Gunzer实验室的研究得到德国联邦教育和研究部（公元0315590）的授权和德国研究基金会（授权号GU769 / 4-1，GU769 / 4-2）的支持。我们进一步感谢IMCES的技术支持和Sebastian Kubat对3D卡通造型的帮助。

所有动物实验均按照欧盟指南进行，并得到了埃森相关地方当局的批准（AZ 84-02.04.2014.A036 - LandesamtfürNatur，Umwelt und Verbraucherschutz Nordrhein-Westfalen，Essen，Germany）。使用动物并在特定无病原体（SPF）条件下饲养。 白喉毒素诱导心肌炎（DTX） <ol><li>通过将DTX储备溶液用磷酸盐缓冲盐水（PBS）稀释到1μg/ mL工作溶液中，制备DTX溶液用于诱导心肌炎。 </li><li> 应用100μL这种溶液气管内（它）到8至10周龄的CD11c.DTR小鼠（ 约 5ng / g体重（bw）），如Hasenberg 等人所示。为了应用真菌孢子悬浮液20 。 注意:腹膜内施用毒素可能导致类似的诱导致命性心肌炎。但是，这种方法必须首先被验证。 <ol><li>对于它的应用，通过腹膜内（ip）注射100mg / kg bw氯胺酮和10mg / kg bw赛拉嗪麻醉动物。 </li><li>达到深度麻醉（脚趾压缩测试）后，通过使用22 G静脉导管通过口腔插管动物。通过使用小型动物呼吸器以每分钟250次呼吸和每次呼吸250μL的吸入体积通气，检查镜片管的正确定位，Hasenberg 等也显示。人。 20 。 注意:正确定位时，动物的呼吸速率将根据应用的通气设置而改变。 </li><li>断开通气系统，并将100μLDTX溶液或等量的PBS作为对照通过导管滴入肺内，并继续对动物进行再通气1分钟。 </li></ol></li><li>让动物从麻醉中恢复，并监测一般健康状况和体重变化4天。 注意:根据遗传背景，小鼠应变或初始体重，心肌炎的严重程度和发作可能有所不同，应首先确定。 </li></ol> 2.光照显微镜的样品制备<ol><li>毒素施用后4天，用蒸发的2％异氟烷/氧气混合物冲洗诱导室，麻醉小鼠。使用后视镜应用途径静脉内（iv），在50μLPBS中注射15μg直接标记的抗CD45抗体（克隆30-F11，AlexaFluor647（AF647）），这确保了非常快速和高度准确的应用正确量的抗体。 注意:当小鼠躺卧并且不对脚趾夹紧测试做出反应时，达到了所需的麻醉深度。 </li><li> 孵育2小时后，通过颈椎脱位处死小鼠d用5 mL PBS / EDTA（5 mM）灌注心脏。 <ol><li>使用21 G导管将心脏暴露于右心室注射灌注溶液。灌注缓慢，恒定的压力，并确保在切开主动脉后可以排出血液。 注意:这种经心脏灌注可能会导致较小的准备工件，这可能会干扰器官的最终3D可视化。因此，先进的动物实验者可以通过下腔静脉进行灌注，切除腹主动脉。 </li></ol></li><li> 对于化学固定，通过与5mL的4％多聚甲醛（PFA）相同的凹处灌注心脏。将导管保持在适当位置，然后轻轻地安装一个装有PFA的新注射器。充满缓慢和恒定的压力。 注意:对甲醛有剧毒性;避免与皮肤，眼睛和其他粘膜接触。 <ol><li>用锯齿镊子轻轻抓住心脏; PU稍微向上;并切割所有组织连接，包括进入和输出的动脉和静脉。将器官在4％PFA中另存4 h，以进一步固定。 </li></ol></li><li>为了使器官脱水，以50％，70％和100％的上升乙醇系列将心脏孵育12小时，在黑暗中4℃孵育。在这里，使用黑色的5毫升聚丙烯反应管，使其能够以较慢的转速在管旋转器中恒定旋转，以防止气泡形成。 </li><li>通过化学清除完成样品制备。为了使心脏透明，将其在98％二苄醚（DBE）中孵育，同时不断旋转过夜。 注意:DBE刺激眼睛，皮肤和呼吸系统。 </li></ol> 3.光照显微镜<ol><li>使用光照显微镜进行LSFM（参见材料表）。将样品放在显微镜的标准样品架上放入DBE填充的铜中vette，适用于高达30 mm x 30 mm x 15 mm的样品。 </li><li> 在使用脱水和清除的琼脂糖块的图像采集过程中将样品牢牢固定在适当位置。 <ol><li>为了制备琼脂糖块，将2％熔融琼脂糖倒入模具（15 mm x 15 mm x 5 mm）。让琼脂糖冷却下来，直到凝固。 </li><li>以升序乙醇系列（50％，70％，100％100％）脱水。