这项工作得到了其他 kr-基金会和 DFG (德意志 Forschungsgemeinschaft, 德国研究基金会) SFB 854 (Sonderforschungsbereich, 合作研究中心) 的支持. 特别感谢汉斯-Holger Gärtner, Audiovisuelles Medienzentrum, 奥托-冯-奥托·冯·格里克大学马格德堡, 马格德堡, 德国, 技术支持。

根据德国动物保护法8节的规定, 我们的研究是在萨克森-安哈尔特、Landesverwaltungsamt 哈雷州的许可下进行的。(和 #167; 8, 德国法律的1段为动物保护从 18.05.2016-BGBI。我的 1206, 1313, 和 #167; 31 TierSchVersV 从 13.08.2013). 
 注意: 在这里的实验中, 使用了8到12周的老雄性 balb/c/c 小鼠, 从献血者的人单核细胞通过活体显微镜将其用于单核细胞的可视化. 
 1. 单元格准备 注意: 为了隔离单核细胞, 请参阅我们以前发布的关于朱庇特的视频说明: 和 #34; 小鼠骨髓来源的分离与静脉注射单核细胞和 #34; 由瓦格纳 et al. 4 注意: 在处理单元格时, 所有步骤都必须是无菌的, 以避免污染. 
<ol> <li> 33 和 #39 的细胞染色;-Dioctadecyloxacarbocyanine 高氯酸盐 <ol> <li> 重血清自由培养基中的细胞, 密度为 1 x 10 6 细胞/毫升. 
		注: 只有血清游离培养基能够有效染色, 因为脂溶性染料本来已经被血清的亲油性成分所捕获. </li>
		<li> 添加5和 #181; 1 mM 33 和 #39;-Dioctadecyloxacarbocyanine 高氯酸盐在胺1毫升的细胞悬浮和重仔细. </li>
		<li> 在37和 #176 孵育单元解决方案; C 为 20 min. </li>
		<li> 在37和 #176 上离心细胞; C 和 500 x g, 5 分钟. </li>
		<li> 用37和 #176 将上清液取出并重; C. 温胎小牛血清补充培养基. </li>
		<li> 重复步骤1.1.4 和1.1.5 两次. </li>
		<li> 使用公式计算单元格数: 
		<img alt="Equation 1" src="/files/ftp_upload/56290/56290eq1.jpg"/> </li> <li> 重150和 #181 的单元格, 不育 0.9% NaCl 溶液. </li>
		<li> 将细胞注入尾静脉. </li>
	</ol> </li> </ol> 2。麻醉 <ol> <li> 吸入麻醉 <ol> <li> 在一个化学安全罩下的一个密闭容器中使用浓度为5% 的蒸发器帮助汽化异氟醚. </li>
		<li> 小心地处理鼠标并将其放入纸盒中. </li>
		<li> 在停止移动后, 在后颈部的皮肤上处理该动物. </li>
	</ol> </li> <li> 腹腔麻醉 注: 使用2.1 步中描述的异氟醚麻醉进行腹腔注射。随着异氟醚麻醉的快速恢复和短效麻醉效果, 对小鼠进行腹腔麻醉更容易处理。
	<ol> <li> 使用2.4 毫升氯胺酮 (10%)、0.8 毫升嗪 (2%) 和6.8 毫升氯化钠 (0.9%) 进行腹腔注射的解决方案. </li>
		<li> 在应用麻醉前称其为动物. 
		注: 麻醉药的配方是: <img alt="Equation 2" src="/files/ftp_upload/56290/56290eq2.jpg"/> </li> <li> 使用1毫升的胰岛素注射器, 用30克针将溶液注入左下腹. </li>
		<li> 将鼠标放在笼子里, 等待麻醉效果. 
		注: 如果注射成功, 麻醉效果通常在5分钟内出现。正确的麻醉深度可以由没有眼睑反射或对脚趾捏的反应, 以及正常的, 控制的呼吸率来确定. </li>
	</ol> </li> </ol> 3。基底膜样基质的植入 注意: 本组采用该方法研究单核细胞注射后肿瘤血管生成。根据实验, 生长因子可添加到基底膜样基质中。我们进行了股动脉结扎术, 然后在老鼠的侧面注射肿瘤。该矩阵必须有一个温度为4和 #176; C 的注射。在这个温度下, 矩阵是流体;在体温 (37 和 #176; C) 中, 凝胶硬化成固体。为更好的可见性皮下基质插头, 剃须在注射现场的鼠标的皮肤. 
 注意: 可选: 添加 100 ng 碱性成纤维细胞生长因子, 300 ng 血管内皮生长因子, 26 单位肝素在无菌条件下对基底膜样基质. 
<ol> <li> 将1毫升的基质装入30克胰岛素注射器并在冰上储存, 直到使用. </li>
	<li> 将动物放在桌子上, 并在侧面的注射部位旁边按住鼠标的皮肤. </li>
	<li> 注入500和 #181; 基底膜状基质的 L. 
	注: 由于实际的原因, 有必要将基底膜样基质紧密地注入一个位置, 以避免皮下分散。在实验结束后, 将人工肿瘤从组织中取出, 将会更容易. </li>
</ol> 4。尾静脉注射 注意: 在试验前, 在实验动物身上练习尾静脉和氯化钠溶液。如果单核细胞不能在尾静脉中充分注入, 就不会对抵押有系统性影响。在这个协议中, 我们注射了250万单核细胞。试着注入不超过5和 #181; 体重的 L/克. 
