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>

<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 border="0" cellpadding="0" cellspacing="0" width="1433">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10% fetal calf serum (FCS)</td>
			<td style="width:495px;">Sigma Aldrich, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% penicillin/streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1mL Omnifix -F insuline syringe</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml syringe&#160;</td>
			<td>Fresenius Kabi AG, Bad Homburg, Germany</td>
			<td style="width:179px;"></td>
			<td>Injectomat- syringe 50 ml with canule</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well-ultra-low-attachement-plates</td>
			<td>Corning Incorporated, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8- 12 week old, male, C57BL/6, BalbC mice&#160;</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive tape</td>
			<td>TESA SE, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition Software</td>
			<td>Leica, Wetzlar, Deutschland&#160;</td>
			<td></td>
			<td>Leica Application Suite Advanced Fluorescence (LAS AF); Version: 2.7.3.9723</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Canules</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td style="width:179px;"></td>
			<td>29G, 30G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture medium</td>
			<td>Manufactured by our group with single components</td>
			<td style="width:179px;"></td>
			<td>Medium199, 10% Fetal calf serum, 1% Antibiotic (penicillin/streptomycin)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter GmbH, Krefeld, Germany</td>
			<td style="width:179px;"></td>
			<td>Allegra X-15R centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream</td>
			<td>Veet, Mannheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiO</td>
			<td>Invitrogen Eugene, Oregon, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disinfection agent</td>
			<td>Sch&#252;lke&#38;Mayr GmbH, Norderstedt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable scalpel No.10&#160;</td>
			<td>Feather safety razor Co.Ltd, Osaka, Japan&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Erlenmeyer flask</td>
			<td>GVB, Herzogenrath, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 70%</td>
			<td>Otto Fischar GmbH und Co KG, Saarbr&#252;cken, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Forceps</td>
			<td>Rubis, Stabio, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flurophor/Rhodamindextran</td>
			<td>Thermo Fischer Scientific, Waltham, MA USA</td>
			<td>Katalognummer: D-1819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gloves</td>
			<td>R&#246;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad&#160;</td>
			<td>Labotect GmbH, G&#246;ttingen, Germany&#160;</td>
			<td style="width:179px;"></td>
			<td>Hot Plate 062</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human macrophage-colony stimulating factor</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td>SRP3110&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Humane leucocyte filters</td>
			<td>Blood preservation</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Baxter Deutschland GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine (10%)</td>
			<td>Ketavet, Pfizer Deutschland GmbH, Berlin , Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leukocyte separation tubes (tubes with filter)&#160;</td>
			<td>Bio one GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope&#160;</td>
			<td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td>
			<td style="width:179px;"></td>
			<td>Axiovert 40 C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lymphocyte separation medium LSM1077</td>
			<td>GE Healthcare, Pasching, Austria</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel&#160;</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium M199&#160;</td>
			<td>PAA Laboratories GmbH, Pasching, Austria</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microbiological work bench</td>
			<td>Thermo Electron, LED GmbH, Langenselbold, Germany</td>
			<td style="width:179px;"></td>
			<td>Hera safe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope slide</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe</td>
			<td>Art. Nr. 1879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope stand with incubator and heating unit</td>
			<td>&#160;Leica DMI 6000, Pecon, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monocyte wash buffer</td>
			<td>Manufactured by our group with single components</td>
			<td style="width:179px;"></td>
			<td>PBS, 0,5% BSA, 2mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse restrainer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-photon microscope&#160;</td>
			<td>Leica, Wetzlar, Deutschland&#160;</td>
			<td></td>
			<td>Leica SP5 Confocal microscope, Cameleon, Coherent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (0,9%)</td>
			<td>Berlin Chemie AG, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauer counting chamber&#160;</td>
			<td>Paul Marienfeld GmbH und Co.KG, Lauda-K&#246;nigshofen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective</td>
			<td>Leica, Wetzlar, Deutschland&#160;</td>
			<td></td>
			<td>Leica HC PL APO 10x/0.4 CS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td style="width:179px;"></td>
			<td>ph 7,4 sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll</td>
			<td>Manufactured by our group with single components</td>
			<td style="width:179px;"></td>
			<td>90 % Percoll, 10% 1,5M NaCl, &#961;= 1,064 g cm-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Percoll solution</td>
			<td>GE Healthcare, Bio-Science AB, Uppsala, Sweden</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td>10&#181;L/100&#181;L/200&#181;L/1000&#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes serological</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td>&#160;Cellstar2ml, 5ml, 10ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetting heads</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipetus</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polystyrol tube</td>
			<td>Cellstar, Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissor</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scale</td>
			<td>Mettler PM4800 Delta Range, Mettler-Toledo GmbH, Gie&#223;en, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suction unit</td>
			<td>Integra bioscience, Fernwald, Germany</td>
			<td style="width:179px;"></td>
			<td>Vacusafe comfort</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue solution 0,4 %</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubes with cap</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td style="width:179px;"></td>
			<td>15ml, 50ml Cellstar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (2 %)</td>
			<td>Ceva Tiergesundheit GmbH, D&#252;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

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

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

科研

医学

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

动脉再狭窄小鼠模型:股导线损伤的技术方面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

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

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

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 ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

冠状血管生成体模型

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

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

摘要

探索更多视频

此视频中的章节

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

动脉再狭窄小鼠模型:股导线损伤的技术方面

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

活体显微镜及血栓诱导在无毛小鼠的耳垂

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

评估下肢缺血的Murine模型的治疗血管生成

冠状血管生成体模型

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

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

摘要

探索更多视频

此视频中的章节

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

动脉再狭窄小鼠模型:股导线损伤的技术方面

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

活体显微镜及血栓诱导在无毛小鼠的耳垂

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

评估下肢缺血的Murine模型的治疗血管生成

冠状血管生成体模型

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口