在98％DBE中孵育琼脂糖块过夜。将琼脂糖储存在DBE中直至使用。 注意:由于琼脂糖主要由水组成，每个脱水步骤的孵育时间可以根据块的大小而延长。 注意:DBE刺激眼睛，皮肤和呼吸系统。 </li><li>当需要时，使用锋利的手术刀将制备的琼脂糖块切割成所需的形状。通常，切割棱镜或楔形形状以放置在样品适配器的边缘。 注意:由于琼脂糖块具有弹性，当用力稍微推动时，样品保持原位。 </li></ol></li><li> 用适当的激发和发射滤光片设置检测感兴趣的荧光信号（这里为自发荧光和抗CD45抗体染色）。 <ol><li>为了检测结缔组织来源的自发荧光信号，使用波长488 nm（OPSL:50 mW）的525/50 nm带通滤波器。在668nm（带通滤波器:680/30nm）和647nm的激发波长（二极管激光器:50mW）下检测到具有AF647偶联抗体的CD45染色。 </li><li>选择4μm作为两个单独z平面之间的距离。 </li></ol></li></ol>图像后处理注意:采用科学的3D / 4D图像处理和分析软件进一步处理获取的数字图像数据（参见材料表）。 <ol><li> 打开和预先调整数据文件。 </st荣&gt; <ol><li>开始分析软件后，选择左上角的"Surpass"按钮。要打开图像堆栈，请选择"文件"，"打开"，然后选择包含第一个获取的通道数据的文件夹。选择"编辑"和"添加频道"以添加更多获取的频道。 注意:根据数据大小和计算机系统，此过程可能需要几分钟。所呈现的协议演示了2个采集通道（自发荧光和AF647）的所有步骤。 </li><li>通过单击"编辑"并选择"图像属性"来更正x，y和z体素尺寸。当新窗口打开时，调整参数。 注意:此步骤取决于所用的显微镜​​系统和特定的数据格式。正确的xyz值对于随后的尺寸和远距离测量是至关重要的。在大多数情况下，这些参数作为元数据存储在显微图像文件中。但是，如果文件fo分析软件无法正确解释rmat，手动输入像素大小很重要。 x和y尺寸中的像素尺寸取决于相机像素大小，潜在应用的合并，以及镜头和物镜放大倍数。 z中的像素大小等于自定义的z步长（ 例如 4μm）。 </li></ol></li><li> 频道调整 <ol><li>首先通过打开"显示调整"（"编辑"和"显示调整"）将自动荧光信号转换为灰度。新窗口将列出所有打开的通道和所有显示设置;要更改其默认颜色，选择自动荧光通道并选择"白色"显示样式。点击"确定"以应用。 </li><li>对于黑色电平调整，返回"显示调整"，并调整黑色电平值，以排除不代表样品结构的不需要的背景信号秒。 <ol><li>为此，请使用通道栏上的左上角三角形并将其从左向右拖动。 注意:更改将立即可视化。确保仅抑制不是从样品得到的信号。背景中的最小值不应该是"0"，以避免音调黑色像素，因为可能需要噪声信号进行额外的计算。确保在图像采集后执行所有黑色电平变化。否则，宝贵的样品信息可能会丢失。黑色调整只是代表性的目的。要在不同的样品上使用相同的显示设置，可以在通道列表下面的"最小"数值区域中输入精确的值。这对定量分析非常有帮助。 </li></ol></li><li>执行强度调整，要么通过使用右上角的三角形来调整相应的通道强度，要么将精确值输入到通道下方的"最大"数值范围列表。 </li><li>通过拖动通道条中间的下三角形来进行对比度调整，以增加或减少伽马值或输入精确值。 </li><li>根据自动荧光通道调整AF647通道。不同的是，将AF647信号的可视化设置为热图颜色查找表。通过类似于步骤4.2.1的方法选择频道模式"火"。 注意:由于整体信号强度与自发荧光通道相比显着降低，黑色电平通常调整到更低的值。对于自发荧光通道，该通道的亮度通常增加。 </li><li>选择"混合模式"来细化组织结构。用相同的参数值分析来自一个系列的所有样品。 </li></ol></li><li> 虚拟剪辑 <ol><li>在3D场景展示之前虚拟切割器官rt，在渲染的3D对象上应用裁剪平面。 <ol><li>点击对象列表中的"剪切平面"图标（剪刀）。在图像视图中，将出现黄色框架和操纵器（白色主轴）。用鼠标旋转较薄端的主轴，从而改变裁剪平面的方向，并移动主轴较厚的中间部分，以选择所需的裁剪深度。 </li><li>通过取消选中对象列表下方的相应复选框来隐藏框架和操纵器，并创建所需的快照。 </li></ol></li></ol></li></ol>

