我们感谢博士。矶田渊和:沃尔夫冈·库伯勒（多伦多大学）关于活体显微镜指令。我们感谢安德鲁·卡希尔（尼康仪器）在显微镜的设计和实施的援助。这项工作是由美国国立卫生研究院/ NHLBI拨款的P30 HL101295和K08 HL105538（EPS）。

1。制备手术管，血管导管，胸壁窗口<ol><li> 活体显微镜阶段，我们定制一个的有机玻璃阶段麻醉鼠标是在显微镜。这个阶段可同时接待15厘米10厘米灵活的塑料菜板（鼠标是在麻醉诱导，气管切开的位置，静脉导管），以及一个同样大小的加热元件（菜板下方）。 </li><li> 鼠标胸廓造口术管准备 （ 图1）。一个10厘米长的PE 50的管（Intramedic，内径0.58毫米，外径0.965毫米）的削减。一端被连接到弯曲的23号针的钝端，这个针将被用于通过胸壁（→外内）通过管封闭胸窗口之前。 油管（1.5厘米的长度，连接的23号相反的前端反复的30号针头刺破针），"侧端口"，以便有效地吸入胸腔内的空气。 然后此窗孔部从管的其余部分分离，由几个周4:0丝线缝合循环，这些循环的"塞子"，将作为最终锚固1.5厘米的孔的部分的胸腔内。 </li><li> 颈静脉导管。两个15厘米长的PE管（Intramedic，内径0.28毫米，外直径0.61毫米）被切断。甲手术刀用于锥的管的端部，从而增加易用性静脉穿刺。油管冲洗1ml注射器经由含有6％150 kDa的葡聚糖溶液（在PBS中），连接到非斜削端的油管。 </li><li> 胸壁窗口准备（图2）。切成一个椭圆形的形状（主轴线（6）厘米，短轴4厘米），透明聚偏膜（新吴裹，Kuresha，东京）。一个圆形的5毫米＃1盖玻片（BellcO）是贴的膜，使用α-氰基丙烯酸酯胶（Pattexflüssig，汉高，杜塞尔多夫）。 </li><li> 气胸感应（"吹管"），10厘米长的管道（内径3毫米，外径为5毫米）的金属管被连接到一个5毫升的注射器的另一端将被用于将空气引入动物胸廓前胸壁窗口的植入。 </li><li> 注射器的客观的水浸泡。甲23号针头连接到一个30毫升注射器含有蒸馏水。针尖钝化（使用金属文件），以防止损坏的目标。 </li></ol> 2。鼠标麻醉<ol><li>鼠标是与氯胺酮（10毫克/毫升）和甲苯噻嗪（2毫克/毫升），腹膜内给药在每克小鼠体重的剂量为8微升的混合物麻醉。镇静发生在3 - 6分钟，，不应妨碍自主呼吸。 </li><li>使用一个电动剃须刀，刮脸的喉咙，胸部，腹部和右侧的鼠标。 </li><li>使用磁带，保证鼠标移动到一个薄薄的塑料菜板。头的鼠标指向的操作（ 图3）。温和的紧张所提供的上排牙齿缝线穿过下面的循环，以维持头部后仰。切割电路板放置在一个加热垫后，保持鼠标euthermia的气管切开术和静脉置管过程中。 </li><li>用100％乙醇的湿剃区域。 </li><li>确认适当的麻醉与尾/爪子掐。如果最小的响应，提供了一个额外的静脉推注氯胺酮/甲苯​​噻嗪，如果没有足够的麻醉。 </li></ol> 3。气管切开术<ol><li> 1厘米的切口，滑过喉咙。相关结缔组织解剖，唾液腺分离和侧面反映。前胸骨舌骨肌立即气管被切除。 </li><li>先进的下个4:0缝合的循环Ë气管（ 图4）。循环再切割，创建两个独立的缝合链相关的气管。将用于保护气管切开管的尾部缝合，颅缝将用于气管切开放置在气管上的紧张。 </li><li>用两个手指上的缝合线是掌握和温和的张力被施加到气管。