Name: intravital microscopy and thrombus induction in the earlobe of a hairless mouse
Uploaded: 2017-04-02T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse at JoVE.com

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所有的体内实验（7221.3-1-006 / 15）在符合有关动物保护和NIH指南实验动物的护理和使用（实验动物资源研究所，国家研究理事会）德国法律进行。 1.动物一般饲养<ol><li>执行与年龄4至6周雄性SKH1-HR 小时小鼠实验。使用动物用20和25克的重量。 </li><li>保持动物在无病原体的设施和24至26℃下在标准条件和约60％相对湿度下，以稳定获得水和食物随意 。 </li><li>跟上只雄性动物在笼子里一个。动物的外壳为他们的幸福过程中提供的床上用品和丰富的材料。 </li></ol> 2.动物的预先安排<ol><li>称重小鼠并装入各自的药物（ 例如，大麻素，5米克/千克体重（BW））转换成胰岛素注射器。施用前血栓感应药物30分钟。 </li><li>通过保持拇指和食指和用小指的小鼠的尾之间的小鼠的颈部，舒展动物和注射药物腹膜内（IP）到腹部的左下象限。把动物放回笼子15分钟。 </li><li>用氯胺酮（90毫克/千克体重）和赛拉嗪（25毫克/千克体重）准备麻醉。前血栓诱导15分钟，麻醉小鼠。把鼠标在笼中，稍拉尾部，并与胰岛素注射器注入麻醉剂的ip。 </li><li>把鼠标放回笼子，直到麻醉起效。为了验证足够的麻醉，掐钳尾。 </li><li>负载0.05毫升解冻异硫氰酸荧光素标记的葡聚糖（FITC-葡聚糖; 5％，150 kDa）的成胰岛素注射器。虽然填充注射器，确保没有气泡残留，因为即使小intravenouslY（IV）-administered气泡可能是致命的动物。 </li><li>放置麻醉小鼠上的面朝下位置的加热板。调节加热板，以37℃。 </li><li>把眼膏鼠标的角膜。消毒皮肤，并用消毒器械。 </li><li>缝合聚丙烯7/0的两根缝线到右耳的头部和尾部边缘。放置尽可能靠近边缘和邻近基尽可能（ 图1B）的线迹。 </li><li>移鼠标背的位置。修复所有的腿用胶粘带的acrylglass平台。钩门牙下一个缝合线，并通过粘缝合线与粘合带的acrylglass在背屈定位磁头。 </li><li>转运操作体视显微镜下平台上的动物。使用16X放大倍率。 </li></ol> 3.左颈静脉和FITC-葡聚糖的注射液的制备注:MIC右耳的roscopy，准备左颈静脉。 <ol><li>使用手术刀，创建在脖子上的在头尾方向上的左侧的皮肤上的5毫米切口。解剖用microforceps和microscissors皮下组织。或者结扎血管交叉与聚酯8/0缝合线或与电凝。 </li><li>使用自由和microforceps从microscissors外膜静脉不接触容器。 </li><li>使用制备的胰岛素注射器将荧光染料的注入。小心地抓住与microforceps血管壁，无穿孔静脉。穿透扩张血管壁与注射器和注入FITC-葡聚糖IV。 </li><li>撤销使用棉签，注射器后停止出血。避免血液和染料耳朵的污染。 </li></ol> 4.活体荧光显微右耳的定位<ol><li>在加热板的动物转移到acrylglassÇ。施工与用于加热板的狭槽和用于定位耳0.5厘米高平面。 </li><li>修复动物面朝下放在使用粘合带的加热板。