Name: visualizing leukocyte rolling and adhesion in angiotensin ii-infused mice: techniques and pitfalls
Uploaded: 2018-01-04T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls at JoVE.com

这项工作得到了德国教育和研究部 (BMBF 01EO1003 和 BMBF 01EO1503) 的支持。

在由莱茵兰-Palatinate (授权号 23 177-07/G12-1-002 和 23 177-07/G15-1-051) 的动物实验伦理委员会批准下, 对8岁至12周龄的雄性小鼠进行了实验。
1. 小鼠麻醉和手术准备
<ol>
	<li>手术前, 检查动物的健康状况。手术前, 每天对动物进行2-3 天的监测, 观察它们的一般情况 (外貌、姿势、自发行为) 以及体重、食物和用水量。</li>
	<li>准备麻醉用咪唑安定 (5 毫克/千克体重), medetomidine (0.5 毫克/千克体重), 和芬太尼 (0.05 毫克/千克体重), 以腹腔 (ip) 注射200µL/30 克鼠标在1毫升注射器与26克针。</li>
	<li>在一个39° c 升温平台的操作场上进行渗透泵植入和动物制剂的综合防治实验。在消毒浴中消毒所有工具, 消毒加温平台。</li>
	<li>在手术过程中, 用眼药膏保护动物眼睛。在渗透泵植入前用剃刀除去毛皮。使用脱毛膏和棉签分离动脉和颈静脉导管植入在综合防治实验。用两种皮肤消毒剂对动物皮肤进行消毒: 一种含有聚维酮碘的防腐剂和消毒剂, 一种含有 octenidine 的皮肤防腐剂。</li>
	<li>准备主混合拮抗麻醉 ("antisedan 混合") 与 atipamezol (0.05 毫克/千克体重) 和马西尼 (0.01 毫克/kgbody 重量), 并准备200µL/鼠标在1毫升注射器与 26 G 针是皮下注射 (南卡罗来纳州) 注射。</li>
</ol>
2. 渗透泵的制备和植入
注: 使用渗透泵的皮下 ii 输液详细介绍了鲁et al.17
<ol>
	<li>用无菌盐水重组冻干粉制备 ii, 并以动物重量和泵送率调节浓度。将重组的 ii 在塑料管中 (不要使用玻璃管)。</li>
	<li>用 ii 溶液填充泵, 以提供1毫克/(kg·d) 7 天。准备好后, 将泵在无菌盐水中保持在37° c 的4-6 小时以上。</li>
	<li>在麻醉后用后足反射法对小鼠进行完全麻醉, 并将其置于温热的操作场。麻醉诱导需要10-15 分钟, 如果把动物放在黑暗环境中, 效果会更好。</li>
	<li>剃掉老鼠的下背部, 用酒精性皮肤防腐剂消毒。</li>
	<li>用剪刀将1厘米的切口垂直于脊柱。使用海峡止血为泵创建一个口袋, 并插入泵 (流版主第一)。口袋必须允许泵的自由运动, 在伤口上没有压力。</li>
	<li>用缝线缝合伤口, 而不是夹紧, 以限制炎症;然后用皮肤防腐剂消毒。</li>
	<li>拮抗麻醉的200µL 的 antisedan 混合和返回的老鼠到它的笼子。</li>
	<li>用丁丙诺啡 (0.075 毫克/千克) 进行术后镇痛, 一次手术后根据动物的行为要求。</li>
</ol>
3. 颈静脉导管植入及动脉制剂
<ol>
	<li>对麻醉后的小鼠进行全身麻醉, 用后部食物反应, 在39° c 手术板上用直肠探针将麻醉动物放在背 recumbence 上, 以保持体温并把药膏放在在程序中保护他们的眼睛。</li>
	<li>使用脱毛膏去除颈部毛发。用棉签将奶油涂抹2分钟, 直到头发开始脱落。再加2分钟后, 用刮刀除去奶油和毛发, 用酒精性皮肤杀菌消毒皮肤。</li>
	<li>在显微镜下进行手术。做一个 1-1. 5 厘米长的切口在皮肤纵向地在脖子, 1 cm 在气管旁边。然后使两个其他切口垂直于第一切口的两个肢。</li>
	<li>小心地移除皮肤旁边的组织, 取出覆盖左颈静脉和气管的皮肤块。</li>
	<li>仔细隔离腮腺;舌下和颌下腺腺体从感兴趣区与2弯曲钳。不要割伤或损伤腺体, 让它们进入颈静脉和动脉的最佳通路。</li>
	<li>为了隔离颈静脉, 使用薄钳, 并慢慢打开和关闭它, 轻轻地释放血管从周围的组织。一旦静脉是完全清洁, 定位钳下的船只, 并放置两个7-0 缝合10厘米每下。</li>
	<li>用两节结缝合头部近端。在其他缝合, 准备一个结, 但不要关闭它。</li>
	<li>保持导管 (0.28 毫米内径, 0.61 毫米外径), 用一只手钳填充37° c 无菌生理盐水;而另一方面, 用一把薄薄的剪刀或一条弯曲的26克针, 在容器中做一个小切口。然后将导管插入颈静脉, 并闭合两次结节。在导管上缝合头部近端, 以确保完全固定。</li>
	<li>为了分离和准备颈动脉, 用薄弯曲钳解剖覆盖气管的区域。</li>
	<li>一旦动脉从周围的组织中解脱出来, 从血管中取出迷走神经 (它看起来像一条与颈动脉相邻的白线) (但不要割断它)。薄钳可以关闭, 慢慢打开, 轻轻地调动神经。</li>
	<li>一旦两个动脉都完全清洁, 放置钳下第一颈动脉, 并放置一个小的黑色3毫米宽 x 2.5 厘米长的塑料件下的容器。这是非常重要的, 不过度的船只和检查, 血流存在, 一旦船舶持有人被放置。将第二个动脉放在容器的支架上。如果未将鼠标直接置于显微镜下, 则将腺体放回气管上, 并应用37° c 无菌生理盐水以避免干燥区。</li>
</ol>
4. LysM 和粘附细胞的评价
<ol>
	<li>将吖啶橙从2毫克/毫升的库存溶液中贮存在-20 ° c, 并在1毫升注射器中稀释1:4 的无菌盐水 (0.5 毫克/毫升最终浓度)。</li></ol>保护注射器免受光照, 每只老鼠使用200µL。<ol>
		<li>测试不同条件: (1) 用颈静脉导管注射吖啶橙;(2) 将吖啶橙直接注入尾侧静脉 (无颈静脉导管植入);(3) 无吖啶橙的测量使用荧光 LysMCre+表意文字+小鼠 (这里再次没有颈静脉导管植入)。</li>
	</ol>
	
