经过细胞分离整个协议大约需要2小时。 1。显微准备<ol><li>从供体骨髓和分离的亚群中的荧光标记，根据标准化的协议9,12允许细胞时进行动物操作的休息时间进行干细胞分离。对于荧光标记，我们建议CFDA（二醋酸盐琥珀酰亚胺酯）。 </li><li>麻醉雄性小鼠体重20-25克用氯胺酮（75毫克/千克）和赛拉嗪（2.5毫克/千克）。在整个手术过程中和实验方案，麻醉应由补充剂根据需要（通常每30-60分钟）保持（初始注入的10％，IP）。 </li><li>轻轻地刮右阴囊前壁方面以及右大腿和腹股沟。收集所有失去的头发用浸湿的棉签。将鼠标腹侧的有机玻璃观看舞台和固定费t使用弹性悬垂性。该阶段是定制的，包括一个底板和一个第二板的底板为两脚和阴囊抬高的尾缘。在舞台上放置一个加热垫，以保持体温在37°C。固定在舞台上后，将动物在手术显微镜下，用10执行下列操作步骤 - 到16倍的放大倍率。 </li><li>使用皮肤剪刀在右大腿，从膝盖股骨血管只是直到前向腹股沟进行初始切口。 </li><li>确定股动脉，并按照其近端，从周围的软组织动员动脉。识别和使用thermocautery切一个大的分支，以左侧睾丸。此后表面上放置一个留缝在左睾丸。温柔的牵引将有利于进一步调动近端股动脉。 </li><li>近端动员完成后，识别和切一个大的分支运行mediallŸ在大腿由thermocautery。此后结扎的股动脉在右大腿的远端部分。在结扎温柔的牵引力，将有助于进一步的准备。 </li><li>将注意力现在的股骨近端动脉再次并将动脉周围缝合的近端越好。准备一个结，但不打领带下来呢。适用温柔的牵引缝线，营造动脉扭结。 </li><li>建立扭结后，切开动脉小心使用microscissor。要特别小心，不要损坏股静脉。 </li><li>通过切口插入准备好的微导管（内径0.28毫米）。导管必须被强制脱气（0.9％氯化钠，NaCl）中，并连接到一个1ml注射器之前插入。 </li><li>导管插入成功后，小心地松开扭结，以利于导管的通道。动脉的血流量，应立即可见导管内。轻轻将导管几毫米用言语以前的扭结。此后，领带下来的导管固定的准备结扎。 </li><li>轻轻冲洗导管（0.9％NaCl）中并吸出，以确认正确的位置。用一块胶布，以进一步确保导管的操作阶段。 </li><li>经过充分固定，把导管留做以下准备步骤，提睾肌。为了防止血块形成适用定期冲洗和导管每隔5-10分钟的愿望。 </li><li>关闭关注，现在到右边阴囊和切割皮肤和筋膜以上的阴囊右侧的腹侧方面做一个切口。小心不要触摸下面的组织与任何仪器。 </li><li>延长切口至腹股沟折起和尾端到阴囊的前端。 </li><li>仔细分离底层软，结缔组织上面提睾麻袋，而不触及它。确定提睾肌的远端，并放置一个留缝线这里SLightly延长肌肉。轻轻拉动肌肉和尾端现在前方切除剩余的软组织下方的肌肉麻袋。 </li><li>从结缔组织完全分离后，打开提睾肌袋在一个点上用剪刀颅边缘。从该开口向下的肌肉的远端延伸的切口。 </li><li>密封小血管连接睾丸和副睾使用thermocautery，其后用剪刀剪断肌肉和睾丸之间的剩余连接。 </li><li>放下睾丸，出了显微镜领域。 </li><li>打开提睾现在在舞台上平放。将在组织的边缘的四个6-0普理灵缝线逗留并修复它们与弹性悬垂性，以分散提睾肌组织。从现在起，连续地润湿所述组织用磷酸盐缓冲盐水（PBS），使用1ml注射器（ 图1）。 </li><li>现在让准备休息15分钟。此后注入0.05-0.1毫升1％的若丹明-葡聚糖通过左侧股动脉的微血管的对比度增强。如果计划在应用程序的任何趋化因子或消炎药，你现在应该局部滋润肌用1毫升注射器各自的试剂。在对照实验的情况下，继续用PBS润燥。 </li></ol> 2。活体显微镜<ol><li>保持鼠标固定在操作阶段和修改后用于落射照明，并连接到一个CCD（电荷耦合器件）摄像机，在荧光显微镜下移动。请特别注意不要打乱股动脉导管。 </li><li>使用水浸泡的目的进行实验。 </li><li>调整客观上为足够的提睾肌微循环的可视化和细胞注射前，后，随机确定6毛细血管静脉为公司干细胞的粘附，以后的分析。 </li><li>辖荧光标记的干细胞，溶解于200μlPBS中，在每次注射0.4×10 6个细胞，总的利用股动脉导管5连续注射液和1ml注射器。细胞样品仔细撼动第一次注射之前。 </li><li>记录干细胞细胞注射后立即滚动，从而确定"滚动"作为电解槽的速度超过50％，沿血管内皮衬里与典型的蜂窝组合"大棒和释放"的动作。进行轧干细胞计数和所有的干细胞通过一个预定义的小静脉的距离在观察1分钟以下每单次注射。干细胞滚动的量表示为所有通过细胞的百分比。不要指望细胞表现出随机简短圈养现象滚动细胞。 </li><li>以下通过计算最后细胞注射决定牢牢附着的干细胞的数量和表达有关次方计算的内皮细胞表面的量Ë六个预定义的小静脉（直径×长×π）为贴壁细胞/毫米2。牢固粘结被认为是在没有细胞的运动是可检测的，持续30秒。 </li><li>在活体显微镜完成后小心地取下提睾肌，并将其对应储存到以后的分析（免疫组化和分子生物学）。通过颈脱位牺牲动物。的活体显微镜录音为干细胞的滚动和坚定的内皮细胞的粘附，以及微血管血流动力学，如红细胞速度（mm /秒），血管长度（微米），血管直径（μm），墙分担率的定量分析（秒-1）;应脱机蒙蔽利用图像处理软件各自的实验治疗组织的调查员进行。 </li></ol>