一水平切口是在气管之间的上部和下部的缝线。这个切口交叉的气管周长大约三分之二。法兰气管切开术管（哈佛仪器，外径1.22毫米）的插入远端气管和到位的尾部气管缝合固定。 </li><li>气管切开术是连接到一个音量控制的小动物呼吸（吸气，哈佛仪器），和鼠标通风与吸入氧浓度40％和9毫升/公斤潮气量（设置优化，在我们的实验室，以维持足够的氧/通风）。位置在这一点上，郑颖人呼气末正压（PEEP）开始。值得注意的是，呼吸机设置应个别实验室内优化，以得天独厚的条件。冗余管（呼吸机管Y形连接件和气管）之间的不同的长度可以被用于调节死空间，从而确保稳定的肺泡通气量的任何选择的潮气量。 </li></ol> 4。静脉插管<ol><li>的交界处的内部和外部的颈静脉，可以识别通过跟踪近侧远侧静脉分支。颈外下方发现所反映的唾液腺，这可以追溯到近端外部内部的颈交界处。 </li><li>使用温和的钝性分离，分离出颈交界处周围的结缔组织。 </li><li>使用4:0缝合，结扎颈外静脉和颈内静脉，颈交界处远端（颅）。 </li><li>做一个小切口进入隆突颈交界处出血，应是最小的。 </li><li>两根导管可以增量前进通过切口和进入颈中继线。温柔的愿望，以确保血液回流后，导管静脉内使用4:0缝合固定。 </li><li>磁带静脉导管的菜板，以防止意外坠下。 </li></ol> 5。活体小鼠肺显微外科（改编自田渊等人。18） <ol><li>菜板（含内敛，麻醉小鼠以及录音静脉的导管）转换到活体显微镜阶段，将进行剩余的外科手术。直肠温度探头放在这个接口的自适应加热系统（菜板下），允许鼠标euthermia维护。 </li><li>一个颈静脉导管连接到注射泵，提供了一个氯胺酮（10毫克/毫升） - 二甲苯胺噻嗪（2毫克/毫升）混合物在200微升每小时。再次证实了足够的麻醉尾/爪子掐。 </li><li>正中切口从颈部延伸至剑突的过程，进而向右侧的侧面（ 图5）。 </li><li>使用电，胸部的肌肉，露出胸廓。护理是采取措施，确保彻底止血。 </li><li>交叉鼠标的右后腿在左侧和磁带的。导致腹部的扭转旋转的胸部稍微，改进易用性的手术。 </li><li>将舞台上一个45度角（ 图6），这种定位使肺离心离德的胸壁一次引起气胸。 </li><li>的第一肋（最低劣肋骨）的钳子与掌握，并提出一个弯钳直言不讳地推下的肋骨。这将壁层胸膜胸壁分离。胸膜保持unpunctured。 </li><li>使用喷吹管和注射器，空气被强行引入对壁层胸膜。这导致破裂的胸膜表面和气胸不损坏底层的肺的情况下。底层的肺将落在离胸壁，使引进的电烙镊子不破坏肺。在呼吸机的潮气量的减少通常不需要在这一步中。 </li><li>使用电烙镊子，解剖胸壁肌肉和跨越的第 5和第 6肋骨/壁层胸膜，1〜8毫米的圆孔到胸壁。至关重要的是，保持完全止血，出血的存在下将掩盖显微镜（ 图7）。 </li><li>使用针驱动程序，插入胸腔管进入胸腔壁孔。针应穿刺胸壁和退出胸腔胸窗口（ 图7）的下方及侧面。请小心不要刺破隔膜。 "管然后轻轻地拉出的胸壁，直到它的阻力发生从位于该管的窗孔部的边缘处的缝合线"挡块"。 </li><li>放置在舞台平坦。 </li><li> 3厘米H 2 O呼气末正压呼吸机协助肺复张。 </li><li>胶（Pattex胶，汉高）放在圆周周围的胸部窗口。该膜附着，与玻璃盖玻片面临外部的胸腔。