这项研究是由INSERM，艾克斯 - 马赛大学和HFSPO由CL弗赖斯获得职业发展奖的支持。

伦理声明:所有的动物研究是按照批准了埃克斯 - 马赛UNIVERSITE，法国的机构动物护理和使用委员会的指导方针和协议执行。雌性C57BL / 6小鼠在8 - 10周，根据DécretN°8 87-848，1987年10月19日，巴黎的规则购得和处理。全部采用L.实验原虫 LD1S寄生虫根据来自法国和欧洲联盟立法生物安全法规进行。 1.鼠标感染L.原虫鞭毛体寄生虫<ol><li>制备培养基进行体外维持L的原虫鞭毛体寄生虫，LD1S（MHOM / SD / 62 / 1S-CL2D）。 <ol><li>使用M199中。补充M199培养基，用10％胎牛血清（加热灭活在56℃，30分钟），25毫米的HEPES pH值6.9，12mM的碳酸氢钠 ，1mM的谷氨酸tamine，RPMI 16040维生素混合物，10毫叶酸，100mM的腺苷，7.6毫血红素，50U / ml青霉素和50mg / ml链霉素。 </li></ol></li><li>培养LD1S寄生虫在10ml完全M199培养基的在26℃在25 cm 2的通风烧瓶中。 </li><li>连续使用差速离心步骤，准备一个富含亚循环前鞭毛体形式的寄生虫人口。 <ol><li>收获了数中期寄生虫文化。指望一个hemocytomer。重悬5×10 5个寄生虫/ ml的在10ml完整的M199培养基中培养。生长的寄生虫培养在厌氧条件下，直到它们到达晚固定相（在约5-6天）。 </li><li>旋LD1S培养于1200×g离心10分钟，在26℃，以沉淀非亚循环寄生虫，回收上清液，并在2,500 xg离心再次旋转它在26℃下进行10分钟。 注意:此最终沉淀高度富集亚循环前鞭毛体寄生虫。 </li><li>悬浮在1颗粒00微升PBS。删除一个小等份固定用25％戊二醛1/100体积:体积和使用血球计数的寄生虫。 </li></ol></li><li>稀释富集-亚循环寄生虫种群至1×10 一百分之七微升PBS中。注入寄生虫悬浮到尾静脉麻醉小鼠的（见下文），使用1ml注射器与地下30针。对于对照动物，注入100μl的PBS中至尾静脉。 </li></ol> 2.外科手术<ol><li>消毒用杀生物喷雾消毒的工作空间和所有的手术器械用70％乙醇30分钟。 </li><li>制备的麻醉剂溶液，根据动物的体重，含有氯胺酮125毫克/公斤和12.5毫克/千克甲苯噻嗪的在PBS中稀释。 </li><li>称重小鼠并通过腹膜内用1ml注射器与地下30针注射麻醉剂的适当剂量。 </li><li>按住鼠标温暖和检查鼠标是完全anesthe通过继续之前捏垫脚级分配。重新注入在小鼠每隔60分钟的麻醉剂溶液的一半剂量。 </li><li>刮胡子鼠标的腹部，并与Vetedine清洁。使一个小切口在腹部的左侧胸廓下皮肤和肌肉。 </li><li>躺在刚打开面积腹部下方的一块湿纱布。暴露肝脏，将它放在纱布。躺在另一块纱布湿润肝上的。 </li><li>放置一个24×60毫米2到150微米厚的盖玻片，氰基丙烯酸酯，结合到金属框架，在显微镜下肝脏进行可视化。 </li><li>保护的小鼠的眼中，用湿纱布。 </li><li>成像会话后，安乐死颈椎脱位的麻醉动物。 </li></ol>肝脏结构的活体3显微成像<ol><li>开展活体显微镜实验上的倒置共聚焦显微镜配备有hermostatic受控室，复消色差63X油甘油浸没物镜（NA 1.4），氩（488纳米）和氦氖（543纳米，633纳米）激光器和蓝色二极管（405纳米）。 </li><li>将鼠标与盖玻片覆盖肝脏面朝下放在客观的显微镜阶段。设置在腔室的温度升高到29℃。使用肝自发荧光，以允许血窦可视调整焦点。 </li><li>为了显现血管，制备500微克的BSA-Alexa的647中的溶液稀释在100μlPBS中，每只小鼠。