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Cell Sci. 129 (2), 444-456 (2016).</li><li>Pan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shrinkage-mediated+imaging+of+entire+organs+and+organisms+using+uDISCO.">Shrinkage-mediated imaging of entire organs and organisms using uDISCO.</a> Nat. Methods. 13 (10), 859-867 (2016).</li></ol>

作者没有什么可以披露的。

在现代生命科学中，生物过程的微观可视化起着越来越重要的作用。在这种情况下，在过去两个世纪里已经取得了许多发展，帮助回答到目前为止无法解决的问题。首先，从根本上提高光学显微镜的分辨能力有明显的趋势。使用结构照明显微镜（SIM） 21,22 ，受激发射耗尽（STED）显微镜23和光激活定位显微镜（PALM） 24,25或随机光学重建...

在光学显微镜的进化过程中，出现了许多专门的形式，它们都被开发出来，以最小化特定标本的可视化过程的限制。一种这样的方法是光片荧光显微镜（LSFM）。首先在1903年由Siedentopf和Zsigmondy 1开发 ，并在20世纪90年代早期找到了其第一个基本的生物应用2 ，LSFM已经成为迄今为止最大的微观工具，可以显示大样本，如完整的小鼠器官，具有荧光信号分辨率下降到细胞水平。由于这些优点，结合其活细胞成像的潜力，自然方法将LSFM命名为"2014年度方法3 "。 顾名思义，LSFM中的样品照明由光片进行，光片垂直于所用物镜的轴定向或发射光收集和随后的图像形成。光板通常通过将宽的准直激光束与柱面透镜聚焦或者通过在仅一个水平或垂直平面4,5中的窄的聚焦激光束的快速侧向运动来产生。以这种方式，只有成像光学器件的焦平面被照亮，通常具有1-4μm的厚度。因此，对于放置在照明平面中的荧光样品，消除或大大抑制6,7的散射光的产生和来自焦平面上方或下方的区域的光漂白的影响。由于所有失焦的飞机都不被照亮，所以在这些区域中不会出现光漂白效果。与标准共聚焦或多光子显微镜相反，照明和发射光的路径彼此分离，因此最终图像质量<html>不依赖于通过目标的激发光束的完美聚焦。根据潜在的问题，因此可以使用具有巨大视场（FOV）的物镜，使得照射平面的最大可能面积可被成像，而没有任何部件在xy方向上移动。 在现代LSFM系统中，产生的光学部分的荧光图像被捕获在能够获得整个视场（FOV）的高灵敏度电荷耦合器件（CCD）或互补金属氧化物半导体（CMOS）照相机上，微秒。因此，通过将样品移动通过光片并通过以确定的z步骤获取图像，可以在合理的时间量8,9获得样本的完整3D信息，使得该技术适用于活细胞研究10 ，ass ="xref"&gt; 11，12。 然而，尽管LSFM提供了一种快速，灵敏和荧光友好的方法，但通过样品的透光性仍然是一个主要问题，特别是当大型生物样品成为3D分析的目标时。通过吸收的物理方面和具有不同折射率的结构的界面处的光散射来严格地改变透光性。因此，当大小成像采样数毫米时，LSFM主要与清除协议相结合，使样本光学透明。这些技术基于从相应的生物组织中除去水并将其与基于溶剂的浸渍介质（基于溶剂的浸渍介质）进行交换的想法，所述浸渍介质被选择为与特定目标组织组分的折射率窄度匹配。结果，横向光散射最小化，并且所有的波长的光可以更有效地穿过组织13 。在许多情况下，以这种方式处理的生物标本在宏观上看起来是晶体透明的，即使在整个小鼠器官上，使用长工作距离，低放大倍数的目标，也可以进行LSFM。 在这里，我们提出了在光照片显微镜（参见材料表）中大样本成像的准备方案，我们已经建立了这一方案来研究心肌炎鼠模型中的细胞心脏浸润15 。 CD11c.DTR小鼠在CD11c启动子16的控制下表达灵长类白喉毒素受体（DTR）。因此，与DTR一起表达CD11c的这些小鼠中的细胞对白喉白喉毒素（DTH）的外毒素敏感 ;用DTX对这些动物的系统治疗导致所有CD11c + ce的耗尽LLS。 CD11c是整联蛋白，并且作为各种不同可溶性因子的细胞表面受体，主要参与单核细胞谱系17的细胞的激活和成熟过程。因此，CD11c.DTR小鼠模型已被广泛用于研究树突状细胞和巨噬细胞亚群在许多不同免疫学问题背景下的作用。随着时间的推移，据报道，用DTX系统治疗的CD11c.DTR小鼠可能会​​产生不良副作用，并可显示强烈升高的死亡率18,19 。最近，我们能够确定死因的原因15 ，描述了在这些动物中气管内DTX应用后暴发性心肌炎的发展。毒素挑战引起细胞破坏，包括在心脏刺激传播系统的中心部分。伴随着大量的CD45+白细胞浸润，最终导致致命的心律失常。在这种情况下，不仅白细胞群体的出现是重要的，而且也是其心脏内部的空间分布。这个实验问题是现代显微成像的一个挑战，我们已经通过光镜显微镜方法解决了这一点，该方法由活体抗体染色和基于有机溶剂的光学清除方案支持。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>diphtheria toxin</td>
			<td>Sigma Aldrich</td>
			<td>D0564</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Biochrom</td>
			<td>L182-10</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine [50 mg/ml]</td>
			<td>Inresa</td>
			<td>PZN 4089014</td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine [2 %]</td>