小心地（和圆周）近似的膜使用的棉签胶水。 </li><li>当执行一个肺招聘机动（3期间，潮气量呼气末正压呼吸机端口被挡住），-3毫米汞柱吸入被施加到胸腔引流管。应该坚持近似肺膜，而可自由移动在潮气通气（ 图8）期间。 </li><li>右前足的鼠标越过左侧，导致在左侧卧位的鼠标。海绵楔形件可以被用于正确地定位在鼠标使胸部窗口对准显微镜水浸物镜。 </li><li>盖玻片显微镜之前被放置在蒸馏水中，允许使用水浸物镜肺进行可视化。水将需要整个成像要间歇地补充。 </li></ol> 6。肺血管内皮面层厚度的测量<ol><li>紧随胸壁封闭后，加入500μl的FITC-标记的150 kDa的葡聚糖（在PBS中的6％溶液），通过所述第二（非麻醉）的颈静脉导管给药。此丸剂作为容量复苏，以及为ESL测量血管示踪剂。右旋糖酐静脉推注不影响中性粒细胞或肺水肿的形成。 </li><li>水浸泡目标集中在盖玻片。客观的选择是至关重要的可视化ESL厚度，高数值的微小差异需要校准孔（&gt; 0.8），同时仍保持2 - 3毫米的工作距离（允许穿透肺窗口和胸膜表面）。我们使用尼康CFI 75 LWD 16X（NA 0.8）和CFI 75 LWD 25倍（NA 1.1）的目标达到此目的。 </li><li>为了准确测量ESL厚度的移动器官，至关重要的是，明场和荧光血管宽度是同时进行的。这可通过使用图像分离器（双视图，配光），它允许同时捕获的反射光的微分干涉相差（DIC，明场）和FITC标记的图像（ 图9）。 </li><li>在5秒吸气暂停期间，连续执行成像和记录。后来，这些影像可能会审查，以确定对焦框。 </li><li>使用聚焦框，胸膜下微血管（直径&lt;20微米）确定;，至少3微血管通常发现单个帧上。完成后的实验，DICFITC-葡聚糖血管宽度的测量（由不知情的观察者）的平均长度每微血管的三个相互垂直的拦截。假设等于ESL在容器的两个边缘的厚度，可以定义由一个DIC和FITC-葡聚糖，代表结果"一节中描述的血管宽度之差的一半的ESL大小。 </li><li>通常情况下，活体显微镜，可以进行90分钟，没有任何证据的肺损伤或低血压2。在一段时间的观察，应进行初步试验，以确认鼠标的稳定性（血压，氧合，通气，肺损伤）。试验的药物可以通过在操作过程中的任何点处的第二（非麻醉）颈导管引入。 </li></ol> 7。另一种测量肺血管内皮表面层的完整性完整的内皮细胞表面层的功能（部分）排除circulat的元件从内皮表面2。 ESL的完整性，因此，可以测量由循环元件的能力（ 例如，荧光微球）来访问和与细胞表面的粘附分子（如ICAM-1）进行交互。 <ol><li>抗ICAM-1标记的荧光微球前准备手术。抗生蛋白链菌素被覆0.97微米荧光微球孵育与生物素化的抗ICAM-1（YN1/1.7.4克隆，1:50，eBioscience公司）在室温下的30分钟的抗体或同种型对照。微球洗涤三次，悬浮于PBS中在1×10 9微球每毫升。 </li><li>在活体显微镜，微球的悬浮液（100微升）被注入到颈静脉导管。循环15分钟后，在5分钟内被捕获荧光图像。微动5分钟，被认为是贴壁和量化使用图像处理软件。 </li></ol> 8。 "安乐死" &lt;P类=的"jove_content"&gt;麻醉小鼠的程序完成后，直接通过心脏穿刺放血安乐死。安乐死的确认通过后，双侧气胸，肺部的收获和快速冷冻，以供日后分析。