<ol> <li> 使用在包含250万单核细胞的阶梯1.1.8 下制备的核细胞液. </li>
	<li> 使用30克针和1毫升胰岛素注射器注射. </li>
	<li> 小心处理鼠标, 抑制抑制剂中的动物, 确保鼠标不受伤害, 并有足够的呼吸空间. </li>
	<li> 将抑制剂放在加热板上, 使尾部可以与板接触. </li>
	<li> 识别尾部静脉, 它们位于尾部的侧面. </li>
	<li> 转动尾部90和 #176;, 尾部的尾静脉出现在尾部的上部. </li>
	<li> 在注射单核细胞前先消毒注入侧. </li>
	<li> 尝试将单核细胞溶液注入一个平坦的角度与针的斜面朝上. 
	注: 如果出现水疱, 请停止注射, 因为这是注射失败的征兆。再次尝试该过程更接近. </li>
	<li> 在注射部位停止出血, 在尾部施加温和压力约 60s. </li>
	<li> 观察该动物30分钟, 监测系统的副作用, 并把鼠标在其笼子后, 动物已经完全恢复. </li>
</ol> 5。活体显微镜 <ol> <li> 准备 <ol> <li> 将麻醉鼠标放在加热垫 (37 和 #176; C) 上, 以保持恒温, 并将爪子固定在胶带上. </li>
		<li> 在用于显微镜的腿部或侧面的部位进行皮肤消毒. </li>
		<li> 剃须区域的利益, 以更好地处理和避免干扰头发. </li>
		<li> 在 0.5 x 0.5 cm 的正方形区域中使用无菌手术刀和细钳切除皮肤. 
		注意: 重要的是保持该地区的利益湿润;否则, 图像和组织的质量将受到影响。氯化钠溶液可用于滋润该地区. </li>
		<li> 将腿放在两个可调整的邮票之间, 并在邮票顶部放置一个盖玻璃. </li>
		<li> 确保组织湿润并与玻璃接触. </li>
		<li> 注射液ect 50 和 #181; l-葡聚糖后进入静脉系统, 以提高血管的能见度. </li>
		<li> 启动显微镜并根据需要调整腿部的位置. </li>
	</ol> </li> <li> 活体显微镜设置 <ol> <li> 打开显微镜、计算机、电子接口和激光器. </li>
		<li> 打开孵化室和/或加热阶段 (板/垫) 的加热单元, 并将温度设置为37和 #176; C. </li>
		<li> 启动购置软件。如果显微镜配备了共振扫描仪, 请选择此快速扫描模式. </li>
		<li> 等待温度达到恒定水平 (35-37 和 #176; C) 
		注意: 一个稳定的温度是重要的: (a) 鼠标, 这在麻醉下不能控制其体温, 和 (b) 避免或至少最小化焦点漂移。这可能需要 1 h 到几个小时, 这取决于周围的条件 ( 例如 , 空调系统和房间里的热源的数量, 包括人)。如果使用浸入式目标, 透镜与盖玻璃 (或组织) 之间的接触区是不稳定的典型来源。用物镜加热镜片可能有帮助;另外, 强烈建议使用自动对焦系统的显微镜. </li>
		<li> 选择具有高数字光圈的放大镜头, 以满足实验的分辨率要求 (请参阅本节末尾的注释). </li>
		<li> 在将实验动物放在舞台上之前, 优化显微镜设置以获取增益、通道设置、扫描速度、像素分辨率、深度音量、步长和平均值. </li>
		<li> 扫描双向以减少捕获时间. </li>
		在显微镜达到稳定的条件后, 将麻醉小鼠放在 prewarmed 显微镜下, 并在假人的帮助下进行测试. </li>
		<li> 通过使用明亮的光照, 将基底膜状基质内的感兴趣区域带入焦点, 并通过紫外光对毛细管进行简要检查, 并对应用的荧光进行适当的过滤设置. </li>
		<li> 切换到扫描模式; 开始扫描低分辨率 256 x 256 模式, 并增强增益和激光功率, 直到显示器上出现信号. </li>
		<li> 关注感兴趣的结构。放大, 直到所有细节都可见. </li>
		<li> 定义和 #34; 开始和 #34; #34; 结束和 #34; 位置在轴向方向. </li>
		<li> 停止扫描 </li>
		<li> 定义步进大小 ( 例如 、0.5 和 #181; m) 和焦点平面 ( (如 、10-20) 的数目. </li>
		<li> 根据移动的亚细胞结构或单元格的速度切换到最终像素分辨率 (512 x 512 或 1024 x 1024), 并选择最适合运动的扫描速度 (扫描频率)。建议使用双向扫描来固定堆栈的获取. 
		注意: 选择扫描的最高扫描速率, 以提供足够的图像质量。典型的点扫描器提供400赫兹和1.4 赫之间的速度。共振扫描仪提供额外的8赫或12赫。通过减少 y 方向的行数, 可以进一步提高扫描速率 (典型值为 512 x 512、512 x 256 或 512 x 200 像素分辨率)。根据信噪比, 可以通过在2和4之间添加线平均来提高图像质量。通常情况下, 512 x 200 的设置没有平均值, 在双向模式下使用8赫谐振扫描仪获得18毫秒的捕获时间/帧;512 x 200 和4x 平均, 它减慢到每帧56毫秒. </li>
	</ol>
	</li>
</ol>