静脉内注射该溶液到尾静脉麻醉小鼠用1ml注射器与地下30针。 </li><li>为了显现肝细胞核，制备的Hoechst 33342的在PBS中的溶液。注入此溶液在8毫克/公斤的小鼠腹膜内用1ml注射器与地下30针。 </li><li>使用正常的顺序方式，63X浸没物镜和扫描仪在400赫兹到图像肝细胞核和肝脉管。打开405纳米的蓝色二极管激发赫斯特和BSA-647 Alexa的激发633纳米的激光。打开氩488 nm激光上定义大量收购乐队以可视化的肝自体荧光。 </li></ol>肝脏血流速度测量的活体4显微成像<ol><li>打开显微镜（LAS-AF浏览器版本3.1.0版本8587）的LAS软件。打开显微镜软件时选择共振扫描仪模式。 </li><li>单击配置选项卡上设置硬件。点击激光，然后选择氦氖激光633。点击设置并选择12位分辨率。 </li><li>点击获取菜单上。在光束路径设置窗口中，选择目标63X，并成立了633激光功率为100％。激活信号增益和关闭设定的参数从PM1-3下拉菜单中选择Alexa的-633。 </li><li>在采集选项卡中，选择"XYT"收购模式。设置格式宽1024×1024。在8000赫兹选择速度。选择3艾里单位针孔。选择3，缩放因子不要选择双向模式。 </li></ol> RBC速度的使用XYT图片5.定量分析（方法1） <ol><li>点击"在线"图标，在可视化的实时模式的血流量。选择感兴趣的区域，并选择一个特定的血管进行分析。通过调整USB控制面板上的"X"，"Y"和扫描磁场旋转坐标设定感兴趣的水平位置的容器中。 </li><li>停止"现场"模式，一旦感兴趣的容器设置。更改格式设置宽度为1024×256的采集模式设置窗口，平均线= 1，帧平均= 1，选择"栈"150点击"开始"，以获取图像。 </li><li>对于使用红细胞速度的定量分析"XYT"图片打开ImageJ的软件。选择"文件"在ImageJ的选项卡，单击"打开"，然后选择感兴趣的文件。 </li><li>转至ImageJ的"插件"菜单，选择基因座和生物格式导入选项。在这个窗口中，选择查看栈参数"hyperstack"。在堆叠顺序选项，选择该选项:"xyzct"。注意:这将打开一个窗口，所有的一系列收购。 </li><li>点击ImageJ的菜单上的"插件"，然后选择MTrackJ选项。点击"添加"图标上的小窗口，点击RBC跟踪。点击相同的红细胞中的每个以下框架，然后单击"度量"图标来测量速度。 注:生成可以保存在.exl格式的文件。 </li><li>跟踪至少每容器5 RBC和分析至少相同大小的三个单独的容器中。 </li></ol> RBC速度的使用XT线路图像6.定量分析（方法2） <ol><li>点击"直播"进行可视化血˚F低在实时模式。选择一个特定的血管进行分析。设定感兴趣的水平位置的容器中。停止"现场"模式，一旦感兴趣的容器设置。 </li><li>选择"XT&#39;扫描模式，并设置沿行扫描所选容器的中心内腔中。定格宽1,024×512，速度= 8,000Hz，平均线= 32，时间= 512。点击"开始"，并获得XT线路图像。 </li><li>通过打开.lif文件，并打开感兴趣的XT线图像产生条纹的红细胞速度的定量分析。在LAS-AF软件，选择"实验"，打开.lif并选择图像。注意:它会自动打开一个kymograph。 </li><li>测量所需的颗粒条纹（表示暗反射）的时间（t，获得的在Y轴）的旅行有关此kymograph一定距离（d，所得的X轴）。选择工具"画线"水平画一条线条纹。注意在远处微米，在图像的底部中的结果标签产生秒的时间。 </li><li>计算速度为使用从kymograph获得的值:V =距离/时间。 </li><li>量化最少每XT图像5颗粒条纹和最小的尺寸相同的三个单独的容器中。 </li></ol>

