Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00.000+00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

所有的动物实验和程序由美国杜兰大学的机构动物护理和使用委员会（IACUC）的批准。 1.手术过程设置<ol><li>高压灭菌仪器，手术用品，并在手术前文化用品。每只大鼠手术耗材包括:1悬垂性好，悬垂1与预切孔（0.5英寸×1.5英寸）的中心，纱布垫，1个吸水焊。手术器械包括:1手术刀用10号刀片，2双镊子和一对细剪刀。培养用品包括:1的悬垂性，1镊子，并准备用的聚碳酸酯过滤器6孔板插入。 </li><li>消毒一个有机玻璃平台，手术阶段和用70％乙醇的外科工作台空间。保持手术分期在无菌碗中待用。 <ol><li>在100毫米培养皿的中心钻孔在大约2通过1创建一个手术阶段。接下来，用砂纸顺利任何尖锐的边缘，并添加硅胶胶水层到孔的边缘，以创建用于组织中升高的表面。 </li><li>另外，采用CAD软件设计手术阶段和3-D打印（ 图1）做出。 </li></ol></li><li>将无菌吸水焊下来，躺在它上面一个有机玻璃平台。放置悬垂未经预切孔，在旁边的吸湿焊加热垫。 </li><li>预暖的无菌磷酸盐缓冲盐水（PBS），介质和盐水至37℃。地方媒体和PBS单独培养皿在50mL锥形旁边的外科设置管加热垫和地方盐水顶上。 </li><li>确保所有软件包之前，手术开始打开，确保所有材料的消毒处理。在此过程中使用的常用工具的完整列表中列出 具体的手术材料和工具表 。 </li></ol> 2. Mesenterÿ组织收获<ol><li>使用成年雄性Wistar大鼠（350±25克; 6 - 8周龄）。其它菌株和大鼠的年龄可以被取代。 </li><li> 通过氯胺酮（80毫克/公斤体重）和赛拉嗪（8毫克/公斤体重）的肌内注射麻醉大鼠。确认大鼠处于麻醉状态的脚趾之间掐来检查反射反应;应该没有。此终端过程先发制人镇痛是没有必要的。 </li><li>剃须腹部和使用脱毛膏去除剩余的头发。用70％的异丙醇接着碘伏擦拭腹部皮肤的两倍。用于擦拭物的外科医生应该开始在手术部位的中心，并移动到圆形方式制备的区域的外侧，以不重叠的那些具有相同的一块无菌纱布或无菌棉签预先洗涤的区域。然后动物转移到无菌手术的设置，然后将有机玻璃高原之上形成。 </li><li>使用手术刀刀片，使在一个0.75 - 1.25在切口在胸骨下方开始1肠道。小心不要刺破肠或肠系膜（1层皮肤，1层结缔组织，1层肌肉）。 </li><li>放置一个悬垂超过切口一个预切孔和放置无菌手术阶段悬垂的顶上。确保与切口对齐开幕。用消毒棉签涂抹定位，并通过手术分期开盘拉出回肠。 </li><li>用棉签涂抹通过阶段8肠系膜窗口，并注意不要触碰窗口（ 图1） -拉6。组织通常由小肠盲肠附近开始的回肠区域采收。保持暴露组织湿润使用无菌注射器滴下的溶液根据需要加热的无菌盐水。 </li><li>安乐死通过戊巴比妥钠的心内注射鼠（每只鼠0.2mL）中。在卸下我senteric窗口，确保老鼠被触诊心脏安乐死;应该没有脉冲。 </li><li>通过使用镊子夹住脂肪垫和精细剪刀剪开窗口中删除所需的肠系膜组织。离开的脂肪（2mm）的窗口周围的边框。媒体在温暖无菌PBS洗一次组织一次。 </li><li>返回形象化回肠到腹腔，并根据机构指南动物的处理。 </li></ol> 3.肠系膜组织培养时间推移研究<ol><li>传输蒸压文化用品（参见1.1节）和组织无菌层流罩。 </li><li>用镊子每个组织转移聚碳酸酯过滤膜上面。由脂肪垫抓组织，以避免损坏血管。 </li><li>使用快速脂肪垫，小心不要碰到窗口传播组织。倒置与组织插入到6孔板的底部，并用3毫升培养基（ 图1覆盖</st荣&gt;）。用于这一过程的典型介质包括极限必需培养基（MEM）中，用1％青霉素链霉素（PenStrep）和10％胎牛血清（FBS）。媒体可辅以其它血清和/或生长因子刺激血管生成。 </li><li>重复步骤3.2 - 3.3长达5天的标准培养箱条件的每个组织和培养（5％CO 2，37℃）。 </li></ol> 4.肠系膜组织的时间推移成像<ol><li>上成像的当天，用共轭BSI-凝集素补充媒体在每个孔和标准培养条件下温育30分钟。提供免费凝集素媒体组织洗净两次。 BSI-凝集素染色将肠系膜组织保持可见在培养3天。 </li><li>板块转移到显微镜阶段。标识基于其形态和网络结构的血液和淋巴管。 </li><li>找到每个组织所需的网络区域，并拍摄图像。记下成像位置，以确保在同一区域将被捕获为后续图像。如果使用机动阶段，记录的坐标。 </li><li>返回组织的孵化器，并继续培养至所需的终点。根据所需实验时间点需要4.3 - 重复步骤4.1。 </li></ol> 5.组织免疫标记<ol><li> BSI-凝集素标签 <ol><li>在37°C孵育组织30分钟，在媒体后跟两个漂洗与媒体1:40 FITC-缀合凝集素（在6孔板每孔2.5毫升抗体溶液）。