放置相对较强，而凸软骨在耳根0.5厘米高平面旁边的耳（ 图1B），使得耳朵的顶端部能够在平面上被定位平面。 </li><li>室温的0.9％NaCl一滴添加到acrylglass平面以便放置耳。将右耳，与面临的凹腹侧预先布置的缝合线向下，在0.9％的NaCl的下降。用棉花棒，吸收氯化钠的下降，让毛细力附着耳朵平面到acrylglass。 </li><li>大盘缝合到acrylglass修复耳朵的位置。 </li><li> 0.9％室温下的NaCl一滴添加到耳的凸背侧。小心地把一个盖玻片（直径为0.5厘米）在耳朵上而不压缩进入第基底容器Ë耳朵。使用棉签，以便最小化盖玻片和耳靶血管之间的距离从除去盖玻片下尽可能多的NaCl越好。 </li></ol>右耳的活体5荧光显微镜和诱导血栓<ol><li>调整FITC-葡聚糖可视化活体荧光显微镜（450 - 490纳米; FT:510; LP:520）。使用可变100-W汞灯作为光源。高分辨率，黑色和白色的CCD相机连接到DVD刻录机。 </li><li>转移在包含与所述固定绑架耳朵到活体荧光显微镜的桌子加热板的acrylglass动物。 </li><li>在直径和与400的顺行血流60微米 - - 600微米/秒使用20X放大倍率（20X / 0.95的数值孔径）和20％的光强度，搜索静脉容器50。 </li><li>室温水一滴加入盖玻片为63X magnificatio的水浸泡Ñ物镜（63X / 0.95的数值孔径）。用注射器以1毫米直径的套管，并放置在显微镜的物镜的下降。加入足够的水接触盖玻片，客观与水滴。 </li><li>水滴的应用后，立即开始与直径和血流的离线测量20％的光强度的记录为20秒的容器中。 </li><li> FITC-葡聚糖的注射后开始血栓诱导5分钟。为此目的，提高的光强度为100％。 </li><li>在血栓感应，关闭时间为30-S内的显微镜的2秒的孔来检查血液流动。在持续的血流量的情况下，再次打开光圈。在停止血液流动的情况下，观察容器30秒。 注意:作为闭塞该容器被分类如果流静止30秒或更多，或如果血液逆行流动。如果orthograde血流重新开始，完全开放光圈和CONTIN<html>如上所述UE发生血栓感应直到血管闭塞。在早期血栓感应，确保当光圈被关闭以检查血液流动的时间是为了维持几乎连续的落射照明尽可能短。后来，血栓生长期间，将容器灌注有更少的荧光染料，因此可以连续地被观察到。 </li><li>选择并堵塞每穗5艘船。限制血栓诱导时在显微镜下大约1小时，FITC-葡聚糖的注射后。 </li></ol> 6.后续活动<ol><li>执行使用经皮聚丙烯6/0缝合颈部的伤口闭合。 </li><li>在从麻醉中恢复，把鼠标放回笼子，并使用红外光暖动物。 </li><li>从DVD记录器传送所记录的数据到软件允许血管和血液流速的直径的测量。 </li></ol>_title"&gt; 7。左耳的检查<ol><li>让动物恢复和消除所有注射FITC葡聚糖48小时。 </li><li>重新运行此时间制备右颈静脉和左耳上述步骤。 </li></ol> 8.组织Asservation <ol><li>左耳的活体荧光显微镜后，样品0.5毫升的来自眼睛的眼球后静脉丛血液用玻璃毛细管。仔细穿透与旋拧运动的内眼睑角度，直到静脉血液流经毛细管。收集在乙二胺四乙酸（EDTA）血管探头。 </li><li>血液取样后，通过注射500毫克/公斤体重氯胺酮到尾静脉牺牲动物。 </li><li>计数使用血液分析仪用于白细胞，红细胞，血小板，血红蛋白和血细胞比容的定量评估的血细胞。 </li><li>离心剩余的EDTA血液在2500×g下和室温下，10分钟英寸吸管和冷冻血浆作进一步调查。 </li><li>用剪刀，剪耳和4％的甲醛作病理检查修复。 </li></ol>