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作者声明他们没有竞争的金融利益。

本文所述方法揭示了侧枝动脉的发展、这些血管中单核细胞的行为以及 arteriogenesis 的过程。应用该协议的步骤很容易学习, 可以用于其他科学领域。尽管有这些优点, 但也有一些缺点。例如, 需要显微设备来执行所描述的技术。为一个实验获取设备是不可持续的, 因此与其他机构合作共享这些设备是很重要的。
与此协议相关的其他困难也可以在实践中避免。一开始, 在显微镜下...

心血管疾病, 包括冠心病或周围动脉疾病, 是全球最常见的死亡原因1。细胞疗法是治疗心血管疾病的一种很有前途的方法, 特别是对于那些不能接受手术治疗的人。有几种方法使用细胞或其分泌物质作为治疗工具2,3, 其总体目标是改善缺血性和 underperfused 组织的灌注和维持功能。实现这一目标的一个尝试是改善 arteriogenesis, 从而提高侧枝动脉的发育。单核细胞是一种与抵押相关的重要单元类型。我们的小组专注于研究在炎症区域的单核细胞的影响4,5, 特别是使用后肢缺血模型诱导缺血和随后的炎症6。单核细胞的家到炎症的地区, 并导致复杂的系统性反应, 导致抵押的发展7。
随着活体显微镜的使用, 我们可以研究这些细胞的行为在体内, 并观察被注射的单核生物归巢到炎症区域。大多数以前的研究只描述了验尸分析, 它的缺点包括组织学文物的引入和准备工作所需的大量动物。通过我们的方法, 我们可以通过实时成像在多个时间点来研究免疫过程和侧枝形成。
除了在缺血地区发展侧枝动脉外, 单核细胞还会影响肿瘤的生长。为了研究这些过程, 我们注入了一个基底膜样基质从 Engelbreth-栎-群小鼠肉瘤, 一个富含细胞外基质蛋白的肿瘤8, 并使用活体显微镜进行分析。该矩阵用于筛选血管内皮细胞网络形成或通过血管生成抑制的抗癌治疗的实验分子;在这种情况下, 我们将评估肿瘤血管生成潜能的单核细胞治疗9,10,11。
本议定书的目的是证明一个简单和有效的方法来研究的免疫过程中造成的缺血在一个体内模型。我们可以产生一个更现实的测试环境与组织学检查的验尸肌肉组织。

