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 11,12个体红细胞速度。此处所使用的方法依赖于等离子体荧光团的小鼠静脉内注射，如荧光牛血清白蛋白之间的对比，和红细胞，这仍然是黑暗， 在体内的血流量测量必须提高我们病亲理解的电位正如事实。 在这里，两种方法被用于比较测量在肝血管的血流量。第一方法是耗时和挑剔数据分析的术语，因为它要求用户在利用MTrackJ插件中的ImageJ软件单个容器手动跟踪红细胞运动。第二方法是简单和快速的，因为它包括从一个线扫描采集与共振扫描仪模式中产生的血管的XT图像的分析。这两种方法给出可比数据类似的船只。在这个阶段，几乎是不可能的我们的数据与其他人相比，因为，据我们所知，血流速度从未量化在这样的小血管和在小鼠正弦毛细管具有大约10μm的宽度。 IVM为基础的方法允许高分辨率为500nm使得正弦曲线的分析用10-50微米13的宽度的xy。与此相反，基于多普勒的技术仅要得甲阶ution为70μm，因此限制了血液流向肝脏动脉和门静脉3的测量。 其中一个主要步骤，是对成功实施这些方法的关键是整个成像会议期间进行了成功的手术和维持麻醉鼠标在良好的生理状态。要做到这一点，必须小心地控制显​​微镜室的温度，并每隔一小时注射麻醉剂。另一个关键步骤是在荧光等离子体试剂到其注射后可能发生的自然内皮衬里的内化。这样的内部会产生难以解释的XT的图像和非XY对比图像。这一现象决定于注入的染料的量，并可以通过注射高剂量的血浆试剂（ 图4）被防止。 重要的是，肝脏血流成功活体显微镜成像需要第Ë考虑的几个关键环节。这些包括肝脉管系统的复杂性，含小的和大血管具有不同的结构和功能，不同的血液循环以快速和慢速通量和快速红细胞速度血管的不同亚型的存在。是基于一个XT图像的分析方法的主要限制是采集容器和缺乏Z轴分辨率的速度，当图像在三维（XYT）获得的。更具体地说，标准系统的行扫描速率达到灵敏度的限制方面取得大血管（直径高于180微米2），其特征在于非常高的RBC的速度，这是与血管的情况。在大容器中，所述红细胞的轨迹可以是出在成像平面的，并且是垂直于扫描平面的任何组件将被从分析中排除。 该方法提出ħERE表示用于体内和各种病理条件下几个血流参数实时定量的理想工具。肝脏血流动力学的调查将在一个特定的器官揭示疾病过程和寄生虫病机新光源。我们努力洞察施加到利什曼原虫属感染的实验小鼠模型的肝脏微脉管系统开辟的可能性的多个血流参数的相关性与寄生虫的乘法和疾病发展中该器官，在现实的时间。试剂，工具的进一步发展和方法对肝脏的血管系统的感染过程中的改进表征IVM是非常重要的寄生虫殖民的影响对这种生理参数的研究和，相互，血液的影响流上利什曼病机。这种策略可潜在地扩展到其他传染性系统，其中的病原体靶向肝脏。