<ol>
	<li>Negrini, D., Tenstad, O., Passi, A., Wiig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+degradation+of+matrix+proteoglycans+and+edema+development+in+rabbit+lung.">Differential degradation of matrix proteoglycans and edema development in rabbit lung.</a> AJP - Lung Cellular and Molecular Physiology. 290, L470-L477 (2006).</li><li>Schmidt, E. P., 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+pulmonary+endothelial+glycocalyx+regulates+neutrophil+adhesion+and+lung+injury+during+experimental+sepsis.">The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.</a> Nat. Med. 18, 1217-1223 (2012).</li><li>Florian, J. 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=Heparan+sulfate+proteoglycan+is+a+mechanosensor+on+endothelial+cells.">Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.</a> Circ. Res. 93, e136-e142 (2003).</li><li>Chappell, D., 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+Glycocalyx+of+the+Human+Umbilical+Vein+Endothelial+Cell:+An+Impressive+Structure+Ex+Vivo+but+Not+in+Culture.">The Glycocalyx of the Human Umbilical Vein Endothelial Cell: An Impressive Structure Ex Vivo but Not in Culture.</a> Circulation Research. 104, 1313-1317 (2009).</li><li>Potter, D. R., Damiano, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamically+relevant+endothelial+cell+glycocalyx+observed+in+vivo+is+absent+in+vitro.">The hydrodynamically relevant endothelial cell glycocalyx observed in vivo is absent in vitro.</a> Circ. Res. 102, 770-776 (2008).</li><li>Weinbaum, S., Tarbell, J. M., Damiano, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Structure+and+Function+of+the+Endothelial+Glycocalyx+Layer.">The Structure and Function of the Endothelial Glycocalyx Layer.</a> Annual Review of Biomedical Engineering. 9, 121-167 (2007).</li><li>Pittet, M., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging.">Intravital Imaging.</a> Cell. 147, 983-991 (2011).</li><li>Kilpatrick, D. C., Graham, C., Urbaniak, S. J., Jeffree, C. E., Allen, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+tomato+(Lycopersicon+esculentum)+lectin+with+its+deglycosylated+derivative.">A comparison of tomato (Lycopersicon esculentum) lectin with its deglycosylated derivative.</a> Biochem. J. 220, 843-847 (1984).</li><li>Smith, M. L., Long, D. S., Damiano, E. R., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-wall+micro-PIV+reveals+a+hydrodynamically+relevant+endothelial+surface+layer+in+venules+in+vivo.">Near-wall micro-PIV reveals a hydrodynamically relevant endothelial surface layer in venules in vivo.</a> Biophys. J. 85, 637-645 (2003).</li><li>Vink, H., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circ. Res. 79, 581-589 (1996).</li><li>Marechal, X., 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=Endothelial+glycocalyx+damage+during+endotoxemia+coincides+with+microcirculatory+dysfunction+and+vascular+oxidative+stress.">Endothelial glycocalyx damage during endotoxemia coincides with microcirculatory dysfunction and vascular oxidative stress.</a> Shock. 29, 572-576 (2008).</li><li>Presson, R. 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=Two-Photon+Imaging+within+the+Murine+Thorax+without+Respiratory+and+Cardiac+Motion+Artifact.">Two-Photon Imaging within the Murine Thorax without Respiratory and Cardiac Motion Artifact.</a> The American Journal of Pathology. 179, 75-82 (2011).</li><li>Looney, M. 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=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nat. Meth. 8, 91-96 (2011).</li><li>Pearse, D. B., Wagner, E. M., Permutt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ventilation+on+vascular+permeability+and+cyclic+nucleotide+concentrations+in+ischemic+sheep+lungs.">Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs.</a> J. Appl. Physiol. 86, 123-132 (1999).</li><li>Hossain, M., Qadri, S., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+nitric+oxide+synthesis+enhances+leukocyte+rolling+and+adhesion+in+human+microvasculature.">Inhibition of nitric oxide synthesis enhances leukocyte rolling and adhesion in human microvasculature.</a> Journal of Inflammation. 9, 28 (2012).</li><li>Schmidt, E. P., 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=Soluble+guanylyl+cyclase+contributes+to+ventilator-induced+lung+injury+in+mice.">Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice.</a> AJP - Lung Cellular and Molecular Physiology. 295, L1056-L1065 (2008).</li><li>Mead, J., Takishima, T., Leith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+distribution+in+lungs:+a+model+of+pulmonary+elasticity.">Stress distribution in lungs: a model of pulmonary elasticity.</a> J. Appl. Physiol. 28, 596-608 (1970).</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>Gattinoni, L., Protti, A., Caironi, P., Carlesso, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator-induced+lung+injury:+the+anatomical+and+physiological+framework.">Ventilator-induced lung injury: the anatomical and physiological framework.</a> Crit. Care Med. 38, 539-548 (2010).</li><li>Tabuchi, A., Kim, M., Semple, J. W., 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=Acute+Lung+Injury+Causes+Pendelluft+Between+Adjacent+Alveoli+In+Vivo.">Acute Lung Injury Causes Pendelluft Between Adjacent Alveoli In Vivo.</a> Am. J. Respir. Crit. Care Med. 183, A2490 (2011).</li><li>Roebuck, K. A., Finnegan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intercellular+adhesion+molecule-1+(CD54)+gene+expression.">Regulation of intercellular adhesion molecule-1 (CD54) gene expression.</a> J. Leukoc. Biol. 66, 876-888 (1999).</li></ol>

