Name: use of in vivo imaging to monitor the progression of experimental mouse cytomegalovirus infection in neonates
Uploaded: 2013-07-06T13:00:00
Description: Watch this Scientific Journal Video about Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates at JoVE.com

我们感谢李那么Tuddenham（IBMC，斯特拉斯堡）放大和滴定鼠巨细胞病毒的吕克·和托马斯鲍默特的（INSERM U748，斯特拉斯堡）为许可使用病毒学研究所动物设施。 INSERM的，大学和斯特拉斯堡：法新社国立德拉RECHERCHE（ANR-08-冕-005-01）的财政支持被确认。也承认他们的硕士项目“索尼娅Beroud和Laetitia Lelieur的期间初始参与。

1。病毒悬液的制备<ol><li>获得史密斯株的MCMV表达荧光素酶（MCMV -吕克·15）从乌尔里希Koszninowski的实验室。在这种重组体，荧光素酶基因插入在IE2鼠巨细胞病毒基因组的基因座。 </li><li>为了放大MCMV -吕克·鼠巨细胞病毒感染小鼠骨髓基质细胞线（M2-10B4，ATCC＃CRL-1972），在不同的感染复数（MOI，0.001〜1）16。对于这一点，在无血清培养基中培养细胞的病毒在37℃下一小时后?...

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场地 Using in 体内的成像技术，，，以监察MCMV的-吕克·传播在小鼠新生儿中的，，我们分别为能够观察到病毒式的传播到的大脑突变体的的的的动物，作为反对到野生-型。进一步解剖动物和体外成像的大脑确认发光病毒的存在下，在中枢神经系统。另外，我们也可以进行免疫组化实验（图中未示出）对脑薄切片在鼠巨细胞病毒E1早期蛋白质的特异性抗体，并观察到，实际上，M...

人类巨细胞病毒（HCMV/HHV-5）是β-疱疹病毒家族的成员。巨细胞病毒是一个非常普遍的，条件致病菌，通常在生命早期获得的无症状感染1。像所有的疱疹病毒，巨细胞病毒仍然存在的主机，其免疫系统严格控制病毒复制的整个生命。发作的病毒复发大多发生在免疫力低下的个体如移植患者接受药物，以防止移植排斥反应2。巨细胞病毒在成人中，也一直与胶质母细胞瘤3。此外，巨细胞病毒是一个突出的病原体为新生儿不成熟的免疫4-6。主要在发育中的胎儿或新生儿感染可有严重的后果。巨细胞病毒感染是最常见的先天性缺陷，以及在发达国家的儿童疾病感染的原因。据估计，新生儿巨细胞病毒感染的发病率影响0.5-1％F所有活产婴儿，其中5％至10％会患上严重的症状，如小头畸形或小脑发育不全。此外，10％被感染的亚临床病毒感染的婴儿将后来发展后遗症导致智力低下，听力损失，视觉缺陷或发作和癫痫7,8的 。 相对于其他的人类疱疹病毒，如单纯疱疹病毒1（HSV-1/HHV-1）可接种到小鼠通过不同的路线。注射9，巨细胞病毒复制具有物种特异性。此功能已严重阻碍了HCMV发病机理是不同的动物模型中进行的调查（小鼠，大鼠，豚鼠，恒河猴）及彼等各自真正的主机特定CMVs。 ，所有CMVs表现出显着的相似之处，基因组大小与组织，组织嗜性和基因表达调控。他们在各自的主机也引起类似的病症。尽管基因组多样性之间HCMV和小鼠巨细胞病毒（MCMV）（50％的存在于人类病毒的ORF中确定的小鼠巨细胞病毒），在小鼠模型最近已被证明是有利的，主要是因为他们有能力控制病毒，突变体菌株可以进行测试在体内复制。这导致了一个基因的屏幕，使小鼠基因表达在成年阶段组成的的“resistome”这种病毒10的数量估计。总之，这表明，鼠巨细胞病毒感染的小鼠在成人为研究宿主 - 病毒相互作用模型代表一个有吸引力的。先天性CMV感染的探索更复杂，因为人类和老鼠之间的差异，胎盘层组织损害母亲向胎儿传播的病毒感染小鼠。最近，直喷妊娠12.5天的鼠巨细胞病毒在胎盘使脑部感染小鼠而导致新生儿听力障碍11。然而，我最现在nvestigations使用4-20的HR岁新生儿腹腔注射全身性病毒的传播可能导致脑部感染血，一个模型，更比颅内注射有关。此协议的巨细胞病毒发病机制提供了重要的见解，更特别的是，它表明MCMV感染的新生儿的查询结果，分布在与单核细胞，例如巨噬细胞12浸润的炎症灶，在神经元和神经胶质细胞在病毒复制过程。该报告还伴有减少颗粒神经元的增殖和迁移和多个干扰素刺激基因诱导的小脑形态发生改变。 CD8 + T细胞在中枢神经系统的控制的MCMV的重要作用，也有报道由同一组13中 。的病理效果的微生物分析时要考虑的一个重要方面是动态的感染根离子。在鼠巨细胞病毒的情况下，它是病毒传播的进展探索和量化到发育中的大脑，以了解和预测未来的神经生物学受伤的幅度尤为关键。传统上，量化感染的进展，需要经常牺牲滴定的病原体感染的动物组织，如脑，否则无法进入。现在这种类型的协议是必要的改善动物福利和3R原则（减少，缩小，替换）原则14提出质疑。 在体内成像技术的使用可允许这是必要的，在体内感染实验动物的数量急剧减少。在这里，我们报告，并描述了一个时间过程分析病毒传播到大脑后腹腔注射MCMV - 吕克·鼠标新生儿。使用相同的动物，我们体内的跟踪和监控网站的激烈六拉尔在2周期间的复制。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Fisher Scientific</td>
			<td>W3523A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Methylene blue</td>
			<td>Sigma Aldrich</td>
			<td>319112</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insulin needles</td>
			<td>VWR</td>
			<td>613-4897</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine</td>
			<td>CentraVet</td>
			<td>Ket 201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine/Vetranal</td>
			<td>Sigma Aldrich</td>
			<td>46995</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DPBS</td>
			<td>DUTSCHER</td>
			<td>P0436500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Luciferin</td>
			<td>Caliper</td>
			<td>760504</td>
			<td></td>
		 </tr>
		 <tr>
			<td>gentamycin</td>
			<td>Sigma Aldrich</td>
			<td>G1272</td>
			<td></td>
		 </tr>
		 <tr>
			<td>penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>carboxymethylcellulose</td>
			<td>Sigma Aldrich</td>
			<td>C4888</td>
			<td></td>
		 </tr>
		 <tr>
			<td>formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		 </tr>
		 <tr>
			<td>crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C3886</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>IVIS 50</td>
			<td>Caliper/Perkin Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