<table><tbody><tr><td>10% fetal calf serum (FCS)</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>1% penicillin/streptomycin</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>1mL Omnifix -F insuline syringe</td><td>B. Braun, Melsungen AG, Melsungen, Germany</td><td></td><td></td></tr><tr><td>50 ml syringe&amp;nbsp;</td><td>Fresenius Kabi AG, Bad Homburg, Germany</td><td></td><td>Injectomat- syringe 50 ml with canule</td></tr><tr><td>6-well-ultra-low-attachement-plates</td><td>Corning Incorporated, NY, USA</td><td></td><td></td></tr><tr><td>8- 12 week old, male, C57BL/6, BalbC mice&amp;nbsp;</td><td>Charles River, Sulzfeld, Germany</td><td></td><td></td></tr><tr><td>Adhesive tape</td><td>TESA SE, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Acquisition Software</td><td>Leica, Wetzlar, Deutschland&amp;nbsp;</td><td></td><td>Leica Application Suite Advanced Fluorescence (LAS AF); Version: 2.7.3.9723</td></tr><tr><td>Canules</td><td>B. Braun, Melsungen AG, Melsungen, Germany</td><td></td><td>29G, 30G</td></tr><tr><td>Cell culture dish</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Cell culture medium</td><td>Manufactured by our group with single components</td><td></td><td>Medium199, 10% Fetal calf serum, 1% Antibiotic (penicillin/streptomycin)</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter GmbH, Krefeld, Germany</td><td></td><td>Allegra X-15R centrifuge</td></tr><tr><td>Depilatory cream</td><td>Veet, Mannheim, Germany</td><td></td><td></td></tr><tr><td>DiO</td><td>Invitrogen Eugene, Oregon, USA</td><td></td><td></td></tr><tr><td>Disinfection agent</td><td>Sch&amp;uuml;lke&amp;amp;Mayr GmbH, Norderstedt, Germany</td><td></td><td></td></tr><tr><td>Disposable scalpel No.10&amp;nbsp;</td><td>Feather safety razor Co.Ltd, Osaka, Japan&amp;nbsp;</td><td></td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Erlenmeyer flask</td><td>GVB, Herzogenrath, Germany</td><td></td><td></td></tr><tr><td>Ethanol 70%</td><td>Otto Fischar GmbH und Co KG, Saarbr&amp;uuml;cken, Germany</td><td></td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Fine Forceps</td><td>Rubis, Stabio, Switzerland</td><td></td><td></td></tr><tr><td>Flurophor/Rhodamindextran</td><td>Thermo Fischer Scientific, Waltham, MA USA</td><td>Katalognummer: D-1819</td><td></td></tr><tr><td>Gloves</td><td>R&amp;ouml;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td><td></td><td></td></tr><tr><td>Heating pad&amp;nbsp;</td><td>Labotect GmbH, G&amp;ouml;ttingen, Germany&amp;nbsp;</td><td></td><td>Hot Plate 062</td></tr><tr><td>Human macrophage-colony stimulating factor</td><td>Sigma Aldrich, Hamburg, Germany</td><td>SRP3110&amp;nbsp;</td><td></td></tr><tr><td>Humane leucocyte filters</td><td>Blood preservation</td><td></td><td></td></tr><tr><td>Incubator</td><td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter Deutschland GmbH, Unterschlei&amp;szlig;heim, Germany</td><td></td><td></td></tr><tr><td>Ketamine (10%)</td><td>Ketavet, Pfizer Deutschland GmbH, Berlin , Germany</td><td></td><td></td></tr><tr><td>Leukocyte separation tubes (tubes with filter)&amp;nbsp;</td><td>Bio one GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Light