具"中的"类型"选择"测量投资回报率。 </li><li>在该"投资回报工具"点击"圆"工具，选择投资回报率的数量从下拉菜单画 - 理想"3"三只老鼠。 </li><li>在图像中，右击每个ROI和下"属性"的宽度和高度调整至1.25公分。在被量化的大脑区域中的每个ROI的位置。 </li><li>在图像的左上角，所述单元面板设置为"辐射（光子）"。 </li><li>在该"工具面板"，选择"图像调整"和调整最低和最大的"色阶"，例如，距离0.20e6到1.00e6。 </li><li>在"文件"保存"保存"的数据。 </li></ol> 6.生化分析<ol><li>根据已建立的方案18,19隔离脑和脊髓。 </li><li>均质化全脑和整个脊髓样品分别在PBS中在蛋白酶和磷酸酶抑制剂（在PBS中稀释至1×）的两个30秒周期的存在下与在组织匀浆器6000转。确保脑和脊髓样品均质化至20％（重量/体积）的最终浓度。 </li><li>声处理样品的两倍，而以10个s的恒定脉冲和振幅设定为50％使它们保持在冰上。 </li><li> 1用1.5M NaCl的PBS:通过稀释它们1调整到匀浆在PBS和750mM的NaCl的10％。 </li><li>为了分离细胞碎片，离心匀浆物在1,000×g于5分钟4℃。保持下一个步骤的上清液并丢弃颗粒。 </li><li>测量根据制造商的说明书，使用二辛可宁酸（BCA）测定法匀浆中的蛋白质浓度。以10％（重量/体积）在冰上15分钟的最终浓度从孵育的脑匀浆1.0毫克总蛋白或从在N- laurylsarcosyl脊髓匀浆0.8毫克。 </li><li>叠加到匀浆在蒸馏水中的10％（重量/体积）3mL的蔗糖垫，并在4℃以465000×g下超速离心它们1个小时。 </li><li>重悬粒料在含4％十二烷基硫酸钠（SDS），2％β巯基乙醇，192 100mM甘氨酸，25毫摩尔Tris，和5％（重量/体积）蔗糖的缓冲液。 </li><li>煮沸样品在100℃下5分钟，并将它们加载到SDS-聚丙烯酰胺凝胶用于在吗啉乙磺酸（MES）缓冲系统电泳。 </li><li>转移分离的蛋白质以聚偏二氟乙烯（PVDF）膜中的SEMidry印迹系统。 </li><li>孵育所述膜在0.4％（体积/体积）甲醛在室温下在PBS中的溶液30分钟，在转子与35rpm下以交联与膜蛋白质。 </li><li>方框在含有0.05％（体积/体积）的TBS缓冲液吐温20和在室温下1个小时5％（重量/体积）奶粉的膜。 </li><li>孵育与初级抗体的印迹在4℃过夜封闭缓冲液。 </li><li>洗涤该膜三次在TBS缓冲液中，并用在室温下1个小时，在TBS过氧化物酶或荧光团偶联的二级抗体孵育它们。 </li><li>根据已建立的方案20通过化学发光或荧光显现结合的二次抗体。 </li></ol> 7.免疫荧光分析<ol><li>脱水解剖脑和脊髓组织中的加工工位，并且使用石蜡站将它们嵌入到石蜡中。 </li><li>切石蜡包埋TIS在8个微米厚的冠状切片切片机起诉并安装在载玻片上的章节。 </li><li>通过在两个单独的二甲苯浴5将它们孵育至10分钟Deparaffinize的组织切片，并通过一系列梯度乙醇浴（100％，90％，70％，50％），最后用H 2 O再水化它们</li><li>孵育在0.01M柠檬酸盐缓冲液（pH 6.0）的部分，在室温下5分钟，并另外煮沸用于在微波炉中10分钟。 </li><li>让部冷却到室温，并用30分钟3％过氧化氢溶液抑制内源性过氧化物酶孵育它们。 </li><li>用20％（体积/体积）正常山羊血清，1％（体积/体积）牛血清白蛋白（BSA）和0.5％的Triton X-100的PBS在室温下1个小时阻断由孵化的组织切片。 </li><li>孵育切片用1％（体积/体积）正常山羊血清，1％（体积/体积）BSA，和0.25％Triton在PBS X-100过夜，在4℃稀释一级抗体。 </li> &lt;LI&gt;用0.25％（体积/体积）的Triton X-100的PBS并用PBS洗涤一次的部分两次。 </li><li>染色与相应的荧光团偶联的二级抗体和在1％稀释的核染料DAPI（体积/体积）正常山羊血清，1％（体积/体积）BSA和PBS中于室温下1个小时的章节。 </li><li>用0.25％（体积/体积）的Triton X-100的PBS并用PBS洗涤一次两次后，盖玻片嵌入媒体的幻灯片和可视化用共焦激光扫描显微镜的染色。 </li></ol>