use of in vivo imaging to monitor the progression of experimental mouse cytomegalovirus infection in neonates

人巨细胞病毒（HCMV或HHV-5）是一种危及生命的病原体的免疫能力较差的个人。先天性或新生儿感染后，该病毒可以感染和复制在脑发育，这可能会导致严重的神经系统损害，包括耳聋和智力迟钝。尽管有潜在的症状的严重程度，治疗的选择是有限由疫苗和特定的抗病毒治疗的情况下不可用。此外，感染中枢神经系统（CNS）期间发生的分子事件仍然缺乏一个精确的描述，因为观测大多来自受感染儿童的尸检。几种动物模型，如猕猴巨细胞病毒，已开发提供了重要的见解为巨细胞病毒的发病机制中的中枢神经系统。然而，尽管它的进化与人类接近，这种模式被限制用于的颅内接种程序，感​​染动物和缺点istently引起中枢神经系统感染。此外，伦理方面的考虑，促进发展替代型号，其中新生儿感染新生小鼠的鼠巨细胞病毒（MCMV）最近导致了显着的进步。例如，据报道，MCMV腹腔注射免疫Balb / c新生儿导致在特定的大脑区域的神经元和神经胶质细胞的感染。这些结果表明，实验小鼠接种HCMV感染引起的儿童复述赤字。然而，动态分析鼠巨细胞病毒感染的新生儿是很难执行，因为经典方法需要一个重大的牺牲动物的数量在不同的时间点来分析病毒负荷和/或免疫相关的参数。要绕过这个瓶颈，使未来调查的罕见突变的动物体内成像技术，我们采用维拉执行时间过程分析MCMV的重组表达荧光素酶C57BL / 6新生儿外周注射后大脑中升传播。

一位代表实验， 如图1所示。 MCMV - 吕克·50个PFU（图A显示了类似注射亚甲蓝进行形象化的皮下注射针）腹腔注射后，新生儿麻醉，并同时接收荧光素，荧光素酶底物（钳）0.3毫克。十五分钟后，动物被置于腹侧收购的IVIS 50室（钳）在活体成像系统，在10秒的曝光，整个动物发出的光被抓获。面板B显示快照拍摄的图像分析专用软件（生活图片3.2，卡尺）7天，9天，11和14。图像具有相同...

Watch this Scientific Journal Video about Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates at JoVE.com

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

人类巨细胞病毒（HCMV）感染新生儿智力低下的一个重要原因，但仍然知之甚少的分子事件导致病毒引起的发病。要调查脑部感染的动态，我们适应整个动物<em&gt;在地体内</em&gt;成像执行时间过程分析与荧光素酶的重组病毒感染的新生儿。

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

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Immunology and Infection

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

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Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

分离中枢神经系统组织和相关脑膜，用于免疫细胞的下游分析

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...