	<li>一旦导管到位, 两个动脉被隔离, 将鼠标置于显微镜下。使用远距离冷凝器和 10X (NA 0.3) 水浸泡物镜, 用单色仪、分束器和电荷耦合器件摄像机对高速宽场荧光显微镜进行测量。利用 real-time 成像系统进行图像采集和分析。</li>
	<li>将显微镜物镜集中在血管内皮表面的颈动脉中央。缓慢注入50µL 吖啶橙 (0.5 毫克/毫升) (考虑到第一次注射的导管死体积)。将软件设置为记录100图像, 每个图像的曝光时间为120毫秒。</li>
	<li>每一个视频, 每颈动脉 (左, 右) 在不同的位置制作四视频。如果荧光信号减弱, 则再注入50µL 吖啶橙。</li>
	<li>录制完所有的视频后, 安乐的动物被颈椎脱位。</li>
	<li>将视频速度设置为每秒10图像, 并在每个视频的容器中间放置一个200µm x 250 µm 矩形视图中的滚动和粘连单元格的数量。计数轧制和黏附细胞;黏附细胞被定义为在十年代视频中不移动或脱离内皮的细胞。为了简化量化, 用红色荧光去除通道, 只使用绿色荧光计数轧制和粘附细胞。</li>
	<li>为实验使用吖啶橙, 做一个手动阈值的荧光, 以消除背景, 由内皮细胞。</li>