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有对成功血栓诱导SKH1-HR 人力资源小鼠的耳垂几个关键步骤。进行故障排除，协议的各个步骤中括号表示。 考试条件是幼龄动物的理想在4岁 - 6周，与表皮的角化低。在较老的动物中，血管的可视化的质量要差少可比由于皮肤表面和靶血管（步骤1.1）之间的距离更高。 为了防止在考试区域FITC葡聚糖外渗，固定缝线必须尽可能勉强放置越好。在接?...

本制品的目的是描述施加到无毛小鼠的耳廓用于直接观察和微循环的分析，血栓形成，和血栓进化活体显微镜的技术。随着1在1000的发病率，静脉血栓形成仍是发病的常见原因。虽然诊断，预防策略和治疗已在近几年得到了发展，静脉血栓形成的三分之一表现为肺动脉栓塞1。动脉血栓形成在心血管疾病，这是在工业化国家死亡的最常见原因至关重要的作用。基于粥样硬化斑块的破裂动脉血栓形成涉及心脏发作，肠系膜梗死，中风和。每个手术暴露属下的结构中血液成分，改变血流动力学，并固定了病人。在下肢，器官的T人工关节手术ransplantation和皮瓣手术血栓并发症的常见原因。尤其是微血管血栓形成经常导致不可逆的损害，因缺乏临床症状。同样，微血管栓塞起着至关重要的规则在几种疾病，包括血栓性血小板减少性紫癜，败血症，弥漫性血管内凝血，抗磷脂综合征，慢性静脉功能不全，等等。 用于治疗和预防血栓形成的一些新的药物，近年来被开发，但抗血小板药物和抗凝药物仍然有副作用，缺乏拮抗剂，并配备持续时间长的效果。这些缺陷导致紧急医疗问题。因此，需要更多的研究来发现血栓形成，这很难在体外模拟过程中发生的复杂过程。 无毛SKH1-HR 人力资源小鼠在伦敦动物园里发现了1926年。由于14号染色体上的基因缺陷，动物失去了它的皮毛10日龄后，这使得对血管的活体显微镜访问的良好血管耳廓。耳的平均厚度为300μm。它由真皮的两层，其通过软骨隔开。对软骨的凸背侧，3个维管束进入耳垂。心尖血管弧和基础分流连接三个包。小静脉具有200微米（基础）和10微米（心尖）之间的直径。关闭网状毛细血管周围的空毛囊2。无毛SKH1-HR 人力资源鼠标的剖析，使耳廓血栓形成研究的强大和高性价比机型。

<table><tbody><tr><td>SKH-1/hr mice</td><td>Charles River</td><td>477</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>standard laboratory food</td><td>ssniff Spezialdiaeten</td><td>V1594-0&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>operation stereomicroscope</td><td>Leica&amp;nbsp;</td><td>M651/M655&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>intravital microscope</td><td>Zeiss</td><td>Axiotech Vario 100&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>objective (20X/0.95)&amp;nbsp;</td><td>Zeiss</td><td>20x/0,50 W; Plan-NEOFLUAR&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>objective (63X/0.95)</td><td>Zeiss</td><td>63x/0,95 W; ACHROPLAN&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>black and white CCD-camera&amp;nbsp;</td><td>Pieper&amp;nbsp;</td><td>FK 6990 IQ-S&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>DVD-recorder</td><td>Panasonic</td><td>DMR-EX99V&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Braun</td><td>5/12612055/1011</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Ketamine 10%</td><td>Bela pharm</td><td>F3901-6</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Xylazine 2%</td><td>Bayer</td><td>6293841.00.00</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>FITC-dextran 5%</td><td>Sigma&amp;nbsp;</td><td>46945-100MG-F</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>dexapanthenol 5% eye ointment</td><td>Bayer</td><td>6029009.00.00</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>formaldehyde 4%</td><td>Sigma</td><td>HT501128-4L</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>DMSO</td><td>Sigma</td><td>472301</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>coverslips 5 x 5 x 1 mm</td><td>Menzel</td><td>L4339</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Adhesive strips</td><td>Leukosilk</td><td>4683400</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>centrifuge</td><td>Beckman Coulter</td><td>CLGS 15</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>hematology analyzer</td><td>Sysmex</td><td>KX-21 A6980</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>EDTA-blood tube</td><td>Sarstedt</td><td>201,341</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>cotton swabs</td><td>Sanyo</td><td>604-A-1</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>infrared light</td><td>Beurer</td><td>5/13855</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>single use synringe</td><td>Braun&amp;nbsp;</td><td>2020-08</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>insulin syringe</td><td>Braun</td><td>9161502</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>disposable hypodermic needles</td><td>Braun</td><td>465 7640</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>end-to-end capillary</td><td>Sarstedt</td><td>19,447</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>heating plate</td><td>Klaus Effenberg</td><td>OP-T 185/03</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>scissors 14.5 cm</td><td>Aesculap</td><td>BC259R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>needle Holder</td><td>Aesculap</td><td>BM081R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>microforceps</td><td>Aesculap</td><td>BD331R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>microscissors</td><td>Aesculap</td><td>OC496R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>scalpel 21</td><td>Dahlhausen</td><td>11.000.00.511</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Prolene 7-0</td><td>Ethicon</td><td>XNEH7470</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Prolene 6-0</td><td>Ethicon</td><td>XN8706.P33</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>electrocautery</td><td>Servoprax</td><td>H40140</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>acrylglass pad</td><td></td><td></td><td>integrated heating, 0.5&amp;nbsp;cm high plane</td></tr></tbody></table>