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The authors have nothing to disclose.

小鼠肝脏的活体显微镜的最近发展开辟了在体内和实时5,9,10生理响应于感染的调查新的可能性。器官的血流量是一个关键的生理参数是经常改变的许多疾病。然而，生理和传染性条件下肝脏血流动力学的状态仍然是一个探索不良的区域。在这项研究中，IVM为基础的方法，这在以前适于脾和肿瘤脉管系统的研究中，实施了以量化在单个容器内肝脏血流速度，并测量在体内 11...

器官特异性血流动力学是任何哺乳动物器官重要的生理功能。血流异常可能是炎症的后果和器官功能障碍1的标志。因此，血液流动的组织，结构和功能出现生理和病理条件下进行分析的关键参数。已普遍用于分析在特定器官的血流量的技术包含一些限制，包括技术本身的分辨率极限（ 例 ​​如血流的多普勒成像），为的绝对血液流量只（体积的血液每次测量的能力单元服务器官）（ 例如光学相干断层扫描）和在速度在大量异质群体血管2,3的平均变化的测量。肝脏的循环系统链接不同容器亚型是异质在它们的大小，结构和功能。在这项研究中，活体显微镜（IVM）成像技术被应用到评估肝脏血流动力学体内，实时，在高分辨率和在平行于揭露的组成该肝器官的个体的血管的特征。这个强大的光学成像技术的最新发展使研究人员收集的动物生活在高时空分辨率的动态数据。通过允许直接可视化和体内特异性和快速生物过程的实时监控，IVM提供了一个独特的机会，研究者将图像的个别血管，并测量和量化单个红血球内的特别选定的速度（RBC）肝血管。 在这项研究中，我们已经实现了在小鼠肝脏的IVM技术研究小鼠感染的肝血流动力学的嗜肝寄生虫利什曼原虫的影响。L.原虫负责内脏利什曼病，严重的疾病，其特征急性上慢性炎症反应和病理学，其存在于多个器官，包括脾和肝脏中的试剂。内脏利什曼病的实验小鼠模型中，肝感染是自我解决，而脾感染是渐进4。 利什曼原虫属感染的相对于该个体的器官，这些结果仍然没有完全了解。肝，脾血流动力学病理状态下调查将揭示宿主相互作用的寄生虫和疾病发病机制的新光源。 我们的实验模型系统是基于曝光和成像接受静脉注射特异性的荧光染料对肝intravasculature的标记麻醉小鼠的肝脏。肝脏是一个有利的器官内活体显微镜。执行一个小incisio后中的n腹部，肝脏轻轻外在并置于湿纱布，然后在与减少由于心跳和呼吸的任何运动伪影的目标盖玻片。肝脏，然后放置在显微镜透镜的视图内。相比于需要使用两个光子显微镜对IVM研究脾和淋巴结，肝的优势在于其均质3D架构/解剖学，允许使用常规共焦显微镜，具有最大穿透深度约50微米，对于活体显微镜成像5-8。 这项研究描述了两个独立的成像方法是在个别血管红细胞流速和血流速度的定量测量。在第一种方法中，使用的是XY二维的模式随时间获取肝脏血流量。所得XYT数据使用MtrackJ插件在自由的ImageJ软件，其允许individ的跟踪分析UAL红细胞随着时间的推移。在第二种方法中，一个单一的血管被选择及其相应的血流是利用共焦激光扫描显微镜的线扫描快速采集模式进行分析。感兴趣的容器被扫描在沿其中心轴的高频通过的轴线。血流速度，然后根据在未标记暗红细胞和荧光标记的等离子体之间的对比度的差量化。沿着线扫描中获得的红细胞和血浆的荧光强度对时间作图，得到的条纹，角度，其中成比例的单个红细胞的速度。 本文的目的是提供肝个别血管内的简单和可再现的方法进行成像并测量血流速度，并备妥的基本工具小鼠手术，IVM和速度的定量分析的成功表现个别红细胞。 Ť他的做法将允许研究人员获得病理状态下新的见解血流速度。