没有利益冲突的声明。

与体内显微镜扩大使用一致，有规模庞大的ESL以及其众多的血管功能的不断升值。然而，这些新兴的数据，主要来自全身血管的研究。事实上，在肺在体内显微镜技术挑战，由于显着的肺和心脏运动伪影。 一些最近的技术进步允许稳定的肺鼠标移动，给予活体技术更好地应用到肺微循环12，13。然而，这些方法有可能混淆了肺动的生理后果。由于肺是目?...

内皮糖萼层衬在血管内膜蛋白多糖和相关的葡糖胺聚糖是一种细胞外， 在体内，糖萼高度水合，形成大量的内皮细胞表面层（ESL），调节多种内皮细胞功能，包括透液性1，嗜中性粒细胞-内皮粘附2，和机械力的流体剪切应力3。 从历史上看，糖萼一直怀才不遇由于其在培养的细胞制剂4，5和其降解过程中的标准的组织固定和处理6的畸变。越来越多地使用活体显微镜（IVM） 在体内的显微镜，也伴随着高度的科学兴趣在健康和疾病中的血管功能的重要性，ESL。 ESL是不可见的光镜，并不能容易地标记体内的荧光腊梅糖结合凝集素的倾向，导致红细胞凝集和致死性肺动脉栓塞（未发表意见）。因此，一些间接的方法推断出ESL在非移动，如提睾和肠系膜微循环血管床的厚度（和推而广之，蛋白质复合物的完整性）。这些技术包括在循环微粒速度从内皮膜（微粒图像测速9）以及笨重，荧光标记的血管标记物（ 如葡聚糖）的排除测量从内皮表面的距离的函数的测量的差异（的葡聚糖排除技术10，11）。这些技术中，只有葡聚糖排斥是能够推定的ESL从在一个单一的时间点测量的厚度。通过同时测定血管宽度使用明视野显微镜（宽度ESL厚度CLUSIVE"看不见"的ESL）和排除从ESL血管示踪剂的荧光显微镜，可以计算为一个血管的宽度2之间的差的一半。 的瞬时测量的ESL厚度的使用是非常适合于研究的肺糖萼。活体显微镜的肺是具有挑战性的，显着的肺和心脏运动伪影。虽然最近的进步使固定的小鼠肺在体内 12 13，关注方面存在肺瘀血的生理影响。肺动与减少内皮型一氧化氮信号14信号转导通路，同时影响中性粒细胞黏附15和肺损伤16。此外，固定肺公开周围移动肺泡损害剪切力（即所谓的"atelectrauma"）的区域，按照与经典的生理概念肺泡相互依存17。 2008年，矶田渊沃尔夫冈·库伯勒和他的同事开发出一种手术技术，使活体显微镜可自由移动的小鼠肺18。呼吸的神器所产生的这种技术可以通过使用高速成像，包括明场和荧光显微镜的同时测量被否定。在这份报告中，我们详细介绍了如何瞬间右旋糖酐排除成像可以用来测量ESL厚度可自由移动， 在小鼠体内的肺胸膜下的微循环。这种技术可以很容易地修改，以确定糖萼函数具体地，一个完整的ESL排除循环从内皮细胞表面的元素的能力。最近，我们利用这些技术来决定的重要性，肺ESL完整的全身性炎症性疾病如败血症2急性肺损伤的发展。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂名称 </td></tr><tr><td> FITC-葡聚糖（150 kDa的） </td><td>西格玛</td><td> FD150S </td><td></td></tr><tr><td> TRITC-葡聚糖（150 kDa的） </td><td>西格玛</td><td> T1287 </td><td></td></tr><tr><td>链霉亲和素包被荧光微球</td><td>刘海实验室</td><td> CP01F/10428 </td><td>龙绿色荧光（类似FITC） </td></tr><tr><td>氯胺酮</td><td>摩尔医疗</td><td></td><td></td></tr><tr><td>二甲苯胺噻嗪</td><td>摩尔医疗</td><td></td><td></td></tr><tr><td>抗ICAM-1生物素化的抗体</td><td> eBioscience公司</td><td>克隆YN1/1.7。