intravital microscopy and thrombus induction in the earlobe of a hairless mouse

血管疾病的血栓并发症是工业国家的发病率和死亡率的一个主要原因。由于在血栓形成的细胞和非细胞的血液成分之间复杂的相互作用，生理学和血栓形成的病理生理学的研究可靠只能在体内进行。因此，本文提出了在无毛小鼠耳模型，并专注于微循环，血栓形成，血栓和进化的体内分析。通过使用活体荧光显微镜和相应的荧光染料的静脉内（iv）应用程序，能够容易地进行微循环在耳廓的重复分析，而不需要外科手术制备。此外，该模型可以适合于体内的不同问题的研究，包括伤口愈合，再灌注损伤，或血管发生。综上所述，无毛小鼠的耳朵对于在利沃夫的理想模型在生理学或病理生理学条件和它的不同的全身或局部治疗反应的评估皮肤微循环邻研究。

在血栓形成大麻素治疗的影响在0.05 ml的FITC-葡聚糖的的注入，光毒性血栓诱导导致内皮损伤和血小板壁层插头的形成（ 图2和3）。在本研究中，血栓感应大麻素的腹膜内注射（5 mg / kg体重）或载体后导致所有小静脉血栓形成的血管闭塞（ 图4）。在载体处理的动物中，时间到血栓形成为430秒?...

Watch this Scientific Journal Video about Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse at JoVE.com

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

无毛SKH1-HR hr小白鼠的耳朵模型使而不在检查微血管床之前手术准备微循环和光毒性血栓诱导的活体荧光显微镜。因此，无毛小鼠的耳朵是一个极好的体内模型微血管血栓形成，血栓进化，与溶栓期间学习复杂的相互作用。

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

intravital-microscopy-thrombus-induction-earlobe-hairless

Research

JoVE Journal

Medicine

9.6K 次观看.  Rostock University Medical Center. 无毛SKH1-HR hr小白鼠的耳朵模型使而不在检查微血管床之前手术准备微循环和光毒性血栓诱导的活体荧光显微镜。因此，无毛小鼠的耳朵是一个极好的体内模型微血管血栓形成，血栓进化，与溶栓期间学习复杂的相互作用。 

视频: Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

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, ...

科研

神经科学

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 ...

免疫与感染

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

生物工程

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 ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

医学

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

使用打开的血管窗口和活体荧光显微镜测量小鼠耳蜗中的心室血流

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

在维沃 小鼠头骨骨髓中巨细胞和前列腺的双光子成像

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

生物学

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 ...

Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Source: Str&#252;der, D., et al. <a href="https://app.jove.com/v/55174">Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse.</a> J. Vis. Exp. (2017)
This video demonstrates the intravital fluorescence microscopy technique to study thrombus formation in a mouse model. The mouse is injected with a fluorophore-labeled polysaccharide to monitor blood circulation. Upon illumination with a high-intensity light, the phototoxic induction of an injury in the microvasculature of the ear leads to the formation of a thrombus, which is confirmed by visualizing the obstruction in the blood flow.

活体显微镜及血栓诱导在无毛小鼠的耳垂

摘要

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此视频中的章节

活体鼠脑微循环显微镜使用一个封闭的颅窗口

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

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

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

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

使用打开的血管窗口和活体荧光显微镜测量小鼠耳蜗中的心室血流

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

在维沃小鼠头骨骨髓中巨细胞和前列腺的双光子成像

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

活体荧光显微镜研究微血管血栓形成

活体显微镜及血栓诱导在无毛小鼠的耳垂

摘要

探索更多视频

此视频中的章节

活体鼠脑微循环显微镜使用一个封闭的颅窗口

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

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

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

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

使用打开的血管窗口和活体荧光显微镜测量小鼠耳蜗中的心室血流

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

在维沃 小鼠头骨骨髓中巨细胞和前列腺的双光子成像

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

活体荧光显微镜研究微血管血栓形成

在维沃小鼠头骨骨髓中巨细胞和前列腺的双光子成像