			<td>Ceva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>syringe</td>
			<td>Braun</td>
			<td>REF 9161502</td>
			<td>Braun Omincan F&#160;</td>
		</tr>
		<tr>
			<td>indwelt venous catheter&#160;</td>
			<td>Becton Dickinson</td>
			<td>REF 381923</td>
			<td>BD Insyte Autoguard</td>
		</tr>
		<tr>
			<td>small animal respirator</td>
			<td>Harvard Apparatus</td>
			<td>73-0044</td>
			<td>MiniVent</td>
		</tr>
		<tr>
			<td>anit-CD45 antibody (AF647)</td>
			<td>BioLegend</td>
			<td>103124</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td>Carl Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>catheter (21 G)</td>
			<td>BD Biosciences</td>
			<td>REF 387455</td>
			<td>BD valu-set</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PE tube (5 ml)</td>
			<td>Carl Roth</td>
			<td>EKY9.1</td>
			<td>Rotilabo</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Carl Roth</td>
			<td>9065.4</td>
			<td></td>
		</tr>
		<tr>
			<td>dibenzyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>108014</td>
			<td></td>
		</tr>
		<tr>
			<td>tube rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>MACSmix</td>
		</tr>
		<tr>
			<td>agarose (low gelling)</td>
			<td>Sigma Aldrich</td>
			<td>A4018</td>
			<td></td>
		</tr>
		<tr>
			<td>mold (15x15x5 mm)</td>
			<td>Tissue Tek</td>
			<td>4566</td>
			<td>Cryomold&#160;</td>
		</tr>
		<tr>
			<td>light sheet microscope system</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td>Ultramicroscope&#160;</td>
		</tr>
		<tr>
			<td>microscope body</td>
			<td>Olympus</td>
			<td></td>
			<td>MVX10&#160;</td>
		</tr>
		<tr>
			<td>objective</td>
			<td>Olympus</td>
			<td></td>
			<td>0.5&#160;NA MVPLAPO 2XC, WD 5.5&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>sCMOS camera 5.5MP</td>
			<td>Andor Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm OPSL (50mW) laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>647 nm&#160; diode laser (50mW)</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D image processing &#38; analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>IMARIS Ver. 8.3</td>
		</tr>
		<tr>
			<td>transgenic mouse strain</td>
			<td>Steffen Jung et al.</td>
			<td></td>
			<td>CD11c.DTR</td>
		</tr>
		<tr>
			<td>wt mouse strain</td>
			<td>Envigo</td>
			<td></td>
			<td>Balb/c Ola Hsd J</td>
		</tr>
		<tr>
			<td>Laser Module</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MVPLAPO 2XC Objective Lens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative visualization of leukocyte infiltrate in a murine model of fulminant myocarditis by light sheet microscopy

