The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

注:在本协议中描述的所有动物的相关手续已在杜克大学机构动物护理和使用委员会（DUIACUC）事先批准。 1.癌症细胞培养和注射<ol><li>培养荧光标记的转移性肿瘤细胞（ 如人力MDAMB231-GFP乳腺癌细胞，从帕特里夏Steeg的博士，NCI和YFP标记的小鼠肉瘤细胞，从大卫·基尔希博士，美国杜克大学医学中心放射肿瘤学系的礼品赠送）在适当的培养基（ 例如 Dulbecco氏改良的Eagle培养基（DMEM）中，用10％胎牛血清和1％青霉素/链霉），在37℃，直到大约90％汇合。 </li><li> Trypsinize细胞，用PBS冲洗2次，计数，然后将它们注入尾静脉异氟烷麻醉10周龄女性裸体老鼠每动物5000000细胞，使用带有27G的针头的注射器。手术水平anesthesIA是由缺乏反应脚趾捏的验证。 </li></ol> 2.监控转移使用显微的<ol><li>用微型CT /微辐射器，以检测转移灶的直径大于约2mm的直径的存在下，每周检查一次大鼠。在显微CT委托如前所述12。 <ol><li>受试者大鼠成像之前的3％异氟烷麻醉。确认通过脚趾捏深麻醉。 </li><li>发病麻醉后，迅速大鼠在成像腔室转移至成像支架和经由鼻锥在2.5-3％连接到一个异氟醚 - 空气混合物。调整在其胸部被定位在显微扫描仪的光子束的方法的摇篮的大鼠的位置，使用在成像支架的外部位置的控制和激光定界符。确保门成像室是封闭的，以从伽马射线遮蔽研究者。 </li><li>控制动物的再次使用位置彩色摄像机。执行低分辨率CT成像试运行，并使用所产生的图像，以视场调整到胸腔的XYZ尺寸。 </li><li>图像使用2毫米的Al滤波器大鼠胸廓在40 kVp的，2.5毫安，并0.008体素在7 FPS和返回动物笼子。一只动物的成像应不超过15或20分钟。不返回已经历手术给其他动物的公司，直到完全恢复的动物。不要让动物无人看管，直到它恢复足够的意识，保持胸骨斜卧。 </li><li>通过相对不透射线的物体的外观不能由胸内的血管进行说明确认转移（ 图1A） </li></ol></li></ol> 3.窗口商会外科<ol><li>麻醉，生命体征和尾静脉插管<ol><li>转移性疾病的存在选择的动物。注入动物腹膜内ðOSE的50毫克/公斤戊巴比妥。出发前确认所捏脚趾手术级别的麻醉。 注:麻醉的协议应该匹配到相应的实验装置。戊巴比妥这里被选为长效麻醉剂，以诱导麻醉深为冗长的程序，同时提供容易的重新给药的选项。但是，动物的损失用药过量​​是一种常见的问题戊巴比妥麻醉。它保留自主反射到更大的程度比巴比妥另一种选择是氯胺酮结合镇静剂如赛拉嗪或美托咪定，仅在单次再给药周期然而其允许。 </li><li>剃上具有转移性疾病，并且在颈部区域中的主体的侧面的动物，使用一个削波器。擦去从皮肤所有剩余松散的头发。后松散的头发被删除，适用兽医药膏眼睛，以防止它们干燥。 注:无胸腺裸鼠体内可能有残留的头发requi在进行外科手术前，水库搬迁。这是非常重要的，以彻底去除所有的头发的，因为它可以与外科手术和成像干涉。 </li><li>修复的动物在被放置在37℃的水中循环加热垫的金属板仰卧位置。前面和后肢肢体被固定在板上用胶带。 注:它是非常有用的控制，并通过使用一个脉冲血氧计，在整个手术和实验程序记录生命体征，如心脏速率和动脉血液充氧。 </li></ol></li><li>气管插管<ol><li>在以设置一个导管的动物的通风，首先使横向颈部皮肤切口，接着沿纵向肌肉腹侧气管的中位数的分离。 </li><li>使用重复打开到关闭动作用尖锐的镊子通过气管的背侧以创建一个通道的缝合。 </li><li>做一个小切口，插入吨气管他腹侧，不超过半圆形，大致在第二和第三气管环之间。留足够长的暴露在背表面气管部，以使所述气管导管的固定。 </li><li>将2.5〜3.0mm的"Y"气管插管进入气管，并拧紧了4-0单丝缝合。确保套管连接到一个压力循环通风机与连接到期满导管填充有6厘米水的瓶子，以维持正肺动脉压。流入的气体应是100％的氧气，除非实验所需的除外。 </li><li>插入一个导管具有25-27ģ针，并且填充有肝素生理盐水到尾静脉大鼠中的一个，并代替用胶带固定。 注:确保尾静脉导管的通畅整个过程通过重复注入少量肝素的生理盐水到尾静脉。此外，奥罗气管插管，即气管指导经由麻醉动物的嘴和过去的喉部进入气管管，是一种可能的替代方案是这里描述的气管切开术过程。