<ol>
	<li>Madlambayan, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+stem+and+progenitor+cell+contribution+to+neovasculogenesis+is+dependent+on+model+system+with+SDF-1+as+a+permissive+trigger.">marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger.</a> Blood. 114, 4310-4319 (2009).</li><li>Lapidot, T., Dar, A., Kollet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+stem+cells+find+their+way+home?.">How do stem cells find their way home?.</a> Blood. 106, 1901-1910 (2005).</li><li>Lehr, H. A., Vollmar, B., Vajkoczy, P., Menger, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+bone+marrow-derived+mast+cells+roll+on+P-selectin+under+conditions+of+flow+in+vivo.">Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo.</a> Lab Invest. 74, 634-643 (1996).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cremaster+muscle+preparation+for+intravital+imaging+of+the+microcirculation.">The mouse cremaster muscle preparation for intravital imaging of the microcirculation.</a> J. Vis. Exp. , e2874 (2011).</li><li>Kaminski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+NOS+is+required+for+SDF-1alpha/CXCR4-mediated+peripheral+endothelial+adhesion+of+c-kit++bone+marrow+stem+cells.">Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.</a> Lab Invest. 88, 58-69 (2008).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGB-1+induces+c-kit++cell+microvascular+rolling+and+adhesion+via+both+toll-like+receptor-2+and+toll-like+receptor-4+of+endothelial+cells.">HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.</a> J. Cell Mol. Med. 16, 1094-1105 (2012).</li><li>Furlani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+intravascular+administration+of+mesenchymal+stem+cells+safe?+Mesenchymal+stem+cells+and+intravital+microscopy.">Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy.</a> Microvasc. Res. 77, 370-376 (2009).</li><li>Donndorf, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysing+migratory+properties+of+human+CD+133(+)+stem+cells+in+vivo+after+intraoperative+sternal+bone+marrow+isolation.">Analysing migratory properties of human CD 133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.</a> Cell Transplant. , (2012).</li></ol>

所有的动物实验，这个稿子进行已获当地动物护理和使用委员会。 作者宣称，他们有没有竞争的财务权益。

<html>活体荧光显微镜可以直接可视化和干细胞与血管内皮细胞相互作用的定量分析。提睾肌模型是非常有用的，因为显微曝光和技术活体可视化已经确立的白细胞 - 内皮细胞相互作用和各种化学组分的研究过程中可以选择性地施加到目标组织通过简单灌流技术。由于没有运动伪影和严重的图像噪声，这种特殊的模式，相较于其他的活体，镜设置，提供了用于细胞 - 细胞相互作用的可视化的最佳条?...