光谱荧光显微镜（LSFM）与化学清除方案相结合，已经成为分析大型生物标本中荧光标记结构的黄金标准，并且归功于细胞分辨率。同时，不断改进的底层协议和增强的专业商业系统的可用性使我们能够调查整个小鼠器官的微结构，甚至允许表征各种活细胞成像方法中的细胞行为。在这里，我们描述了空间整体可视化和发射小鼠心脏CD45 +白细胞群体定量的方案。该方法采用最近已被证明可用作研究暴发性致命性心肌炎发展的鲁棒诱导型模型的转基因小鼠株（CD11c.DTR），其特征在于致死性心律失常。该方案包括心肌炎诱导，玻璃体内抗体介导的细胞染色，器官制备和随后的计算机辅助图像后处理的LSFM。尽管该方案作为我们特殊科学问题的高度适应性方法，但协议代表了易于调整的系统的蓝图，也可以在其他器官甚至其他物种中完全不同的荧光结构。

所提出的LSFM方法分析诱导重度心肌炎时小鼠心脏中的白细胞分布和量。 图1A说明转基因CD11c.DTR小鼠心肌炎诱导的预处理方案。该步骤代表将白细胞募集到心肌的必要触发因素。 DTX应用成功后，动物会发生严重的疾病症状，如2-4天的全身无力，厌食，体重减轻。 4天后，白细胞被iv染色。在经心脏灌注之前应用荧光染料偶联的抗CD45抗体以从循环中除去血?...

Watch this Scientific Journal Video about Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy at JoVE.com

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

在这里，我们描述了在无菌暴发性心肌炎的鼠模型中可视化心脏CD45 +白细胞浸润的光照片显微镜方法，其由气管内白喉毒素治疗CD11c.DTR小鼠诱导。

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 ...

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7.7K 次观看.  University of Duisburg/Essen. 在这里，我们描述了在无菌暴发性心肌炎的鼠模型中可视化心脏CD45 +白细胞浸润的光照片显微镜方法，其由气管内白喉毒素治疗CD11c.DTR小鼠诱导。 

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

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

一个小鼠模型，通过神经肌肉电刺激肌肉训练

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

科研

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

心脏异常的实验性自身免疫性心肌炎的无创性评估磁共振成像显微镜在鼠标

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.