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作者没有透露

LysM+单核细胞曾被证明与高血压的发展有牵连5。在这里, 我们表明, LysM 的免疫细胞的+ , 并坚持到内皮层, 以响应 ii 输液。此发现是使用 LysMCre+表意文字+小鼠获得的, 从我们以前的研究中探索免疫细胞在高血压中的作用在体内通过对吖啶橙注射液的可视化白细胞,6,10。因此, 细胞类型的黏附在内皮的歧视是不可能的。
这是一个非常有用的工具的血管研究和体内观察细胞直接在血管, 但注射染料的影响, 在一个外科插入导管的帮助下, 高血压炎症的背景仍然未知。为了评价导管和吖啶橙注射液的潜在作用, 我们利用了 LysMCre+表意文字+鼠标。ii 输液增加了滚动的 LysM+细胞的数量到内皮。插入导管进入颈静脉放大的效果和更多的滚动和粘附白细胞检测 ii 注射小鼠导管与无导管植入的小鼠相比。这表明, 植入颈动脉导管导致全身炎症反应的伤口愈合的情况下覆盖与免疫反应看到的高血压18。此外, 由于颈静脉靠近颈部动脉, 另外的免疫活化可能是通过影响颈静脉而发生的。由于这种作用是不存在的, 当注射直接进入尾部静脉, 我们可以假设, 该效应不是由于染料, 而是插入导管的程序。我们在这里展示了转基因的报告鼠的重要性, 表达荧光蛋白, 以不干涉在体内过程中的实验。此外, 尾部静脉注射荧光示踪剂可能是一个可能的替代静脉导管注射。
该过程的一个关键步骤是 ii 输液。尾袖评估的血压可以作出, 以控制的有效性, ii 交付的泵。2−3天后血压应增加。为了限制可能影响 LysM+细胞活化的炎症, 在植入泵的切口处, 应该用缝合线而不是夹子来做。必须注意的一个限制是尾静脉注射: 为了使最好的数据获取, 4 视频通常是为每一个颈动脉。如果荧光信号下降, 50 µL 吖啶橙注射, 但与尾部注射更难进行多次注射和保持动脉稳定在显微镜下的目标。颈静脉导管存在的持续时间也可能调节免疫细胞的活化作用, 因为它影响了在导管植入后4小时内制备的富含血小板血浆的凝血活化19。这方面的导管植入需要进一步的调查。
最后, 即使我们赞赏颈静脉导管植入对 LysM 的影响, ii 输液后, 我们不能完全估计的准备, 皮肤移除和颈动脉隔离在潜在的 LysM +细胞的作用激活.我们的结论是, 使用荧光报告鼠像 LysMCre 的+表意文字+鼠标建议, 以避免破坏船只的完整性在动物准备为在体内成像。

动脉高血压增加了心血管疾病和死亡的风险, 促进动脉粥样硬化, 冠心病, 动脉或静脉 thromboembolisms 的发展1。高血压的发展取决于环境、遗传、内分泌和血流动力学因素的相互作用。目前, 免疫及相关炎症在高血压病因中起重要作用2。
在免疫细胞, T 淋巴细胞以及单核和巨噬细胞被发现是因果关系的 ii 诱导的血管炎症和高血压, 部分与他们的能力触发活性氧的物种3。巨噬细胞集落刺激因子缺乏小鼠表现出减少对 ii 的反应血压升高和血管炎症4。在以前的工作中, 我们可以证明 LysM + 单核细胞驱动血管功能障碍和炎症的 ii 诱发高血压5。最近, 我们描述了一个新的途径, 凝血因子 XI 配合血小板和血管壁诱导凝血酶依赖性血管炎症6。目前有关免疫系统在高血压中的作用的知识最近总结和回顾了罗德里格斯-Iturbe et al。7
由于免疫细胞参与高血压的发展变得明显, 研究血管与免疫细胞相互作用的模型和技术成为必要。荧光的血管综合病是一个有用的工具, 观察体内循环血细胞和内皮之间的相互作用,8,9,10。用这种技术, 注射染料插与脱氧核糖核酸 (例如吖啶橙色) 能形象化有核的细胞 (循环和从内皮)。在动脉或静脉损伤模型中, 可以注入 rhodamin-6G 或ˊ (现金流量) 的分离血小板, 以显示血小板丰富的血栓. 
通常, 颈静脉导管用于注射示踪剂或标记血小板。改变内皮和随后活化的凝血叶栅都知道有影响单核细胞活化。内皮损伤立即导致血小板活化通过内基质分子密封的组织, 随之而来的单核细胞吸引和激活11。相反, 一个完整的内皮细胞是已知的抗凝血特性 (例如,通过组织因子途径抑制剂或血栓调节蛋白)12和直接抑制作用的单核,例如, 通过分泌胞外囊泡含有 rna13。单核细胞是已知的产生组织因子, 外源性的凝血级联和表达蛋白酶活化受体 (部), 可以激活凝血酶和参与单核激活14,15.因此, 任何血小板活化或由于血管损伤引起的凝血级联, 可能对单核细胞活化产生意想不到的影响, 并干扰观察到的现象。在表意文字转基因 LysM 转基因小鼠的帮助下, 一个 double-fluorescent 的报告鼠 (LysMCre+表意文字+), 我们建议详细研究注射用导管的效果和替代方法在 LysM+粒细胞在小鼠动脉高血压模型中的作用16。