<ol>
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作者什么都没有透露。

外周注射的α-突触核蛋白原纤维的成的Tg（M83 +/-:GFAP -luc +/-）的小鼠腹膜表示简便方法来诱导神经系统疾病伴有神经炎症概括突触核蛋白病的重要特征。类似地，舌注射表示在转基因小鼠为神经侵染另一个路径由α-突触核蛋白prionoids但效率较低。我们选择在注射后420天终止我们的实验，我们不能排除多个或所有的纤维注射的小鼠可在稍后的时间点已经发展疾病的可能性。另外，使用的TG（M83 +/-:GFAP -luc +/-）小鼠星形胶质细胞增生能够非侵入性的可视化和表示超过的整个寿命监视引起α-突触核蛋白的神经侵染炎症反应的简单方法老鼠。 的关键步骤是在制备的接种。看似微不足道因素，如聚合时间，超声时间，丰度的内毒素，或原纤维的用于注射可通过影响错误折叠的α-突触核蛋白21的播种倾向影响的实验的结果的量。因此，注射接种准备时，总是使用相同的条件是很重要的。对于intraglossal注射它必须考虑，不仅舌下神经，还包括其他颅神经，如舌咽神经支配舌头和可参与α-突触核蛋白prionoids到大脑的输送元间。因此，针对舌头的不同区域可能会导致不同的传播动力学到大脑。我们并没有直接注入舌下神经，并有可能针对舌下神经可以改善α-突触核蛋白prionoids的CNS传输。对于α-突触核蛋白原纤维腹膜内注射是critiCAL的接种被正确注射到腹膜，不小心进样肠道或盲肠，这可能神经侵染负面影响内脏器官。注射D-萤光素时，这也是生物发光成像非常重要的。 D-荧光素，去内脏的误炸可以减缓其分布到大脑，从而导致较低的生物发光。于成像它在获得均匀的结果也是很关键的，以始终新鲜制备D-荧光素溶液注射前和让它准确孵育成像前腹膜内注射后10分钟。 生物发光成像是随时间的星形胶质细胞增生非侵入测量的变化的有用方法和不仅腹膜内或intraglossal注射后也是每一个类型的治疗，包括后可应用于，例如，脑内或静脉内注射。为监视从所述周边的α-突触核蛋白的prionoids蔓延到CNS和随后的神经炎症，我们使用bigenic的Tg（M83 +/-:GFAP -luc +/-）小鼠。因为在这些动物中的α-突触核蛋白的半合子的表达是相对于它们保持无自发疾病为高达22个月的年龄，并具有对α-突触核蛋白的传输的合适的遗传背景研究13纯合动物低40％。但是，关于用于生物发光成像的小鼠模型的选择修饰可以是合乎需要的。一个有趣的选择是使用仅表达荧光素酶的转基因，其允许在背景上的α-突触核蛋白原纤维的prionoid行为的评估，即基本上是野生型，只要小鼠的单基因的Tg（GFAP -luc +/-） α-突触核蛋白而言14。此外，交叉的Tg（GFAP -luc +/-）小鼠对其他的转基因小鼠，例如为Tg（SOD1 G93A）小鼠肌萎缩侧索硬化症或至Tg（APP23）米冰阿尔茨海默病，神经炎症使成像在其他疾病模型22，23。 此协议的一个限制是基于注射部位上注入的接种物不能超过一定的体积。因此注射到榫舌只能与除到腹膜，这可能影响传输时间到CNS较小的体积来进行。另一个限制是，GFAP -luc转基因只允许星形胶质细胞增生的检测但不小神经胶质细胞，其是在神经炎症同样重要。最后，这种模式不提供真知灼见，以腹腔注射后α-突触核蛋白prionoids如何到达中枢神经系统。 通过注射外源α-突触核蛋白prionoids的应用已被证明是研究在扼要重述synuclei的特性中起关键作用的传输路径的有效方法nopathies。重组α-突触核蛋白原纤维或疾病相关的小鼠或人α-突触核蛋白种类的脑内注射入动物模型已在几个研究中用于分析随时间5，7，17，24，25，26其播种性能和传输效率。由于立体定向注射到大脑手术后总是引起局部炎症反应很快，这种传播途径可能影响扩散而引起脑平衡的改变的接种感染。最近的研究已经改变了焦点外围或全身应用，而不是主要是为了避免手术过程，而是要分析周，肠壁，骨骼肌，或血液，因为从那里神经侵染到CNS可能合作的初始点mmence 6，9，14，27。对于朊病毒它已经表明，传输通过腹膜或舌导致神经侵染和神经系统疾病;舌头感染后，只有两个星期内扩散到大脑中的舌下神经核朊病毒干11。由于α-突触核蛋白已经讨论有像朊病毒neuroinvasive和播种特性，我们发现，通过榫舌和腹膜传输代表α-突触核蛋白prionoids侵入CNS 14进一步入口点。通过生物发光成像星形胶质细胞增生的量化便于神经炎过程的跟踪随着时间的推移，并且表示一种非侵入性的替代组织学分析。 为了获得一个更深入的了解中枢神经系统如何响应α-突触核蛋白prionoids的神经侵染并导致神经炎症其他治疗，这将是有益的，以生成小鼠模型也允许小神经胶质细胞的非侵入性监控， 例如与从特定于小胶质细胞蛋白表达的启动子驱动的萤光素酶表达的转基因。

