Name: bioluminescence imaging of neuroinflammation in transgenic mice after peripheral inoculation of alpha-synuclein fibrils
Uploaded: 2017-04-13T13:00:00
Description: Watch this Scientific Journal Video about Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils at JoVE.com

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<sup ...

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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。?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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-gauge syringe</td>
			<td>VWR</td>
			<td>613-4900</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal Healthcare</td>
			<td>PZN&#160; 4831850</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Secureline lab marker&#160;</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+/-) 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&#160;</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>TLA-110 rotor&#160;</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&#160;</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&#160;</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20&#160;</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)&#160;</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&#160;</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&#160;</td>
			<td>10516495</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys 24-Dual homogenizer&#160;</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&#160;BCA&#160;Protein&#160;Assay&#160;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...

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 ...

Research

JoVE Journal

Neuroscience

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

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

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

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, ...

Live Imaging of Dorsal Root Axons after Rhizotomy

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

可溶和不可溶α-突触核蛋白从帕金森脑连续提取

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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

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 ...