<table><tbody><tr><td>Hoechst 33342</td><td>Sigma Aldrich</td><td>B2261</td><td></td></tr><tr><td>BSA-Alexa 647</td><td>lifetechnologies</td><td>A34785</td><td></td></tr><tr><td>Dextran-FITC 500 mol wt</td><td>SIGMA</td><td>46947</td><td></td></tr><tr><td>Ketamine</td><td>PanPharma</td><td>20434</td><td></td></tr><tr><td>Xylazine</td><td>Bayer</td><td>KP07KEU</td><td></td></tr><tr><td>Vetedine</td><td>Pharma Animal</td><td>6869029</td><td></td></tr><tr><td>Cyanoacrylate liquid</td><td>Cyanolit</td><td>5833300005</td><td></td></tr><tr><td>Coverslip frame: Membrane slide for microdissection part N&amp;deg;: 5013</td><td>Molecular machines</td><td>50103</td><td></td></tr><tr><td>Coverslip DiaPath 24x60 ep: 1.6 mm</td><td>DiaPath</td><td>61061</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica&amp;nbsp;</td><td>TCS-SP5</td><td></td></tr><tr><td>LAS-AF viewer&amp;nbsp;</td><td>Leica software</td><td>Version 3.1.0 buid 8587</td><td></td></tr></tbody></table>

intravital microscopy imaging of the liver following leishmania infection: an assessment of hepatic hemodynamics

活体显微镜（IVM）是已经成为可能在实时和在活体动物各种生物事件的可视化，监测和量化一个强大的光学成像技术。该技术极大地提高了我们的生理过程，并在特定器官病原体介导的现象的理解。 在这项研究中，IVM施加到小鼠肝脏和协议设计为图像在体内肝脏的循环系统，并测量在个体肝脏血管的红血细胞（RBC）的速度。为了显现不同容器亚型表征肝器官和执行血流速度的测量，之C57B1 / 6小鼠进行静脉内注射一种荧光等离子体试剂标签的肝相关脉管。 IVM能够在体内实时，红细胞速度的测量中感兴趣的特定容器中。建立这一方法将使得能够研究生理和病理条件下肝血流动力学。最终，这种基于成像的方法将是非常重要的研究L的影响原虫感染对肝脏血流动力学。 这种方法可以适用于其它感染模型和小鼠的器官和可能进一步扩展通过量化其对血流量的影响预临床的炎症的药物的效果的测试。

在肝血窦的特定的架构组织可以基于这个器官（ 图1，小图B和C，绿色），腹膜内注射的Hoechst的肝细胞核的标记的自身荧光属性可视（ 图1B，蓝）和静脉注射荧光牛血清白蛋白为肝循环系统（ 图1C，红色）的染色。肝脏是由若干不同容器亚型具有不同的功能和结构以及尺寸范围从40-700微米2直径（D）的（小血管D是间40-80微米2和大血管D是大于300...

Watch this Scientific Journal Video about Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics at JoVE.com

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染:肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

intravital-microscopy-imaging-liver-following-leishmania-infection-an

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

11.5K 次观看.  University of Aix-Marseille. This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

视频: Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神经科学

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Antileishmanial筛选对细胞内的寄生虫救援和转化实验<em&gt;杜氏利什曼原虫</em无鞭毛体在THP1人急性单核细胞白血病细胞株

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...