然而，该方法需要特别的训练和经验，以避免损坏气管，并且也排除食道的意外插管。 </li></ol></li><li>肺窗应用<ol><li>从胸部其中转移性疾病位于的一侧去除皮肤，通过创建一个切口，然后用钝器解剖分离皮肤。 注:皮肤被切除，并去除随后支队</li><li>通过解剖重叠肌肉（胸，锯，以及背阔肌）的两个层，但留下肋间肌完好进行。在约1.5厘米胸腔直径创建一个穿孔，通过除去通常两个相邻的肋的某些部分。理想情况下，找到穿孔在第六和第七r的地区肠易激综合征。 </li><li>截骨术: <ol><li>为了最大限度地减少出血和损害肺表面上，紧紧地保持肋在切割期间被切割带齿外科钳子。用手术剪，切内侧肋的第一，在大约45°的角度，余下的肋骨指向外面的尖端侧。 </li><li>随后，切开肋骨的侧面以类似的方式，再次离开肋骨指向外部，以防止损坏的肺表面的尖端侧。 </li><li>重复该过程的相邻肋骨，然后切开肋间肌肉，去除切一块。在此过程中，维持的方式，肺表面和肋笼之间的机械相互作用被最小化肺压力。通过适当地调节呼吸机呼吸压力做到这一点。 </li></ol></li><li>插入窗口的: <ol><li>插入定制肺窗口，包括一个玻璃罩即是连接到一个有机玻璃插座（ 图1B）。通过胶粘，或者通过施加非常少量的真空润滑脂附着窗口套接字。插入窗口的方式，表面的转移都位于靠近窗口的中心。如果需要的话，调整所插入的孔通过稍微扩大孔向各个侧带来的微转移到窗口的中心。 注:在胸膜表面转移性疾病可以被识别为肺表面的粉红色，否则对鲑鱼的彩色健康部分出现主要沿裂隙内清楚地辨认白色点或区域。虽然微可以发生在胸腔外的其他领域，被调查的细胞系几乎总是显示浮现在微穿孔区域，一旦转移性疾病可放射学检测。 </li><li>插入插座插入穿孔，以及创建与所述升的内脏胸膜直接接触后UNG，缝合窗框的边缘向周围肋间肌，使用4.0单丝缝合（ 图1C）。使用呼吸机呼吸压力略有增加，以帮助残留的空气逸出，并创建一个密封。 注意:大鼠的呼吸速率可有很大变化，这取决于麻醉，兴奋或焦虑的状态中，吸入空气中的氧浓度（氧合指数2）等的状态，是值得推荐的，调整70和90次之间的呼吸速率。吸气压力应调节谨慎和不被设置为大于大约8厘米的H 2 O（0.6毫米汞柱），以避免损坏肺表面。 </li></ol></li><li>定位动物中，其被设计来消除Z方向运动（ 图1D）上被定位在恒温（电）加热毯的钢板定制设计的阻 ​​挡器，在荧光显微镜下。控制动物的身体温度用直肠热敏电阻。调节动物限制器的螺丝，和呼吸机上的吸气压力来实现横向移动的最佳控制。 注:自然呼吸的哺乳动物包括三个方向运动肺和胸部延伸:双边，背腹和头尾。为了保存自然呼吸运动尽可能，它以最小化Z方向压缩到必要的程度是重要的。因为在Z维约束具有引入可能影响血液流动等参数工件的电位，最好是串联在同一动物重复测量过程中保持恒定的条件。 </li></ol></li></ol> 4.成像测量血液微循环和<ol><li>通过心脏穿刺收集的红细胞和如先前所描述的DiI（1,1 = -dioctadecyl -3,3,3 =，3 =四甲基吲哚羰花青高氯酸盐）标记它们，&lt;SUP&gt; 10。 </li><li>注入300微升标记的红血细胞到尾静脉的大鼠的窗口腔手术完成之前，为了避免首过效应的粘附到玻璃窗口。消除任何气泡在注射器或导管，如引入空气进入静脉将抑制任何进一步的注射。 </li><li>图像血流用显微镜CCD照相机在-40℃冷却的芯片温度，和大约100X总分辨率（ 即用10X显微镜物镜，和10X预相机目镜）。使用标准的罗丹明/ TRITC滤光片组（激发波长450-490，发射&gt; 515纳米）。记录所得到的图像序列的实际帧速率和像素分辨率。每个堆栈记录至少200（最好约300）的图像，以确保流速的成功分析。 </li><li>通过注入每一个小时约1毫升生理盐水知识产权的补充在动物体液丢失。 注:涉及干预实验设置， 如一种药物，用于修改在肺癌转移的血流速度，需要血流速度的肺转移癌的重复测量。对于这些扩展实验，以补充足够的流体，动物是非常重要的。 </li><li>安乐死的动物通过输注3毫升3N氯化钾到尾静脉</li><li>评估采用发表，公开可用的基于Matlab的计算机算法，将创建流速灰度和彩色编码图所有的血流痕迹10,11图像栈。随后评价所形成的灰度图像通过使用商业或公开可用的图像分析软件，如的Image J，阈值化的折扣值，表明血细胞的无运动， 即没有活性脉管之后。 </li></ol>