有越来越多的证据表明，α-突触核蛋白具有类似于那些朊病毒蛋白，特别是在其能力，以自种子和细胞之间，并沿着神经通路传播错误折叠特性。 α-突触核蛋白的这个属性也被称为"朊病毒状"或"prionoid"，并通过观察在移植实验，这表明从患病神经元错误折叠的α-突触核蛋白的传递率支撑到新移植健康的神经元1,2 ，3，4。还直接注射错误折叠的α-突触核蛋白的进入大脑或周围， 例如后肢肌肉或肠壁，导致α-突触核蛋白病理到的远端部分的扩展的CNS 5，6，7，&lt;SUP类= "外部参照"&gt; 8，9，10。我们分析了通过外周途径α-突触核蛋白prionoids的传输中更详细和处理的问题的错误折叠的α-突触核蛋白是否可以在单个intraglossal或腹膜内注射后neuroinvade CNS中，先前已被证明为朊病毒但不能用于错折叠的α-特征突触核蛋白。朊病毒于舌的注射后，CNS的神经侵染经由传播沿着通向舌下神经，它位于脑干11的细胞核中的舌片的舌下神经实现。作为小鼠模型，我们选择的Tg（M83 +/-:GFAP -luc +/-）小鼠过表达来自朊病毒启动子的人α-突触核蛋白的A53T突变体，和下GFAP启动子控制萤火虫萤光监测由星形胶质细胞活化生物发光，如先前在大脑中所示朊病毒感染的小鼠12。在我们的手中bigenic TG（M83 +/-:GFAP -Luc +/-）如已被他人13所示的小鼠没有发生疾病直到晚上11个月的年龄。经由intraglossal或腹膜内途径诱导的神经学疾病与大脑病理学和Tg为脊髓人类α-突触核蛋白原纤维的单次注射（M83 +/-:GFAP -luc +/-）支持这一假设小鼠α-突触核蛋白prionoids分享朊病毒14的重要特征。