microscope&amp;nbsp;</td><td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td><td></td><td>Axiovert 40 C</td></tr><tr><td>Lymphocyte separation medium LSM1077</td><td>GE Healthcare, Pasching, Austria</td><td></td><td></td></tr><tr><td>Matrigel&amp;nbsp;</td><td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td><td></td><td></td></tr><tr><td>Medium M199&amp;nbsp;</td><td>PAA Laboratories GmbH, Pasching, Austria</td><td></td><td></td></tr><tr><td>Microbiological work bench</td><td>Thermo Electron, LED GmbH, Langenselbold, Germany</td><td></td><td>Hera safe</td></tr><tr><td>Microscope slide</td><td>Carl Roth GmbH + Co. KG, Karlsruhe</td><td>Art. Nr. 1879</td><td></td></tr><tr><td>Microscope stand with incubator and heating unit</td><td>&amp;nbsp;Leica DMI 6000, Pecon, Germany</td><td></td><td></td></tr><tr><td>Monocyte wash buffer</td><td>Manufactured by our group with single components</td><td></td><td>PBS, 0,5% BSA, 2mM EDTA</td></tr><tr><td>Mouse restrainer</td><td>Various</td><td></td><td></td></tr><tr><td>Multi-photon microscope&amp;nbsp;</td><td>Leica, Wetzlar, Deutschland&amp;nbsp;</td><td></td><td>Leica SP5 Confocal microscope, Cameleon, Coherent</td></tr><tr><td>NaCl (0,9%)</td><td>Berlin Chemie AG, Berlin, Germany</td><td></td><td></td></tr><tr><td>Neubauer counting chamber&amp;nbsp;</td><td>Paul Marienfeld GmbH und Co.KG, Lauda-K&amp;ouml;nigshofen, Germany</td><td></td><td></td></tr><tr><td>Objective</td><td>Leica, Wetzlar, Deutschland&amp;nbsp;</td><td></td><td>Leica HC PL APO 10x/0.4 CS</td></tr><tr><td>PBS</td><td>Life technologies GmbH, Darmstadt, Germany</td><td></td><td>ph 7,4 sterile</td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Percoll</td><td>Manufactured by our group with single components</td><td></td><td>90 % Percoll, 10% 1,5M NaCl, &amp;rho;= 1,064 g cm-3</td></tr><tr><td>Percoll solution</td><td>GE Healthcare, Bio-Science AB, Uppsala, Sweden</td><td></td><td></td></tr><tr><td>Pipettes</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td>10&amp;micro;L/100&amp;micro;L/200&amp;micro;L/1000&amp;micro;L</td></tr><tr><td>Pipettes serological</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>&amp;nbsp;Cellstar2ml, 5ml, 10ml</td></tr><tr><td>Pipetting heads</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Pipetus</td><td>Eppendorf AG, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Polystyrol tube</td><td>Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td></td></tr><tr><td>Scissor</td><td>Word Precision Instruments, Inc., Sarasota, USA</td><td></td><td></td></tr><tr><td>Scale</td><td>Mettler PM4800 Delta Range, Mettler-Toledo GmbH, Gie&amp;szlig;en, Germany</td><td></td><td></td></tr><tr><td>Suction unit</td><td>Integra bioscience, Fernwald, Germany</td><td></td><td>Vacusafe comfort</td></tr><tr><td>Surgical scissors</td><td>Word Precision Instruments, Inc., Sarasota, USA</td><td></td><td></td></tr><tr><td>Trypan blue solution 0,4 %</td><td>Sigma Aldrich, Hamburg, Germany</td><td></td><td></td></tr><tr><td>Tubes with cap</td><td>Greiner Bio-One GmbH, Frickenhausen, Germany</td><td></td><td>15ml, 50ml Cellstar</td></tr><tr><td>Xylazine (2 %)</td><td>Ceva Tiergesundheit GmbH, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr></tbody></table>