<ol>
	<li>Billingsley, K. G., 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=Pulmonary+metastases+from+soft+tissue+sarcoma:+analysis+of+patterns+of+diseases+and+postmetastasis+survival.">Pulmonary metastases from soft tissue sarcoma: analysis of patterns of diseases and postmetastasis survival.</a> Ann Surg. 229, 602-610 (1999).</li><li>Rashid, O. M., Takabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+role+of+surgery+in+the+management+of+breast+cancer+lung+metastasis.">The evolution of the role of surgery in the management of breast cancer lung metastasis.</a> J Thorac Dis. 4, 420-424 (2012).</li><li>Mayer, A., Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia,+lactate+accumulation,+and+acidosis:+siblings+or+accomplices+driving+tumor+progression+and+resistance+to+therapy.">Hypoxia, lactate accumulation, and acidosis: siblings or accomplices driving tumor progression and resistance to therapy.</a> Advances in experimental medicine and biology. 789, 203-209 (2013).</li><li>Okunieff, P., O'Dell, W., Zhang, M., Zhang, L., Maguire, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+oxygen+measurements+and+personalized+medicine.">Tumor oxygen measurements and personalized medicine.</a> Advances in experimental medicine and biology. 765, 195-201 (2013).</li><li>Palmer, G. M., 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=In+vivo+optical+molecular+imaging+and+analysis+in+mice+using+dorsal+window+chamber+models+applied+to+hypoxia,+vasculature+and+fluorescent+reporters.">In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters.</a> Nature protocols. 6, 1355-1366 (2011).</li><li>Palmer, G. M., 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=Optical+imaging+of+tumor+hypoxia+dynamics.">Optical imaging of tumor hypoxia dynamics.</a> Journal of biomedical optics. 15, (2010).</li><li>Funakoshi, N., 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=A+new+model+of+lung+metastasis+for+intravital+studies.">A new model of lung metastasis for intravital studies.</a> Microvasc Res. 59, 361-367 (2000).</li><li>Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+assessment+of+pulmonary+vascular+responses.">Real-time imaging assessment of pulmonary vascular responses.</a> Proc Am Thorac Soc. 8, 458-465 (2011).</li><li>Tabuchi, A., 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=Precapillary+oxygenation+contributes+relevantly+to+gas+exchange+in+the+intact+lung.">Precapillary oxygenation contributes relevantly to gas exchange in the intact lung.</a> Am J Respir Crit Care Med. 188, 474-481 (2013).</li><li>Hanna, G., 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=Automated+measurement+of+blood+flow+velocity+and+direction+and+hemoglobin+oxygen+saturation+in+the+rat+lung+using+intravital+microscopy.+American+journal+of+physiology.">Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy. American journal of physiology.</a> Lung cellular and molecular physiology. 304, 86-91 (2013).</li><li>Fontanella, A. N., 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=Quantitative+mapping+of+hemodynamics+in+the+lung,+brain,+and+dorsal+window+chamber-grown+tumors+using+a+novel,+automated+algorithm.">Quantitative mapping of hemodynamics in the lung, brain, and dorsal window chamber-grown tumors using a novel, automated algorithm.</a> Microcirculation. , (2013).</li><li>Newton, J., 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=Commissioning+a+small-field+biological+irradiator+using+point,+2D,+and+3D+dosimetry+techniques.">Commissioning a small-field biological irradiator using point, 2D, and 3D dosimetry techniques.</a> Medical physics. 38, 6754-6762 (2011).</li><li>Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironmental+physiology+and+its+implications+for+radiation+oncology.">Tumor microenvironmental physiology and its implications for radiation oncology.</a> Seminars in radiation oncology. 14, 198-206 (2004).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> J Appl Physiol. 104, 338-346 (2008).</li><li>Fingar, V. H., Taber, S. W., Wieman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+for+the+study+of+pulmonary+microcirculation:+determination+of+pulmonary+edema+in+rats.">A new model for the study of pulmonary microcirculation: determination of pulmonary edema in rats.</a> J Surg Res. 57, 385-393 (1994).</li><li>Manzoor, A. A., Schroeder, T., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-stop-shop+tumor+imaging:+buy+hypoxia,+get+lactate+free.">One-stop-shop tumor imaging: buy hypoxia, get lactate free.</a> The Journal of clinical investigation. 118, 1616-1619 (2008).</li><li>Evans, S. M., 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=Imaging+and+analytical+methods+as+applied+to+the+evaluation+of+vasculature+and+hypoxia+in+human+brain+tumors.">Imaging and analytical methods as applied to the evaluation of vasculature and hypoxia in human brain tumors.</a> Radiation research. 170, 677-690 (2008).</li><li>Radiloff, D. R., 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=The+combination+of+theophylline+and+endothelin+receptor+antagonism+improves+exercise+performance+of+rats+under+simulated+high+altitude.">The combination of theophylline and endothelin receptor antagonism improves exercise performance of rats under simulated high altitude.</a> Journal of applied physiology. 113, 1243-1252 (2012).</li></ol>