<table><tbody><tr><td>anti-actin antibody</td><td>Merck Millipore</td><td>MAB1501</td><td></td></tr><tr><td>anti-alpha-synuclein, phospho S129 antibody [pSyn#64]</td><td>Wako</td><td>015-25191</td><td></td></tr><tr><td>anti-alpha-synuclein, phospho S129 antibody [EP1536Y]</td><td>Abcam</td><td>ab51253</td><td></td></tr><tr><td>anti-GFAP antibody</td><td>Dako</td><td>Z0334 01</td><td></td></tr><tr><td>anti-IBA-1 antibody</td><td>Wako</td><td>019-19741</td><td></td></tr><tr><td>anti-Sequestosome-1 (p62) antibody</td><td>Proteintech</td><td>18420-1-AP</td><td></td></tr><tr><td>anti-ubiquitin antibody [Ubi-1]</td><td>Merck Millipore</td><td>MAB1510</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Invitrogen</td><td>14190169</td><td></td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Ratiopharm</td><td></td><td>100 mg/kg</td></tr><tr><td>Xylazine</td><td>Ratiopharm</td><td></td><td>10 mg/kg</td></tr><tr><td>27 G syringe</td><td>VWR</td><td>613-4900</td><td></td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Piramal Healthcare</td><td>PZN&amp;nbsp; 4831850</td><td></td></tr><tr><td>Depilatory cream</td><td>Veet</td><td></td><td></td></tr><tr><td>Secureline lab marker&amp;nbsp;</td><td>Neolab</td><td>25040</td><td></td></tr><tr><td>D-luciferin potassium salt</td><td>Acris</td><td>LK10000</td><td>30 mg/mL stock solution</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>5776671</td><td></td></tr><tr><td>Sonopuls Mini20 sonicator</td><td>Bandelin</td><td>3648</td><td></td></tr><tr><td>IVIS Lumina II imaging system</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>Living Image 3.0 Software</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>Tg(M83&lt;sup&gt;+/-&lt;/sup&gt;) mice or B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice</td><td>The Jackson Laboratory</td><td>004479</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-16</td><td></td></tr><tr><td>Narrow pattern forceps</td><td>Fine Science Tools</td><td>11002-12</td><td></td></tr><tr><td>N-laurylasarcosyl</td><td>Sigma</td><td>L5125-100G</td><td></td></tr><tr><td>Optima Max-XP ultracentrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td></td><td>TLA-110 rotor&amp;nbsp;</td></tr><tr><td>Thickwall polycarbonate tubes</td><td>Beckman Coulter</td><td>362305</td><td></td></tr><tr><td>NuPAG Novex 4-12% Bis-Tris Midi Protein Gels</td><td>Thermo Fisher Scientific</td><td>WG1401BOX</td><td></td></tr><tr><td>HRP conjugated antibody</td><td>Cayman</td><td>Cay10004301-1</td><td></td></tr><tr><td>IR Dye 680 conjugated antibody&amp;nbsp;</td><td>LI-COR Biosciences</td><td>926-68070</td><td></td></tr><tr><td>SuperSignal West Dura Extended Duration Substrate</td><td>Thermo Fisher Scientific</td><td>34075</td><td></td></tr><tr><td>Stella 3200 imaging system</td><td>Raytest</td><td></td><td></td></tr><tr><td>Odyssey infrared imaging system&amp;nbsp;</td><td>LI-COR Biosciences</td><td></td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>MP Biomedicals</td><td>TWEEN201</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>SA/T8787</td><td></td></tr><tr><td>Immobilon-FL PVDF membrane</td><td>Merck Millipore</td><td>IPFL00010</td><td></td></tr><tr><td>Xylol</td><td>Sigma</td><td>Roth</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma</td><td>SA/00216763/000500</td><td>working solution 3%</td></tr><tr><td>Bovine serum albumine (BSA)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A3294-100G</td><td></td></tr><tr><td>Goat serum</td><td>Thermo Fisher Scientific</td><td>PCN5000</td><td></td></tr><tr><td>4,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>working dilution 1:50,000</td></tr><tr><td>Fluoromount media&amp;nbsp;</td><td>Omnilab</td><td>SA/F4680/000025</td><td></td></tr><tr><td>LSM700 confocal laser scanning microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>HALT protease and phosphatase inhibitors</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10516495</td><td></td></tr><tr><td>Precellys 24-Dual homogenizer&amp;nbsp;</td><td>Peqlab</td><td>91-PCS24D</td><td></td></tr><tr><td>Alexa Fluor 488 conjugated antibody</td><td>Thermo Fisher Scientific</td><td>A31619</td><td></td></tr><tr><td>Alexa Fluor 594 conjugated antibody</td><td>Thermo Fisher Scientific</td><td>A11005</td><td></td></tr><tr><td>Pierce&amp;nbsp;BCA&amp;nbsp;Protein&amp;nbsp;Assay&amp;nbsp;Kit</td><td>Thermo Fisher Scientific</td><td>10741395</td><td></td></tr><tr><td>Microtome RM2255</td><td>Leica</td><td></td><td></td></tr><tr><td>LSM700 confocal laser scanning microscope</td><td>Carl Zeiss</td><td></td><td></td></tr></tbody></table>

bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils

为了研究错误折叠的α-突触核蛋白的朊病毒样行为，小鼠模型是需要的允许α-突触核蛋白prionoids，其对中枢神经系统（CNS）内的神经病理学快速和简单的传输。在这里，我们描述了α-突触核蛋白原纤维的那intraglossal或腹膜内注射入bigenic的Tg（M83 +/-:GFAP -luc +/-）小鼠中，过表达人类α-突触核蛋白与朊病毒蛋白启动子的突变A53T和萤火虫萤光所述启动子为胶质纤维酸性蛋白（GFAP），足以诱导神经病理学疾病。相较于纯合子TG（M83 + / +）小鼠发展在8个月，杂TG（M83 +/-:GFAP -Luc +/-）一岁开始严重的神经系统症状的动物，直到他们达到一个保持自由自发的疾病22个月的年龄。有趣的是，经由intraperitoneα-突触核蛋白原纤维的注射人路线诱导神经系统疾病与麻痹5的Tg（M83 +/-:GFAP -luc +/-）的四只小鼠用229±17天的中位数的温育时间。患病动物表现出磷酸化α突触核蛋白的严重存款在他们的大脑和脊髓。 α-突触核蛋白的累积是十二烷基肌氨酸钠不溶性且与泛素和P62共定位，并且伴随着导致星形细胞神经胶质增生和小神经胶质细胞的炎症反应。令人惊讶地，α-突触核蛋白原纤维进入舌的接种是引起疾病只有五组注射的动物示出后285天α-突触核蛋白病理的一个不太有效。我们的研究结果表明，通过经由腹膜内途径的intraglossal路线，更使接种是合适的，以诱导神经系统疾病与突触核蛋白病有关的标志中的Tg（M83 +/-:GFAP -luc +/-）小鼠。这为研究朊病毒样发病诱发的新模式由α-突触核蛋白prionoids更细节D。

