Name: method of isolated ex vivo lung perfusion in a rat model: lessons learned from developing a rat evlp program
Uploaded: 2015-02-25T13:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program at JoVE.com

笔者要感谢哈佛仪器，尤其是斯蒂芬妮Pazniokas，MS（ 生理系统和再生医学）的援助，他们在电路组装，修改和灌注电路XVIVO灌注（丹尼尔·马蒂内利，CCP，CTP）的故障排除帮助提供非临床使用肺plegia。

注:所有程序均按照指导线的机构动物护理和国家研究委员会指南的人文关怀和使用实验动物（IACUC）进行，经历了批准俄亥俄州立大学IACUC委员会。 1.初始设置<ol><li>成立EVLP电路和具有温暖（37℃）灌注液通过系统结合了前栽肺前循环（ 图1）。 </li><li>设置的温水浴，用于护套灌注液储，热交换器，以及人工胸部，至37℃，并循环（ 图1）。 </li><li>运行一个脱氧合溶液（ 例如 ，6％O 2，8％的CO 2，84％N 2）计数器目前通过在气体过滤器中的灌注液以确保灌注液具有〜6％的溶解氧实验。 注:此去氧合灌流液使评估ØF根据测量导入灌注液，后器官的氧气的肺功能。 </li><li>打开数据采集程序，并连接肺动脉压力传感器，气管压差传感器，呼吸流量压差传感器，肺重量换能器，以及泵速度换能器到EVLP电路和数据采集/模拟 - 数字转换器盒（ 图2）。 </li><li>设置手术台和操作工具在EVLP电路（ 图3）。 </li><li>成立了一个小容器液氮附近EVLP电路，如果样本将被获得。 注:作者的系统已被修改以收集前器官和后器官灌流不中断压力流动动力学，可能潜在地破坏肺。 </li></ol> 2.准备麻醉药和肝素，大鼠的麻醉<ol><li>把以下个人防护设备（PPE）处理大鼠和大鼠组织前:口罩，手术手套，和一次性的礼服。 </li><li>称量大鼠，​​并记录重量。 </li><li>准备1200 U /公斤肝素。 </li><li>制备两个60毫克/千克氯胺酮和5mg / kg的甲苯噻嗪在同一注射器，首先制备氯胺酮。 </li><li>腹膜内氯胺酮和甲苯噻嗪的混合物注入到大鼠，并允许大鼠5分钟以不省人事。 </li><li>确定合适的麻醉通过检查的脚趾捏反射。如果老鼠不撤回其脚趾，它是不是感觉疼痛。 </li><li>移动老鼠手术台，在仰卧位固定，并喷用酒精消毒。 </li></ol> 3.提取和鼠肺初步通风<ol><li>准备4-20厘米长的丝线缝合（3-0或4-0就足够了）。 </li><li>开始使用数据采集程序记录数据。 </li><li>检查麻醉深度适当，用手术剪进入腹腔CAVITY通过中线剖腹手术和注射肝素进入下腔静脉。 </li><li>颅携带切口越过柄进入颈直到气管露出。不破裂的胸腔（ 图4A）。 </li><li>解剖后对在中线气管和滑动丝线缝合后气管（ 图4B）。 </li><li>提高气管的前部分，使软骨环，高位气管之间的横切口。不通过气管在这一点（ 图4C）后膜质部切断。 </li><li>导管插入与气管插管和安全与丝线缝合（ 图4D）的气管。确保缝线结扎被固定在开槽减轻插管的迁移。 </li><li>气管插管连接到通风电路。 </li><li>打开呼吸机，开始机械通风鲁NGS。 注:被选为初始设置为4毫升2 / kg和积极的呼气末正压（PEEP）潮气量CMH 2 O.这些设置的初始设置，并根据不同的实验条件下，可以调整一次器官是在离体灌注系统。 </li><li>通过胸骨/ xyphoid进入胸腔，并继续对颅胸骨上切迹。小心避免接触肺部。 注:由于大鼠肺是脆弱的，任何意外的操作可导致外伤和肺水肿（ 图5A）。 </li><li>使用2-拉钩，缩回胸腔正确露出解剖（ 图5A）。再次，要小心避免接触肺部。 </li><li>删除略有升高和钝性分离胸腺。 </li><li>移腹部内容到一个侧以暴露要么下腔静脉（IVC）或肠系膜静脉（MV）。 </li><li>切开要么IVC或中压来抽血大鼠，提供安乐死。 </li><li>放置一个丝线缝合后方肺动脉和主动脉中准备用于固定的肺动脉套管（ 图5B）。 </li><li>使在右心室流出道的前表面2-3毫米的切口，并放置在插管中的切口和进入主肺动脉，并与丝缝合（ 图5C）固定。 </li><li>横切心脏的顶点，以允许访问左心室和由流经肺动脉和通过所述心脏的心尖入胸腔冲洗肺血管内的任何凝块约15毫升的低K +电解液（ 图5D）。 </li><li>肺动脉（PA）套管连接到EVLP电路。确保流入线从电路到PA套管未来的底漆与灌流液，以避免进入心脏和肺部的空气。 </li><li>打开主蠕动泵将其设置为低（〜2毫升/分钟）的速度，使灌流液通过肺动脉和出左心室入胸腔运行。**关键步骤**确保PA压力不作为秒杀这是任一堵塞或差的插管（ 图6）的标志。 </li><li>关闭蠕动泵。 </li><li>定位丝线背后的心脏，周围的心室（ 图7）。 </li><li>开始通过插入一个小对外科镊子进入顶点，通过二尖瓣cannulating左心房的过程中，并进入左心房。 注意:这将扩张二尖瓣和促进插管。侵略性扩张，或太深的扩张，可能会无意中划破左心房渲染采购无效。 </li><li>从心脏中取出镊子。 </li><li>通过二尖瓣并进入留在插入左心房套管插入顶点杆菌。 </li><li>固定左心房套管与后面的心脏的丝线缝合（ 图8）。 注:此缝合可以"预捆绑"，以方便插管。 </li><li>肺动脉插管连接到离体肺灌注回路（ 图9A）。不左心房套管连接到EVLP电路直到心脏肺块已经完全从身体移除。 </li><li>夹紧用止血钳食道和切割夹紧下面，使得食道可用于提高心肺结构头侧（夹紧和隔膜之间）。 </li><li>直截了当地解剖周围组织切降主动脉和辅助船只，以释放所述心脏-肺块，因为它正在通过食道（ 图9B）上升。 </li><li>样头侧到气管插管完全免费的心脏，肺块气管。 </li><li>取出心脏，肺块，并将在DESI所述EVLP回路（ 图9C）上gnated位置。 </li><li>左心房套管连接到流出线，并启动主蠕动泵（ 图9D）。 </li></ol>肺部4. 离体灌注<ol><li>快速去除从EVLP装置顶部的通风线，并附加在外壳与压力传感器，然后插入上的EVLP装置的顶部壳体的顶部的通风线。 注意:这将允许通风数据进行记录和压力监测。 </li><li>确保气泡阱充满灌注液的足够量，使得没有气泡（ 即空气栓子）被引入到肺部。 </li><li>在最初的15分钟内慢慢地改变通气和灌注设置到所需的实验水平。另外，在此初始斜坡上升阶段，增加灌注流量为所需的速率和/或压力。 注:程序荷兰国际集团的换气装置，以产生间歇叹息呼吸，这方便的流体移出肺空间，因此延缓水肿的发作，值得推荐。这些可以通过配备有叹息功能通风机来制造。 </li><li>定义"时间0"作为时间时换气参数以4毫升/千克，PEEP潮气量在2厘米的 H 2 O和灌注参数是在它们的预期的水平，并保持恒定。 </li><li>如果需要的话，取灌洗液样品从样品端口，闪存冷冻在液氮中，并记下样品的时间。 </li><li>当实验完成后，隔离在液氮或地方的任何必要的解剖结构件的收集和急骤冻结在确定进一步研究的方案。 </li></ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> United States Organ Transplantation, Organ Procurement and Transplantation Network &amp; Scientific Registry for Transplant Recipients Annual Report 2011. , (2011).</li><li>Maathuis, M. H., Leuvenink, H. G., Ploeg, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+in+organ+preservation.">Perspectives in organ preservation.</a> Transplantation. 83, 1289-1298 (2007).</li><li>Cardoso, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+in+lung+transplantation:+from+conventional+preservation+to+ex+vivo+lung+perfusion+and+lung+reconditioning.">New perspectives in lung transplantation: from conventional preservation to ex vivo lung perfusion and lung reconditioning.</a> Jornal brasileiro de pneumologia : publicacao oficial da Sociedade Brasileira de Pneumologia e Tisilogia. 35, 1057-1059 (2009).</li><li>DeCampos, K. N., Keshavjee, S., Liu, M., Slutsky, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+inflation+volume+for+hypothermic+preservation+of+rat+lungs.">Optimal inflation volume for hypothermic preservation of rat lungs.</a> The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 17, 599-607 (1998).</li><li>Perrot, 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=Report+of+the+ISHLT+Working+Group+on+Primary+Lung+Graft+Dysfunction+part+III:+donor-related+risk+factors+and+markers.">Report of the ISHLT Working Group on Primary Lung Graft Dysfunction part III: donor-related risk factors and markers.</a> The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 24, 1460-1467 (2005).</li><li>Mulloy, D. 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=Ex+vivo+rehabilitation+of+non-heart-beating+donor+lungs+in+preclinical+porcine+model:+delayed+perfusion+results+in+superior+lung+function.">Ex vivo rehabilitation of non-heart-beating donor lungs in preclinical porcine model: delayed perfusion results in superior lung function.</a> The Journal of thoracic and cardiovascular surgery. 144, 1208-1215 (2012).</li><li>Noda, K., 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=Successful+prolonged+ex+vivo+lung+perfusion+for+graft+preservation+in+rats.">Successful prolonged ex vivo lung perfusion for graft preservation in rats.</a> European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 45, e54-e60 (2014).</li><li>Perrot, M., Liu, M., Waddell, T. K., Keshavjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion-induced+lung+injury.">Ischemia-reperfusion-induced lung injury.</a> American journal of respiratory and critical care medicine. 167, 490-511 (2003).</li><li>Steen, S., 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=Transplantation+of+lungs+from+a+non-heart-beating+donor.">Transplantation of lungs from a non-heart-beating donor.</a> The Lancet. 357, 825-829 (2001).</li><li>Perrot, 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=Strategies+to+optimize+the+use+of+currently+available+lung+donors.">Strategies to optimize the use of currently available lung donors.