生物学

In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if not treated; the drugs used in treatment are toxic, and there are cases of resistance. Preclinical tests can represent a bottleneck in discovering new medicines for treatment, depending on the animal model, the strain used, and the inoculum route. The golden hamster stands out for its high susceptibility to subgenera Viannia and Leishmania species, displaying many of the clinical and immunopathological processes observed in human disease.
By hamster anatomy, which has a short tail and limbs, the intracardiac route is usually the choice for intravenous injection of Leishmania. However, it is an inoculum that can lead to bleeding and eventually to animal death. Thus, we standardized an alternative intravenous inoculation route for infection at the gingival vein, which is minimally invasive, allows easy venous access, and causes few local and systemic injuries to the animal. Therefore, hamsters infected by the intraperitoneal (IP) or intragingival (IG) route with Leishmania Infantum expressing luciferase (Luc) were followed up for 22 days by the bioluminescence imaging system and 50 days and 8 months post infection by PCR.
After gingival inoculation of both axenic amastigotes and promastigotes of L. infantum-Luc, bioluminescence was restricted for at least 2 weeks at the site of injection, which is an indicator of infection in the tissues around the gingival plexus. Hamsters infected intraperitoneally with L. infantum-Luc displayed bioluminescence dispersed throughout the abdomen, as expected. However, by the bioluminescence imaging system infection declined until the 50th dpi and was only detectable by PCR.&#160;Axenic amastigotes showed better infection than promastigotes, as evaluated by PCR. Indeed, 8 months after infection, parasites were detected by PCR in the liver of animals inoculated with axenic amastigotes by the intravenous route, which can be a characteristic of the reference strain of L. infantum MHOM/BR/1974/PP75, whose infection progresses slowly and display low parasite burden, below the bioluminescent imaging resolution. Thus, axenic amastigotes can be a better choice for infection and follow-up than promastigotes, and the gingival inoculum is a feasible route for intravenous injection of Leishmania and other pathogens.

Here we present a longitudinal evaluation of golden hamsters infected intraperitoneally (IP) or via intragingival (IG) with L. infantum-Luc by bioluminescent imaging and by PCR. Hamsters were evaluated 1 day post infection (1 dpi), 1 week after infection (8 dpi), and 3 weeks after infection (22 dpi) and euthanized at the 50th dpi and 8 months post infection.

体内研究 通过牙龈接种表达荧光素酶的轴丝无毛体对 婴儿利什曼原虫感染的仓鼠进行成像

American tegumentary leishmaniasis (ATL) and visceral leishmaniasis (VL) are considered neglected by the World Health Organization. VL can be lethal if ...

肝脏活体如下显微成像

摘要

探索更多视频

此视频中的章节

在Live主机转利什曼原虫寄生虫在体内成像

活体鼠脑微循环显微镜使用一个封闭的颅窗口

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

Antileishmanial筛选对细胞内的寄生虫救援和转化实验

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

体内研究通过牙龈接种表达荧光素酶的轴丝无毛体对婴儿利什曼原虫感染的仓鼠进行成像

肝脏活体如下显微成像

摘要

探索更多视频

此视频中的章节

在Live主机转利什曼原虫寄生虫在体内成像

活体鼠脑微循环显微镜使用一个封闭的颅窗口

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

Antileishmanial筛选对细胞内的寄生虫救援和转化实验

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

在 Vivo 感染利什曼病亚马孙病评估小鼠的寄生虫毒性

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

体内研究 通过牙龈接种表达荧光素酶的轴丝无毛体对 婴儿利什曼原虫感染的仓鼠进行成像

体内研究通过牙龈接种表达荧光素酶的轴丝无毛体对婴儿利什曼原虫感染的仓鼠进行成像