本条款的目的是描述活体显微镜的应用对小鼠提睾肌微循环直接观察外周血骨髓干细胞迁移和分析技术（IVM）。 干细胞的当前概念的基础组织和器官再生涉及骨髓来源的干细胞的归巢向受损组织1。对于成功的干细胞迁移的一个关键步骤包括干受伤器官其次是跨内皮迁移，最终器官移植2内与当地内皮细胞间的相互作用。这些干细胞的活体分析-内皮细胞相互作用形成了一个理想的参数用于在体内的病理生理不同设置的干细胞为基础的再生反应的定量。此外，似乎可以设想，即，在将来，特定的干细胞P上的迁移能力的活体分析在标准化的微循环环境opulations，可能会被应用之前推进这些人群进一步临床试验。 活体显微镜已初步开发了白细胞-内皮细胞相互作用在体内的观察和量化在炎症研究3的视场。活体显微镜研究的第一个成功的录音报道了Cohnheim已经在19世纪，研究在光学显微镜下4青蛙的舌头和肠系膜。自从第一次使用IVM发生了翻天覆地的技术发展到今天的某些基本组件构成，以便进行定量IVM的先决条件:（一）组织筹备工作，允许光纤接入，（II），可以通过显微镜来检测分子探针，（三），连接到检测系统及（iv）基于计算机分析系统，可以从图像数据中提取感兴趣的参数的显微镜设置4。 各种组织筹备工作已引入体外成熟的研究，包括肠系膜和小鼠和大鼠5，6小鼠和仓鼠，兔子耳朵和地鼠颊囊，仅举几例背侧皮褶室的肝脏。 然而，在下文中，我们将重点放在鼠标提睾肌，表示用于观察活体的理想的组织，作为制备和可视化是可能的通过公标准化的外科手术过程，和在一般的运动伪影的问题没有发生。开提睾肌的准备进行了第一次在20世纪70年代由贝兹和他的同事7。原本为大鼠描述，已经成功地采用了也鼠标8。经过以前的研究主要集中在与血管壁白细胞的相互作用，我们自己的小组最近推出的鼠标提睾肌整理作为VALUABLE工具直接观察和定义的微环境9内干细胞与血管内皮细胞相互作用的定量分析。各种干细胞亚群进行了研究，利用该模型，包括鼠的c-kit +骨髓干细胞和间充质干细胞，以及人类的CD133 +骨髓干细胞10-12。以下从供体骨髓和可视化的荧光标记细胞的分离，干细胞被选择性地施加到经股动脉，利用动脉注射提睾微循环，从而避免远端器官内的任何细胞截留。此外，由于各种趋化因子的相应设置内潜在介导局部干细胞的迁移，可局部施用于靶组织以简单灌流技术提睾肌模型是特别有用的。

<table><tbody><tr><td>Carboxy-fluorescein diacetate succinimidylester (CFDA)</td><td>Invitrogen, Carlsbad, CA, USA</td><td>C1157</td><td></td></tr><tr><td>Rhodamine 6 G (1%)</td><td>Sigma-Aldrich, Munich, Germany</td><td>83697</td><td></td></tr><tr><td>Ketamin (Ketanest)</td><td>Pfizer, Berlin, Germany</td><td>not available</td><td></td></tr><tr><td>Xylacin (Rompun)</td><td>Bayer, Leverkusen, Germany</td><td>not available</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate - buffered saline (PBS)</td><td>PAN Biotech, Aidenbach, Germany</td><td>P04-36500</td><td></td></tr></tbody></table>

intravital microscopy of the microcirculation in the mouse cremaster muscle for the analysis of peripheral stem cell migration

在在再生细胞疗法的背景下血管内细胞应用协议的时代，干细胞迁移的基本机制，以nonmarrow组织尚未完全阐明。我们在这里描述的活体显微镜应用到小鼠提睾肌微循环在体内外周骨髓干细胞迁移的分析技术。分枝睾的活体显微镜已经在发炎的研究直接观察白细胞相互作用与血管内皮的字段先前介绍。因为充足的外周干细胞和祖细胞的迁移，包括沿与牢固粘附在轧制内皮衬里可以想到应用分枝睾模型intravasculary施用干细胞与内皮细胞的相互作用的观测和量化的相似的初始步骤。作为各种化学组分可以选择性地施加到目标吨问题通过简单的灌流技术，它可以建立必要的微环境的先决条件，初始的干细胞募集到发生在骨髓外的活的有机体。

一般情况下，直接的互动注射干细胞或祖细胞内提睾肌微循环血管内皮是一个罕见的事件，并专门在毛细血管静脉（直径30-80微米）时发生。由于荧光标记的滚动和牢固附着的干细胞可以清楚地量化，在从循环内源性白细胞中所观察到的微静脉（ 图2）的分离。 由血流速度和壁剪切速率表示的微循环条件下通常不显著各实验组之间具有不同的趋化因子治疗（ <strong...

Watch this Scientific Journal Video about Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration at JoVE.com

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

鼠标M.提睾肌微循环活体显微镜提供了一个独特和良好的标准化的体内模型外围骨髓干细胞迁移的分析。

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

intravital-microscopy-microcirculation-mouse-cremaster-muscle-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.2K 次观看.  University Rostock. 鼠标M.提睾肌微循环活体显微镜提供了一个独特和良好的标准化的体内模型外围骨髓干细胞迁移的分析。 

视频: Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

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

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

免疫与感染

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

医学

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis和显微操作的微循环活体荧光成像

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

通过体内显微镜评估的小鼠中白细胞肌渗透

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...