4 </td><td> 1:50稀释</td></tr><tr><td>同种生物素化抗体</td><td> eBioscience公司</td><td> IgG2b的eB149/10H5 </td><td> 1:50稀释</td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>机械通气</td><td>哈佛设备</td><td> INSPIRA </td><td></td></tr><tr><td>气管切开导管</td><td>哈佛设备</td><td> 730028 </td><td></td></tr><tr><td>电灼设备</td><td> DRE医疗</td><td> Valleylab公司的SSE-2L </td><td></td></tr><tr><td>双极电凝钳</td><td>奥尔森医疗</td><td> 10-1200I </td><td> 9.9厘米麦弗逊</td></tr><tr><td>温度控器l系统</td><td>世界精密仪器</td><td> ATC1000 </td><td></td></tr><tr><td>注射泵</td><td>哈佛设备</td><td>泵11精英</td><td></td></tr><tr><td>显微镜（怀德菲尔德） </td><td>尼康</td><td> LV-150 </td><td></td></tr><tr><td>显微镜（聚焦） </td><td>尼康</td><td> A1R </td><td></td></tr><tr><td>图片分离器</td><td>光度</td><td> DV2 </td><td></td></tr><tr><td> CCD相机</td><td>光度</td><td> CoolSNAP HQ2 </td><td></td></tr><tr><td>图像处理软件</td><td>尼康</td><td> NIS元素</td><td></td></tr><tr><td>聚偏膜</td><td>吴WR美联社</td><td></td><td></td></tr><tr><td>圆形盖玻片</td><td> Bellco </td><td> 5CIR-1-BEL </td><td> 5毫米，厚度＃1 </td></tr><tr><td>胶（盖玻片膜） </td><td> Pattex </td><td> flussig（液体） </td><td>盖上盖玻片膜</td></tr><tr><td>胶（盖玻片鼠标） </td><td> Pattex </td><td>凝胶</td><td>附加膜，鼠标</td></tr><tr><td>手术管件</td><td> Intramedic </td><td> PE50，PE10 </td><td></td></tr><tr><td>缝合</td><td>费舍尔</td><td></td><td> 4:0丝</td></tr><tr><td>电动剃须刀</td><td>奥斯特</td><td> 78997 </td><td></td></tr><tr><td>弯曲的手术钳</td><td> Roboz </td><td></td><td></td></tr><tr><td>直手术钳</td><td> Roboz </td><td></td><td></td></tr><tr><td>手术剪</td><td> Roboz </td><td></td><td></td></tr><tr><td>手术microscissors </td><td> Roboz </td><td></td><td></td></tr><tr><td>手术针驱动</td><td> Roboz </td><td></td><td></td></tr><tr><td>外科胶带</td><td>费舍尔</td><td></td><td></td></tr><tr><td>厨房海绵（切块） </td><td>各个</td><td></td><td></td></tr></tbody></table>