对于漂洗，添加媒体，然后立即更换。 </li></ol></li><li> 活/死贴标 <ol><li>在37℃下用1孵育组织10分钟:500 2毫乙锭同型二聚体-1和1:500 1毫钙黄绿素中媒体后跟两个漂洗用媒体的AM（在6孔板每孔2.5毫升抗体溶液）。 </li></ol></li><li> BSI-凝集素/ NG2标签 <ol><li>在显微镜传播组织下滑E（1 - 2组织/幻灯片），并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。用PBS（3×10分钟）冲洗组织。 </li><li>初级抗体标记孵育1在室温下1小时的组织:100兔多克隆NG2抗体和5％正常山羊血清（NGS）。用PBS（3×10分钟）冲洗组织。 </li><li>对于二级抗体标记孵育在室温下1小时的组织以1:100的山羊抗 - 兔CY2共轭抗体（GAR-CY 2）和5％NGS。用PBS（3×10分钟）冲洗组织。 </li><li>孵育在室温下30分钟，组织用PBS中1:40 FITC-缀合凝集素后跟两个漂洗用PBS。对于漂洗，加入PBS，然后立即更换。 </li><li>要安装的幻灯片，覆盖在上面以50:50 PBS组织和甘油溶液和地点盖玻片。使用密封指甲油幻灯片边缘。 </li></ol></li><li> LYVE-1 / PECAM贴标<ol><li>铺在显微镜载玻片组织（1 - 2组织/滑动）并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li> 200小鼠单克隆生物素化的CD31抗体和1:初级抗体标记在室温下用1孵育1小时组织100兔多克隆LYVE-1的PBS + 0.1％皂角苷+ 2％牛血清白蛋白抗体（BSA）+ 5％NGS 。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>对于二级抗体标记，孵育组织为在室温下用1 1小时:500 CY3共轭链霉抗抗体和1:在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS 100 GAR-CY2。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>要挂载幻灯片，封面组织以50:50 PBS和甘油溶液并放置一个盖玻片放在上面。使用密封指甲油幻灯片边缘。 </li></ol></li><li> 的BrdU / BSI-凝集素标签 <ol><li> 1毫克/毫升的BrdU添加到媒体并用BrdU溶液替换组织媒体。在37℃下孵育2小时。 </li><li>铺在显微镜载玻片组织（1 - 2组织/滑动）并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。用PBS（3×10分钟）冲洗组织。 </li><li>变性组织的DNA中的2M HCl中在37℃下1小时。在PBS + 0.1％皂角苷（3×10分钟）冲洗组织。 </li><li>初级抗体标记，孵育组织为在室温下用1 1小时:100单克隆小鼠抗BrdU在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>对于二级抗体标记，孵育在室温下1小时的组织以1:100的山羊抗小鼠的Cy3缀合的抗体（GAM-Cy3标记）在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><lI&gt;孵育在室温下30分钟，组织用PBS中1:40 FITC-缀合凝集素后跟两个漂洗用PBS。 </li><li>要安装在上面滑动，封面组织以50:50 PBS和甘油溶液和地点盖玻片。使用密封指甲油幻灯片边缘。 </li></ol></li><li> BSI-凝集素/ CD11b的标签 <ol><li>铺在显微镜载玻片组织（1 - 2组织/滑动）并晾干。通过稳固地向下按切除脂肪用手术刀去除多余油脂。 </li><li>固定在冷甲醇组织在-20℃下30分钟。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>初级抗体标记孵育在室温下1小时的组织以1:100的小鼠抗大鼠的CD11b在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。洗用PBS + 0.1％皂角苷（3×10分钟）的组织。 </li><li>对于二级抗体标记孵育在室温下用1 1小时组织:100 GAM-Cy3的在PBS + 0.1％皂角苷+ 2％BSA + 5％NGS。用PBS洗涤组织+ 0.1％皂角苷（3×10分钟）。 </li><li>孵育在室温下30分钟，组织用PBS中1:40 FITC-缀合凝集素后跟两个漂洗用PBS。 </li><li>要安装在上面滑动，封面组织以50:50 PBS和甘油溶液和地点盖玻片。使用密封指甲油幻灯片边缘。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

