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，除去上清液，并用10％胎牛血清（FCS）的DMEM培养液更换。五天以后，收获的培养基和存储等分在-80°C，直到进一步利用。 </li><li>滴定斑块检测病毒悬液。 <ol><li>补充有10％FCS含有青霉素（200国际单位/毫升）和steptomycin（200毫克/毫升的拆分3T3细胞（ATCC编号：CRL-1658）在24孔板中以40,000细胞/孔的1毫升的DMEM）和在37℃培养24小时</li><li> MCMV悬浮液的等分试样在DMEM准备的系列稀释液（10 -1〜10 -8），和感染的靶细胞（3T3），去除细胞培养液中后，用200μl的稀释。孵育1小时，在37°C。加入1 ml的常用培养基（DMEM培养液3％FCS，2％（体积/体积）庆大霉素，200国际单位/ ml青霉素，200毫克/毫升链霉素，羧甲基纤维素8克/升），孵育5天，在37℃下</li><li>通过向每孔添加200μl的10％的甲醛（甲醛与水的稀释工作应在化学罩，以避免有毒吸入），并在室温下温育6小时，固定细胞。移除培养基+固定器通过移液，加入200微升细胞染色溶液（1％（重量/体积），在20％乙醇中结晶紫）。在使用前，稀释1份与9份milli-Q水中。在室温下孵育2分钟，洗板3-4X浸泡在水中，让干燥和计数斑块数量与双眼视。 </li></ol></li> &lt;LI&gt; MCMV - 吕克·在DMEM稀释50微升，离开冰，直到进一步利用获得50个PFU。使用更高的病毒接种诱发新生儿前达到2-3周龄的杀伤力。根据我们的经验，500个PFU诱导杀伤力在每一个新生儿注射后5〜7天。 </li></ol> 2。新生儿注射<ol><li>当新生儿（4至20小时的小鼠），操纵的枯枝落叶和粪便笼手套才认真处理幼仔与“外国”的气味会强调母亲和幼崽诱导拒绝，以避免污染。 </li><li>执行在图1A中所示，使用胰岛素注射器（29 G）的腹腔内注射。握住新生儿背部皮肤。前腿下腹侧，介绍了针，轻轻推皮下注射，以达到腹腔（一点点的肚脐下）。目前，我们注入1％亚甲蓝在DPBS稀释和监测生存到practice前执行病毒感染的技术。由于亚甲蓝是清晰可见的，在注射过程中，我们建议执行这个“训练”注射的病毒悬浮液在腹腔内，以确保正确交付。 </li><li>消除可能出现微小的血滴进笼子放置在新生儿背部。 </li></ol> （3） 在活体成像<ol><li>在第7天，如前面提到的，具有相同的警告处理新生儿。注入腹腔注射麻醉剂（4毫克/毫升，0.8毫克/毫升的甲苯噻嗪氯胺酮）和荧光素（0.15毫克/克体重）在50微升在每个动物的混合物。动物的平均体重为1-2克的剂量麻醉药每只动物是：氯胺酮40微克，甲苯噻嗪8微克等待12-15分钟，直到荧光素达到其最大的排放。这是要预先确定的，并依赖于荧光素供应商所使用的成像系统。 </li><li>虽然幼仔anestheti的捷思，他们为未来的识别标记和收获一小块组织，如果突变的动物，然后将其用于未来的基因分型。 </li><li>将幼崽的成像系统中，并执行全身发射的光的采集。感染后七天，病毒复制成无数份的靶器官如肝，肾，肺和唾液腺;因此，暴露的时间应尽量短。在我们的例子中，我们执行10到20秒（ 图1B）的收购。采集的成像装置的腔室中的平台需要被加热，以避免过度冷却麻醉动物的体温。此外，它有利于获得低分辨率图像的几种动物一轮收购，然后，移动平台更接近摄像机的镜头，以专注于一个特定的动物或一个孤立的器官解剖后的一个部分。该软件必须能够快速的变化参数（曝光时间，距离样品/透镜，灵敏度），量化定义感兴趣的区域和未来的出口快照图像。 </li><li>为了获得光信号只能从头部，包括新生儿的身体横向躺在一个厚厚的，黑纸，将掩盖水平出现高病毒复制的器官（肝，肺，唾液腺）发射的光子和执行采集5〜10分钟（ 图1C）。进行采集的动物在相同条件下的相对侧。 </li><li>重新引入小狗入笼，并在上面放置一个加热灯，以避免新生儿麻醉的过度冷却。定期监测麻醉后恢复。随着指示的剂量，麻醉持续30分钟。 </li><li>重复同样的步骤，9日，11日和14日。在11和14天，改变剂量的麻醉药（6毫克/毫升氯胺酮，1.2毫克/毫升甲苯噻嗪）。 </li></ol>

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作者宣称没有竞争的财务权益。 伦理声明动物保持无病原体条件下，在学院d&#39;Immunologie的等d&#39;Hématologie的动物护理设施。按照法国法律保护实验动物小鼠实验程序进行处理。本程序是在获由的服务véterinaire德拉县DU BAS-莱茵（法国）的授权下号码 - 答-67-345批准。

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 ...

use-vivo-imaging-to-monitor-progression-experimental-mouse

Research

JoVE Journal

Immunology and Infection

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

14.8K 次观看.  Université de Strasbourg. 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，我们适应整个动物在地体内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。

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

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

羧基荧光素二醋酸琥珀酰亚胺酯（CFSE）用于监视淋巴细胞增殖

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

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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 ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

李斯特菌单细胞基因感染后小鼠脾脏原位干扰素伽马生产的成像

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...