in vivo measurement of the mouse pulmonary endothelial surface layer

内皮糖萼层衬在血管腔的蛋白多糖和相关的葡糖胺聚糖。 在体内 ，糖复合物是高度水合，形成大量的内皮细胞表面层（ESL），有助于血管内皮功能的维护。由于内皮细胞糖复合物往往是异常失去了在标准组织内固定技术在体外和活体显微镜，研究中需要使用的ESL。为了更好地逼近复杂的生理学的肺泡微血管，是理想的可自由移动的肺进行肺活体成像。这些准备工作，但是，一般会有大量的运动伪影。我们证明如何闭胸，可以使用一个可自由移动的小鼠肺活体显微镜测量糖萼通过ESL排除从内皮细胞表面的荧光标记的高分子量葡聚糖的完整性。此非恢复的手术技术，这需要同步明场和荧光成像的小鼠肺，允许纵向观察胸膜下微血管不引起混淆肺损伤的证据。

步骤1-6中所述的实验方法将允许同时DIC（明场）和荧光图像的多帧捕获。要确定ESL厚度，拍摄的影像进行审查的实验方案完成后，由不知情的观察者。使用聚焦框，胸膜下微血管（直径&lt;20微米）确定至少3微血管通常是一个单一的帧（ 图10）上找到。用图像分析软件（NIS元素，尼康等），血管宽度的测量（由不知情的观察者）的平均长度每微血管的三个相互垂直的拦截。作为ESL是无?...

Watch this Scientific Journal Video about In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer at JoVE.com

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

内皮细胞糖萼/内皮细胞表面层是理想的使用活体显微镜研究。活体显微镜技术上具有挑战性的运动器官如肺。我们展示了如何同时明场和荧光显微镜可用于估计内皮表面层的厚度，在可自由移动<em在体内</em&gt;小鼠肺。

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

in-vivo-measurement-of-the-mouse-pulmonary-endothelial-surface-layer

Research

JoVE Journal

Medicine

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

14.5K 次观看.  University of Colorado School of Medicine. 内皮细胞糖萼/内皮细胞表面层是理想的使用活体显微镜研究。活体显微镜技术上具有挑战性的运动器官如肺。我们展示了如何同时明场和荧光显微镜可用于估计内皮表面层的厚度，在可自由移动

视频: In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

在缺血大鼠肺的血管生成

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

科研

医学

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

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.

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 of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

在小鼠肺压力容积曲线的测量

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

In this article, an economical, optimized, and simple protocol is described which uses the Evans blue dye method for assessing plasma extravasation in the organs of FVBN mice that can be adapted for use in other strains, species, and other organs or tissues.

一种优化的埃文斯蓝协议对小鼠血管泄漏的评估

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

左心房狭窄致大鼠肺静脉动脉化及第2组肺动脉高压

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...