<table><tbody><tr><td>Midazolam</td><td>Ratiopharm GmbH</td><td></td><td>Anesthesia mix</td></tr><tr><td>Medetomidine</td><td>Pfizer Deutschland GmbH</td><td></td><td>Anesthesia mix</td></tr><tr><td>Fentanyl</td><td>Janssen-Cilag GmbH</td><td></td><td>Anesthesia mix</td></tr><tr><td>Alzane</td><td>Zoetis</td><td></td><td>Antisedan mix</td></tr><tr><td>Flumazenil</td><td>Hikma pharma</td><td></td><td>Antisedan mix</td></tr><tr><td>Braunol</td><td>B Braun</td><td></td><td></td></tr><tr><td>Octeniderm</td><td>Sch&amp;uuml;lke</td><td></td><td></td></tr><tr><td>Osmotic pumps</td><td>Alzet</td><td>1007D</td><td>Osmotic pump implantation</td></tr><tr><td>Angiotensin II</td><td>Sigma-Aldrich</td><td></td><td>Osmotic pump implantation</td></tr><tr><td>7-0 prolene suture</td><td>Ethicon</td><td></td><td>Osmotic pump implantation</td></tr><tr><td>Acridine orange</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Catheter</td><td>Smiths Medical Deutschland GmbH</td><td></td><td>Inside diameter, 0.28 mm; outer diameter, 0.61 mm</td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX51WI fluorescence microscope</td><td></td></tr><tr><td>Beam Splitter</td><td>Photometrics</td><td>CMR-DV2-SYS - DualView2 MicroImager</td><td></td></tr><tr><td>Objective</td><td>Olympus</td><td>UMPLFLN10X</td><td>Water immersion objective with a monochromator</td></tr><tr><td>Camera</td><td>Hamamatsu Photonics</td><td>ORCA-R2</td><td></td></tr><tr><td>Image acquisition and analysis software</td><td>Olympus</td><td>Realtime Imaging System eXcellence RT</td><td></td></tr><tr><td>Mice</td><td>The Jackson Laboratory</td><td>Jax 008705</td><td>B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J</td></tr><tr><td>Mice</td><td>The Jackson Laboratory</td><td>Jax 004781</td><td>LysM Cre (B6.129P2-&lt;em&gt;Lyz2&lt;sup&gt;tm1(cre)Ifo&lt;/sup&gt;&lt;/em&gt;/J )</td></tr></tbody></table>

visualizing leukocyte rolling and adhesion in angiotensin ii-infused mice: techniques and pitfalls

荧光活体视频显微镜 (综合防治) 的血管是一个既定的方法来评估免疫细胞的活化作用和他们的能力, 并坚持内皮层。通常使用荧光染料或荧光偶联抗体的注入来可视化循环细胞。另外, 还可以使用荧光报告小鼠。白细胞的相互作用, 特别是溶菌酶 M+ (LysM+) 单核分子, 血管壁在促进血管功能障碍和动脉高血压方面起着举足轻重的作用。我们在这里提出的技术, 以可视化和量化白细胞轧制和粘附在血管紧张素 II (ii) 诱导高血压的小鼠通过综合防治。
导管的植入会损害血管壁, 导致改变的血细胞反应。我们比较了不同的注射技术和管理路线, 以形象化的白细胞在一个 LysMCre 的+表意文字+小鼠广泛表达红色荧光蛋白和绿色荧光蛋白的条件表达在 LysM+单元格。为了研究 LysM 的+细胞活化, 我们使用了 ii 灌注小鼠, 其中白细胞的滚动和粘附增加到内皮。我们要么通过静脉导管注射吖啶橙, 要么直接通过尾静脉, 比较滚动和黏附细胞的数量。我们发现, 颈静脉导管植入本身增加了在假灌输的 LysMCre+表意文字+小鼠与对照组相比滚动和黏附 LysM+细胞的数量。这种活化作用增强了 ii 的小鼠。有趣的是, 直接通过尾静脉注射吖啶橙并没有增加 LysM 的+细胞粘附或在假灌注小鼠中的滚动。因此, 我们证明了转基因小鼠表达荧光蛋白在实验过程中不干扰体内过程的重要性。此外, 尾部静脉注射荧光示踪剂可能是一个可能的替代静脉导管注射。