小鼠（ 表1和图1）:通过舌或bigenic的Tg的CNS（GFAP -luc +/- M83 +/-）腹膜诱导神经病理学α-突触核蛋白prionoids的外周注射。一个单次腹腔注射与α-突触核蛋白原纤维后，五只小鼠的4开发神经疾病具有229±17天的中位数的温育时间。出人意料的是，五只小鼠的一个开发CNS疾病intraglossal注射后285天α-突触核蛋白原纤维后。接种PBS没有实验的420天时间内导致疾病。 通过Western印迹和用针对α-突触核蛋白的Ser129的磷酸化特异性抗体EP1536Y探测生物化学分析表明，两个传输路径的患病动物已经积累的磷酸化和十二烷基肌氨酸钠的聚集体不溶的α-突触核蛋白在他们的大脑和脊髓（ 表2和图2）。与此相反，大脑和PBS注射和非患病动物的脊髓仅包含磷酸化α-突触核蛋白的单体物质。 与磷酸化特异性PSYN＃64抗体腹膜内注射和患病小鼠的脑切片的免疫荧光染色显示磷酸化α-突触核蛋白沉积在整个CNS的宽分布 （ 表2和图3）。此外，磷酸化的α-突触核蛋白沉积共定位与泛素和P62表示的异常蛋白的体内平衡可能有助于沉积物的形成。 在患病动物的α-突触核蛋白聚集物的积累是伴随着神经炎的变化，其通过检测免疫荧光染色为GFAP，星形胶质细胞，可揭示反应性星形胶质细胞增生的标记物（ 图4）。此外，活化的小胶质与由免疫荧光染色丰富的α-突触核蛋白病理为IBA-1（离子化钙结合衔接分子1）的区域也被发现。按照，生物发光成像显示增加辐射（&gt; 2×10 6 P / S /厘米2 / SR）从Tg为大脑发出（M83 +/-:GFAP -luc +/-）与α-突触核蛋白原纤维注射的小鼠，前不久，他们开发的神经系统疾病的迹象。相比之下，用PBS注射的小鼠大脑并没有表现出任何的光芒增加。增加辐射指示反应性星形胶质细胞增生，神经炎症的标志的，由于荧光素酶表达从GFAP启动子驱动。 <img alt="图1" src="/files/ftp_upload/55503/55503fig1.jpg" /><br/&gt; 图1:通过腹膜内或intraglossal路线α-突触核蛋白原纤维注射引起疾病在bigenic的Tg（M83 +/-:GFAP -luc +/-）小鼠。 （A）重组超声处理人类α-突触核蛋白原纤维是的电镜观察腹膜内和intraglossally注射到小鼠中。用乙酸双氧铀负染色揭示的短，棒状聚集多个集群。比例尺= 100纳米。 （B）卡普兰-迈耶存活曲线表明，腹腔注射α-突触核蛋白原纤维四五的Tg（M83 +/-:GFAP -luc +/-）后的小鼠在229±17时代开发神经疾病（紫色实心正方形），而没有PBS注射对照小鼠内420天（紫色空心方块）开发的神经功能障碍的迹象。 intraglossal接种α-突触核蛋白原纤维后的五一后鼠标285天死亡（绿实心圆圈）。相比之下，没有PBS注射对照小鼠的开发神经系统疾病或死亡自发（绿色空心圆）。从Breid 等改性。 2016 14。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55503/55503fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55503/55503fig2.jpg" /> 图2:在外围的CNS磷酸化α-突触核蛋白的生化分析注入的Tg（M83 +/-:GFAP -luc +/-）小鼠。 （A）免疫印迹与抗体EP1536Y，在α-突触核蛋白的Ser129磷酸化识别，表明腹膜内注射和患病小鼠积累高分子量物质的磷酸化α-突触核蛋白的不溶性十二烷基肌氨酸钠-聚集体的在他们的大脑的ð脊髓。与PBS攻击的对照小鼠没有积累磷酸化α突触核蛋白的聚集和显示乐队不仅为它的单体形式。 （B）与α-突触核蛋白原纤维仅一只小鼠，动物121，intraglossal攻击后显示出其脑和脊髓磷酸化α-突触核蛋白的十二烷基肌氨酸钠-不溶性聚集体，而所有其他intraglossally接种的小鼠没有α-突触核蛋白的沉积物保持健康。分子量显示在千道尔顿。在每个泳道上样通过检测肌动蛋白的显示。从Breid 等改性。 2016 14。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55503/55503fig2large.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/55503/55503fig3.jpg" /> 图3:免疫荧光分析显示，存款磷酸化的α-突触核蛋白共定位在大脑和患病的Tg（M83 +/-:GFAP -luc +/-）的脊髓泛素和P62 小鼠。磷酸化的α-突触核蛋白的共染色，用EP1536Y抗体检测，和泛素在脑中发现明显的共定位，如在丘脑核（A），和脊髓观察到，如在灰质（B）检测到的与α-突触核蛋白原纤维腹腔攻击后患病小鼠。类似地，磷酸化的α-突触核蛋白，与PSYN＃64抗体进行检测，也共定位在脑（C）和患病动物的脊髓（D）P62。 （A至D）PBS注射健康对照动物没有显示存款或共存的这些蛋白质。用DAPI核染色显示为蓝色。比例尺=20μm以下。从Breid 等改性。 ，2016 14。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55503/55503fig3large.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/55503/55503fig4.jpg" /> 图4:TG（M83 +/-:GFAP -luc +/-）小鼠与α-突触核蛋白原纤维周攻击后开发的神经胶质增生。 （A）用针对GFAP，星形细胞的标志物，抗体患病动物的脑切片的免疫荧光分析显示反应性星形胶质细胞增生的地区与磷酸化α-突触核蛋白，这是在与所述PSYN＃64抗体脑干检测的沉积物。与此相反，有在PBS注射的对照小鼠的脑中没有反应性星形胶质细胞增生。 （B）同样，与抗体对IBA-1染色，小胶质细胞的标记物，小神经胶质细胞表现出在与患病动物磷酸化α突触核蛋白的存款领域。健康，PBS注射的对照小鼠的大脑没有显示小神经胶质细胞的迹象。 （C）生物发光成像显示从大脑和Tg为（M83 +/-:GFAP -luc +/-）脊髓升高的辐射小鼠α-突触核蛋白原纤维（左图）腹膜内注射，这是由星形胶质细胞的活化引起的前不久，小鼠发育的神经系统症状。在PBS注射的对照小鼠的脑和脊髓的基础辐射不随时间（右图）增加。 intraglossal接种用α-突触核蛋白原纤维后，一个Tg（M83 +/-:GFAP -luc +/-）小鼠，动物121，表明反应性星形胶质细胞增生的迹象前不久在285天（中心面板）死亡。 （D）Tg为腹膜内攻击后（M83 +/-:GFAP -luc +/-）小鼠与α-突触核蛋白原纤维，increa生物发光的SED水平（&gt; 2×10 6 P / S /厘米2 / SR）从四只小鼠（蓝色，绿色，棕色，和橙色圆圈），他们开发疾病的神经病学体征一些周前的大脑测量。一五只动物也表现出生物发光水平升高前不久intraglossal注射α-突触核蛋白原纤维（红色圆圈）后285天死亡。相反，对于健康的，PBS注射的对照小鼠（黑圈;误差棒显示SD [N = 4]）和未腹腔注射α-突触核蛋白原纤维（红色圆圈）后发展疾病的一种动物，生物发光信号保持低于2×10 6个 p / S /厘米2 / SR的观测周期内的阈值。比例尺=20μm以下。从Breid 等改性。 2016 14。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55503/55503fig4large.jpg" target="_blank">请点击此处查看这个更大的版本</a>数字。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>鼠标线</td><td>接种物（微克） </td><td>接种途径</td><td>小鼠的疾病号/无。小鼠接种</td><td>平均存活时间±SD（天） </td></tr><tr><td>的Tg（M83 +/-:GFAP -luc +/-） </td><td>人类α-共核蛋白纤丝（50） </td><td>腹腔</td><td> 4/5 </td><td> 229±四百二十零分之十七</td></tr><tr><td>的Tg（M83 +/-:GFAP -luc +/-） </td><td> PBS </td><td>腹腔</td><td> 0/5 </td><td> 0/420 </td></tr><tr><td>的Tg（M83 +/-:GFAP -luc +/-） </td><td>人类α-共核蛋白纤丝（10） </td><td> intraglossal </td><td> 1/5 </td><td>四百二十〇分之二百八十五</td></tr><tr><td>的Tg（M83 +/-:GFAP -luc +/-） </td><td> PBS </td><td> intraglossal </td><td> 0/4 </tD&gt; <td> 0/420 </td></tr></tbody></table> 表1:接种实验。 *从Breid 等改性。，2016 14 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>目标[抗体克隆] </td><td>主办</td><td>免疫原</td><td>稀释IF </td><td>稀释WB </td></tr><tr><td>肌动蛋白〔C4〕 </td><td>老鼠</td><td> - </td><td> - </td><td> 1:1000 </td></tr><tr><td> α突触核蛋白，磷酸S129 [PSYN＃64] </td><td>老鼠</td><td> pSer129 </td><td> 1:1200 </td><td> - </td></tr><tr><td> α突触核蛋白，磷酸S129 [EP1536Y] </td><td>兔子</td><td> pSer129 </td><td> 1:100 </td><td> 1:1000 </td></tr><tr><td>胶质纤维酸性蛋白（GFAP） </td><td>兔子</td><td> - </td><td> 1:200 </td><td> - </td></tr><tr><td> IBA-1 </td><td>兔子</td><td> - </td><td> 1:500 </td><td> - </td></tr><tr><td> Sequestosome-1（P62） </td><td>兔子</td><td> - </td><td> 1:100 </td><td> - </td></tr><tr><td>泛素[育碧-1] </td><td>老鼠</td><td> - </td><td> 1:500 </td><td> - </td></tr></tbody></table> 表2:用于免疫荧光（IF）和Western印迹（WB）的抗体。 *从Breid 等改性。，2016 14

Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

外周注射的α-突触核蛋白原纤维进入的Tg的腹膜或舌的（M83 +/-:GFAP -luc +/-）小鼠，表达人α-突触核蛋白与家族性A53T突变和萤火虫萤光素酶，可诱发神经病理学，包括神经炎症在他们的中枢神经系统。

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

bioluminescence-imaging-neuroinflammation-transgenic-mice-after

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.5K 次观看.  German Center for Neurodegenerative Diseases (DZNE). 外周注射的α-突触核蛋白原纤维进入的Tg的腹膜或舌的（M83 +/-:GFAP -luc +/-）小鼠，表达人α-突触核蛋白与家族性A53T突变和萤火虫萤光素酶，可诱发神经病理学，包括神经炎症在他们的中枢神经系统。 

视频: Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3, the intermediolateral nucleus of the spinal cord 4 and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8. Here we show in detail two methods used in our previous study 9. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.

Parkinson's disease has been related to the exposure to pesticides. Here we show a method to deliver pesticides using a gastric tube at the desired concentration and a method to analyze their effect in alpha-synuclein accumulation in the enteric nervous system.

鱼藤酮灌胃和图像分析α- synuclein的夹杂物在肠道神经系统的一个使用口服

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

免疫与感染

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

医学

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Bioluminescence Imaging of Neuroinflammation in a Transgenic Mouse

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/55503/bioluminescence-imaging-neuroinflammation-transgenic-mice-after'>Source: Breid, S., et. al. Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils. J. Vis. Exp. (2017)</a>This video demonstrates the bioluminescence imaging of neuroinflammation in transgenic mice, showcasing luciferase-luciferin reactions in activated astrocytes. The protocol enables non-invasive quantification of astrocytic activation, aiding studies on CNS inflammation and therapeutic efficacy.

Source: Breid, S., et. al. Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils. J. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Neurodegeneration & Disease Monitoring

Fluorescence Imaging of Neuro-Immune Dynamics Using a Skull Implant

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/61454/precise-brain-mapping-to-perform-repetitive-vivo-imaging-neuro-immune'>Source:&#160; Bisht, K., et al. Precise brain mapping to perform repetitive in vivo imaging of neuro-immune dynamics in mice. J. Vis. Exp. (2020)</a>This video demonstrates fluorescence imaging of neuro-immune dynamics in a transgenic mouse with a skull implant. Vascular and neuronal stability is imaged alongside dynamic microglial changes, reflecting immune responses to neuronal activity.

Source:&#160; Bisht, K., et al. Precise brain mapping to perform repetitive in vivo imaging of neuro-immune dynamics in mice. J. Vis. Exp. (2020)This ...

Live Imaging & In Vivo Tracking

Induction of Neuroinflammation in Mice via Injection of Alpha-Synuclein Fibrils

Source: Breid, S. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55503">Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils.</a> J. Vis. Exp. (2017).
This video demonstrates the injection of alpha-synuclein fibrils into a mouse to induce neuroinflammation. The fibrils are injected intraperitoneally into a transgenic mouse expressing mutated alpha-synuclein proteins. Upon reaching the central nervous system, the fibrils enter neurons and induce aggregation of the mutated proteins. The aggregates spread to neighboring neurons, microglia, and astrocytes. Recognition of the aggregates triggers microglia to induce neuroinflammation, damaging the neurons.

通过注射 α-突触核蛋白原纤维诱导小鼠神经炎症

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160; ...