作者什么都没有透露。

一个模型提出即是可行的，使用活体显微镜和计算血流分析在血液微循环和大鼠的肺转移其它动态过程，图像的变化。而其他方法存在于啮齿动物的开放ribcages暴露肺部进行显微镜，这种模​​式也是通过胸壁穿孔封闭胸设置的第一个图像肺转移。使用这种方法的可行性被示为测量药理学诱导的变化的微循环血液肺转移的流动。 两种基本方法存在图像的生活啮齿类是由自发血...

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table><tbody><tr><td>Athymic nude rats</td><td>Charles River</td><td>Strain code 316</td><td>Female 10 week-old athymic nude rats</td></tr><tr><td>micro-CT/micro-Irradiator&amp;nbsp;</td><td>Precision X-ray Inc.</td><td>Xrad 225Cx</td><td>Use MicroCT to detect metastases</td></tr><tr><td>DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td><td>Sigma Aldrich</td><td>468495-100MG</td><td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td></tr><tr><td>Rodent ventilator</td><td>Kent Scientific</td><td>TOPO Small Animal Ventilator</td><td>Device is important to maintain positive lung pressure after application of pneumothorax</td></tr><tr><td>Zeiss Axioskop fluorescence microscope upright</td><td>Zeiss</td><td>Axioskop</td><td>Microscope for intravital imaging</td></tr><tr><td>Andor CCD camera</td><td>Andor</td><td>iXonEM 885</td><td>CCD camera for live imaging of blood flow</td></tr><tr><td>Pulse oximeter</td><td>StarrLife</td><td>MouseOx</td><td>Pulse oximeter</td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axioskop</td><td>Fluorescence microscope</td></tr></tbody></table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

在实体瘤的脉管系统是已知的，从正常供血显著不同，表示更大程度的扭曲的，并且更高intervascular距离13。因此，当相比于正常肺微循环（ 图2A，上图）中的实验性肺乳腺癌和肉瘤转移血流磁道具有不规则的形状和大intervascular间隙（ 图2A，下两个面板）。在先前的研究中，肺窗口方法的能力证明，可以执行正常的肺10的变化中的血流速度的自动测量。为?...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

微循环血流速度在大鼠肺转移自动测量

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.4K 次观看.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

视频: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

科研

癌症研究

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

研究癌症转移至肺部的永久窗口

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

使用穆林精密切肺片对肺动脉内下氧肺血管收缩进行视频形态分析

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

医学

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

肱血流介导的扩张和大鼠股浅动脉超声评估

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

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

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

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

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

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

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

微循环血流速度在大鼠肺转移自动测量

摘要

探索更多视频

此视频中的章节

长期高分辨率活体显微镜在肺与真空稳定成像窗口

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

研究癌症转移至肺部的永久窗口

使用穆林精密切肺片对肺动脉内下氧肺血管收缩进行视频形态分析

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

肱血流介导的扩张和大鼠股浅动脉超声评估

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

在离体血液灌注大鼠肺备量化单微血管通透性