该协议记录了使用大鼠肠系膜文化模型作为微血管网络增长的时间推移成像的体外工具的方法。在我们的实验室以前的工作已经建立了使用我们的1模型）血管生成8,2）淋巴管8，3）周细胞-内皮细胞相互作用8，和4）抗血管生成药物测试的9。对于在多个时间点培养的大鼠肠系膜组织成像的能力提供了用于评价组织特异性...

微血管网络的增长和重塑是用于组织功能共同点，伤口愈合，和多个病症和一个关键过程是血管生成，定义为新血管从已有的1，2的生长。组织工程新船或设计基于血管生成疗法，了解参与血管生成的细胞动力学的重要性是至关重要的。然而，这个过程是复杂的。它可以在一个微血管网络内的特定位置而变化，并且涉及多种细胞类型（ 即内皮细胞，平滑肌细胞，周细胞，巨噬细胞，干细胞）和多个系统（淋巴网络和神经网络）。虽然在体外模型已作出了巨大贡献，以检查参与血管生成3种不同的细胞之间的关系，它们的生理相关性可由于兵卫被削弱<html> - [R复杂性有限，事实上，他们没有真实地反映体内的情况。为了克服这些限制，三维培养系统3， 离体组织模型4中 ，微流体系统5，6，和计算模型7已经被开发并引入在最近几年。然而，仍然需要具有时间推移能力的模型来研究在完整微血管网络体外血管生成。血管生成研究建立新的延时模式与复杂性的水平将提供一个宝贵的工具来了解调节血管生成的基本机制，以提高治疗。 使血管跨越一个完整的微血管网络体外调查一个潜在的模型大鼠肠系膜养殖模式&gt; 8。在最近的工作中，我们已经表明，血液和淋巴微血管网络保持培养后存活。更重要的是，大鼠肠系膜培养模型可用于研究官能周细胞 - 内皮细胞相互作用，血液和淋巴管内皮细胞的连接，和延时成像。本文的目的是为我们的时间推移成像方法的协议。我们的代表性的结果文件证明血管生成的刺激与血清后保持存活，并提供使用用于量化组织特定的血管生成反应以及内皮细胞追踪研究该方法的实施例的多种细胞类型。

<table><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</td><td>AbD Serotec</td><td>MCA275R</td><td></td></tr></tbody></table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

血管生成，定义为新的血管从预先存在的血管的生长，包括内皮细胞，周细胞，平滑肌细胞，免疫细胞，并与淋巴管和神经的协调。多小区，多系统的相互作用必要的血管生成在生理相关环境的调查。因此，虽然使用体外细胞培养模型提供了机械的见解，一个共同的批评是它们不概括与微血管网络相关联的复杂性。该协议的目的是证明之前和在培养的大鼠肠系膜组织血管生成的刺激后，使完整的微血管网络的时移进行比较的能力。培养组织含有维持其层次微血管网络。免疫标记证实了内皮细胞，平滑肌细胞，周细胞，血管和淋巴管的存在。在一个ddition，与BSI-凝集素标记的组织使前后血清或生长因子刺激由毛细血管发芽和血管密度后，本地网络区域的延时比较。相比于普通细胞培养模型，这种方法提供了在生理学相关微血管网络内皮细胞谱系研究和组织特异性血管生成药物评价的工具。

后在培养3天后，组织被标记有活/死活力/细胞毒性试剂盒以证明在大鼠肠系膜培养模型（ 图2A）微血管的生存能力。大部分存在于肠系膜细胞保持在血管内皮细胞，根据他们在微血管段位置确定的文化活力。内皮细胞的增殖也被外源凝集素/ BrdU标记（ 图2D）确认。平滑肌细胞和周细胞的存在沿血管被证实与NG2标记（ 图2B）。标记为LYVE...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

an-ex-vivo-method-for-time-lapse-imaging-cultured-rat-mesenteric

Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K 次观看.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

视频: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

科研

免疫与感染

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 ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

生物学

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化：调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

医学

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

微灌技术在调查微血管渗透性的大鼠肠系膜条例

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

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