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微米2 ）。肝脏活体显微镜成像允许可视化在高分辨率肝脉管（ 图1C）的异质性和复杂性。 监测个别贝丝血流速度ELS，两种不同的方法被用于肝脏血管体内图像采集和数据分析。在第一种方法中，使用XYT扫描模式（ 电影1）进行在感兴趣的血管的血液流动的实时成像。使用单个红细胞在单个容器中的速度的定量分析来执行所述MTrackJ插件中的ImageJ软件（ 图2A和2B）。最低每船5颗粒的跟踪在不同的容器，从40-80微米直径2的。用这种方法获得的结果给在不同的小鼠一致和可重复的值，并评估在小肝血管红细胞速度在25-35微米/秒（ 图2C）之间的值。 第二种方法包括沿平行于容器壁中的线反复扫描，与线置于血管腔的中心。图3示出的xy帧扫描（左面板）与相应的XT线扫描（右图）。单个红细胞的速度由比V =距离/时间定和使用上的XT图像获得的条纹的X和Y轴的正交投影计算。在图3D和3E中，每个数据点代表单个红细胞从来自不同的小鼠中选择不同的小或大血管的速度。如从误差线看到的，数据显示红细胞速度之间相对低的变化，并表明血流速度大大加快大血管比在小血管。此外，在来自不同类型的大血管中得到的速度值一显著变化观察到，而血流速度是在不同类型的小血管比较相似。血流量测量，这种新方法生成的数据相媲美，从先前描述获得的数据使用MTrackJ（比较图2C至图3D）D法。在图3C的图像表示一个次优实验，其中在xy帧扫描产生的未可解释XT图像的一个典型的例子。在这种特定的情况下，这个问题从两个i）所述快速内化的BSA进入内皮细胞衬里感兴趣的容器的效果和ii）两个容器是扰乱在获取所选择的区域中的血流之间的结点的存在。等离子体试剂进入内皮衬血管的内化的速率是测量血流速度时要考虑的，因为内化将改变XT图像，显示条纹的质量的关键参数。内化速率主要取决于被静脉内注射染料的剂量。在图4中我们证明了500 BSA-647 Alexa的和/或500 kDa的葡聚糖-FITC的微克是最佳剂量的寿ld的用于血液流动的速度的量化。这些剂量产生一个非常明亮的信号，允许红细胞的鉴定为背景明亮的黑色颗粒，使血流量最少1小时的可视化，而 ​​不注入染料（ 图4，底部面板）的内化。 与此相反，50的BSA-Alexa的647和/或500 kDa的葡聚糖-FITC（未示出）的微克，同时允许对肝脏的血管网的观察，是次优的剂量用于测量血流速度因的非常快的内化染料，其开始于5分钟注射后（ 图4，下图）。 当应用这种方法对受感染动物的肝，我们观察到在血流速度尽快1小时感染后一个显著增加。红细胞速度的改变被保持长达24小时后感染。它最终达到正常值在72小时后的寄生虫注射（ 图5）。因此，该实验验证了使用这种方法来获得新的见解对肝脏血流动力学的病理条件的影响。 <img alt="图1" src="/files/ftp_upload/52303/52303fig1.jpg" /> 图1:小鼠肝脏活体显微镜鼠标（ 一 ）肝脏手术。肝叶的小面积手术后，二湿润纱布片被放置在其周围和滑动被放置在腹部以覆盖之前的小鼠被放置在一个共焦显微镜的阶段的肝脏暴露。未感染的小鼠，演示了肝血窦和不同的尺寸，其包含肝器官的血管的肝脏的代表性区域（B，C）的图像。肝自体荧光显示为绿色。鼠标注射用Hoechst 33342（蓝色）标注的Hepatocyte核（B）和注射用BSA-的Alexa 647（红色）以可视化的肝脉管（℃）。比例尺50微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52303/52303fig1large.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/52303/52303fig2.jpg" /> 图2:血液循环中通过使用快速XY扫描模式下的小鼠肝脏的活体显微镜成像收购了一家小型船只肝脏从使用ImageJ的软件一个XY帧扫描速度红细胞的定量分析 （ 一 ）代表XY图像。暗区对应的红细胞。比例尺，10微米。 （ 二）量化单一RBC的使用ImageJ的，从该MTrackJ插件的速度。线（红，黄，白）表示的轨迹上的个体的RBC的时间。在个别SM（C）RBC速度所有（D约为50微米2）肝血管。该图绘制的跟踪个别肝脏血管五个独立的红细胞从五个不同的小鼠（1-5）中选定的速度的值。 D代表在2微米的血管的直径。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52303/52303fig2large.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/52303/52303fig3.jpg" /> 从获得使用XT线扫描模式肝血流测量由血管的活体显微镜成像标记用BSA-Alexa的647和其相应的XT图像（右图）肝血管的代表性的xy图像（左图）:图3。行扫描单个容器的中心管腔。 （A，B），小（ 一 ）和大肝容器（B）中 。 （C）从一个次优实验示出两个血管的交点与BSA内在化到内皮代表性数据。 （D，E），该图绘制了来自五个不同的鼠标选择每艘三个独立的红细胞（小D和大E）的速度值。 D代表在2微米的血管的直径。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52303/52303fig3large.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/52303/52303fig4.jpg" /> 图4:荧光等离子体染料的剂量上及其内进内皮衬肝窦的影响该图像跟踪BSA和葡聚糖500 kDa的内化到内皮衬里的正弦，作为时间和注射的剂量的函数。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52303/52303fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52303/52303fig5.jpg" /> 图5:L的影响原虫感染对肝脏血流动力学 。这个图形绘制从多个单独的血管获得来自未感染的小鼠（NI）的血液速度的值，小鼠被感染L.原虫并分析1小时，24小时和72小时后的感染（PI）的。每个点代表五红细胞速度测量在约50μm2的单一血管中得到的平均值。在每个条件（NI，1小时​​PI，24小时pi和72小时PI）最少三只小鼠进行了血流速度分析。星表示所得的血流速度显著差1小时和24小时圆周率相比NI小鼠（P &lt;0.01，单向ANOVA）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52303/Movie_1.mp4">电影</a>小鼠肝脏 。 电影1 1。时间推移活体显微镜成像对应于图3的小鼠接受注射BSA-Alexa的647的可视化的血管。显示在三个小血管（50微米2 D）的血流量。黑色区域对应于红细胞。

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 ...

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

11.7K 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 Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

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. ...

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.

<meta charset="utf8"></head><body>活体显微镜研究小鼠肝脏中血小板-白细胞-内皮相互作用

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).

活体显微镜研究小鼠肝脏中血小板-白细胞-内皮相互作用

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

肝脏活体如下显微成像

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在Live主机转利什曼原虫寄生虫在体内成像

腹股沟淋巴结的活体显微镜

活体成像的鼠标使用2 - 光子显微镜的胸腺

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

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

活体显微镜研究小鼠肝脏中血小板-白细胞-内皮相互作用

活体显微镜研究小鼠肝脏中血小板-白细胞-内皮相互作用