动脉的 LysMCre+表意文字的+小鼠注入 ii 的观察使用了综合病媒。用颈静脉导管注射吖啶橙。我们的目的是观察与血管壁接触的 LysM+细胞与所有有核循环细胞的比例 (这也可能与血管相互作用, 因为细胞类型与血管相互作用的区别仍然是一个开放我们以前的工作的问题)。在基线情况下, 颈静脉导管的出现会导致 LysM 的+细胞 (图 2A, D) 的粘连。吖啶橙注射液后, 相同的细胞是荧光的, 而且内皮细胞是荧光的 (图 2B, E)。在减少内皮相关荧光的背景下, 数据表明所有有核的黏附细胞是 LysM 的+ (图 2C, F);这些结果在 ii 输液确认我们以前的结果并证明 LysMCre 的+表意文字+是观察 ii 对 LysM+单元激活的影响的好模型后才成立。
在 LysMCre+表意文字+中, 我们评估了吖啶橙在 ii 输液后的 LysM+细胞活化中的作用。在一周的 ii 输液后, LysMCre+表意文字、+小鼠颈动脉内的白细胞内皮相互作用通过经颈静脉导管或通过尾静脉的吖啶橙注射液进行影像和可视化。没有任何导管或注射, LysM 的滚动的+细胞是显着增加后, ii 输液相比, 未经处理的小鼠, 和粘连显示增加 (图 3)。注射吖啶橙与颈静脉导管增加了 ii 治疗小鼠的黏附力和滚动, 更大程度上与没有导管的小鼠相比 (图 3)。与未接受吖啶橙注射液的小鼠相比, 尾静脉注射吖啶橙会导致类似的粘连和轧制 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56948/56948fig1.jpg"> 
图1。血管紧张素 II. 的输液方案及对 LysM 的评价+细胞在小鼠体内滚动和黏附。LysMCre+表意文字+小鼠注入了7天, ii 和黏附, 以及滚动 LysM+细胞在动脉是量化的, 或没有注射吖啶橙通过颈静脉导管或尾静脉注射.<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56948/56948fig2.jpg"> 
图2。评价 LysM 的+细胞黏附和滚动在颈动脉 LysMCre+表意文字+鼠标。粘附 LysM+ LysMCre 中的细胞 + 表意文字 + 鼠标前 (a, D) 和后 ( B, E) 吖啶注射液通过颈静脉导管。红色和绿色荧光被记录, LysM 的+细胞 (绿色) 和平滑肌细胞 (红色) 是可见的。注射吖啶橙后, 内皮细胞也可在绿色可见, 但去除背景荧光只允许循环有核细胞可视化 (C, F)。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56948/56948fig3.jpg"> 
图3。血管紧张素 II. LysMCre 中荧光染料的管理路线评价+表意文字+小鼠。滚动的量化 (A) 和黏附 (B) LysM+细胞在假操作或 ii 注入动物, 并有或不注射吖啶橙使用颈静脉导管或尾静脉注射。不同条件 (C) 的代表性图片。结果均值平均±标准误差。执行了2路方差分析和 Bonferroni 的post测试, n = 3-12/组;*p和 #60; 0.05。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls at JoVE.com

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

这份手稿描述了使用转基因的记者小鼠和不同的管理途径荧光染料在血管紧张素 II 诱导高血压使用活体视频显微镜的血管评价免疫细胞的活化作用及其能够滚动和坚持内皮。

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 ...

visualizing-leukocyte-rolling-adhesion-angiotensin-ii-infused-mice

Research

JoVE Journal

Immunology and Infection

8.6K 次观看.  University Medical Center Mainz. 这份手稿描述了使用转基因的记者小鼠和不同的管理途径荧光染料在血管紧张素 II 诱导高血压使用活体视频显微镜的血管评价免疫细胞的活化作用及其能够滚动和坚持内皮。

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

成像白细胞粘附到血管内皮细胞在高腔内压力

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

生物工程

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

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 ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

通过体内显微镜评估的小鼠中白细胞肌渗透

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

生物学

Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels is characteristic of inflammation and has been causally linked to capillary occlusion in a variety of tissues and diseases, including diabetic retinopathy. 
Leukostasis has been reported for years as a life-threatening complication of hyperleukocytosis and can only be diagnosed clinically. Given the importance of the phenomenon, intensive research has been done to understand the potential mechanism(s) that lead to its manifestation; however, there is no gold-standard technique in laboratory settings to visualize and quantify the severity of the event.
In the method summarized below, the vasculature is initially perfused with a buffer to remove blood, and then, concanavalin A is perfused into the vasculature where it binds to all exposed cell walls and causes especially bright staining of leukocytes. If the perfusion to remove all unbound blood cells was successful, the remaining fluorescently labeled leukocytes are bound to the vasculature, and they can be manually quantified using any available fluorescence microscope.

We demonstrate a method to label the walls of the retinal vasculature and adherent leukocytes. These adherent leukocytes can then be counted under a fluorescence microscope as a parameter of inflammation or the response of that inflammation to therapies.

视网膜脉管系统中糖尿病诱导的贴壁白细胞的定量

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels ...

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

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白细胞的归位和迁移的非侵入性的成像在体内