intravital microscopy of monocyte homing and tumor-related angiogenesis in a murine model of peripheral arterial disease

外周动脉疾病和缺血性心脏病的治疗目的是增加血流对血液动力学狭窄引起的缺血性区域。在某些病例中, 血管手术是一个可行的选择, 但是对于没有手术适应症的病人, 如进展到休息疼痛, 严重肢体缺血, 或严重的生命或工作中断, 几乎没有可能减轻他们的疾病。通过刺激侧枝形成的单核细胞强化灌注, 是少数非侵入性选择之一。
我们的小组检查 arteriogenesis 后单核细胞移植到小鼠使用后肢缺血模型。在此之前, 我们已经证明了改进的后肢灌注使用破伤风刺激体单核细胞移植。除了对侧枝形成的影响外, 该疗法还可能影响肿瘤的生长。为了研究这些效应, 我们使用了一个基底膜样基质小鼠模型, 通过注射 Engelbreth-栎-群肉瘤的胞外基质到小鼠的侧面, 在股动脉闭塞后。
在人工肿瘤研究后, 我们使用活体显微镜来研究体内肿瘤血管生成和单核细胞在侧支动脉内的归巢。先前的研究已经描述了动物模型的组织学检查, 这是预料随后对宰后伪影的分析。我们的方法直观地将单核细胞归巢到抵押的实时序列, 易于执行, 并研究了 arteriogenesis 和肿瘤血管生成的过程在体内。

活体显微镜检查肿瘤和侧枝血管的生长触发的单核细胞可以帮助揭示新的方面, 分子机制的肿瘤血管生成和 arteriogenesis。必须使用该协议的步骤精心准备和注射细胞。差异可以导致单一实验之间的变化。单核细胞必须注入静脉系统 (图 1), 以保持系统性的影响, 避免栓子, 这可能发生, 如果注射是在动脉系统进行。
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Watch this Scientific Journal Video about Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease at JoVE.com

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

单核细胞是外周动脉疾病中 arteriogenesis 的重要介质。本协议采用基底膜样基质和活体显微镜, 研究单核细胞注射在股动脉结扎小鼠模型中的单核细胞归巢和肿瘤相关血管生成。

小鼠外周动脉疾病模型中单核细胞归巢和肿瘤相关血管生成的活体显微镜观察

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

intravital-microscopy-monocyte-homing-tumor-related-angiogenesis

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.1K 次观看.  University of Magdeburg. 单核细胞是外周动脉疾病中 arteriogenesis 的重要介质。本协议采用基底膜样基质和活体显微镜, 研究单核细胞注射在股动脉结扎小鼠模型中的单核细胞归巢和肿瘤相关血管生成。

视频: Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

科研

癌症研究

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

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

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 ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

血管紧张素 II. 型小鼠白细胞滚动和粘附的可视化技术及缺陷

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

医学

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 ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

小鼠后肢坏死模型中脚垫脉管系统的高分辨率三维成像

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...