</a> The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 23, 1127-1134 (2004).</li><li>Cypel, 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=Technique+for+prolonged+normothermic+ex+vivo+lung+perfusion.">Technique for prolonged normothermic ex vivo lung perfusion.</a> The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 27, 1319-1325 (2008).</li><li>Cypel, 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=Normothermic+ex+vivo+perfusion+prevents+lung+injury+compared+to+extended+cold+preservation+for+transplantation.">Normothermic ex vivo perfusion prevents lung injury compared to extended cold preservation for transplantation.</a> American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 9, 2262-2269 (2009).</li><li>Cypel, 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=Normothermic+ex+vivo+lung+perfusion+in+clinical+lung+transplantation.">Normothermic ex vivo lung perfusion in clinical lung transplantation.</a> The New England journal of medicine. 364, 1431-1440 (2011).</li><li>Cypel, 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=Normothermic+Human+Ex+Vivo+Lung+Perfusion+(EVLP)+for+Improved+Assessment+of+Extended+Criteria+Donor+Lungs+for+Transplantation.">Normothermic Human Ex Vivo Lung Perfusion (EVLP) for Improved Assessment of Extended Criteria Donor Lungs for Transplantation.</a> The Journal of Heart and Lung Transplantation. 28, S126-S126 (2009).</li><li>Sanchez, P. 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=Normothermic+Ex+Vivo+Lung+Perfusion+as+an+Assessment+of.">Normothermic Ex Vivo Lung Perfusion as an Assessment of.</a> Marginal Donor Lungs - The NOVEL Lung Trial. J Heart Lung Transpl. 32, S16-S17 (2013).</li><li>Pego-Fernandes, P. 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=Experimental+model+of+isolated+lung+perfusion+in+rats:+first+Brazilian+experience+using+the+IL-2+isolated+perfused+rat+or+guinea+pig+lung+system.">Experimental model of isolated lung perfusion in rats: first Brazilian experience using the IL-2 isolated perfused rat or guinea pig lung system.</a> Transplantation proceedings. 42, 444-447 (2010).</li><li>Pego-Fernandes, P. 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=Experimental+model+of+isolated+lung+perfusion+in+rats:+technique+and+application+in+lung+preservation+studies.">Experimental model of isolated lung perfusion in rats: technique and application in lung preservation studies.</a> Jornal brasileiro de pneumologia : publicacao oficial da Sociedade Brasileira de Pneumologia e Tisilogia. 36, 490-493 (2010).</li><li>Niemeier, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+lung.">The isolated perfused lung.</a> Environmental health perspectives. 56, 35-41 (1984).</li><li>Zhou, 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=A+pulmonary+hypertension+model+induced+by+continuous+pulmonary+air+embolization.">A pulmonary hypertension model induced by continuous pulmonary air embolization.</a> The Journal of surgical research. 170, e11-e16 (2011).</li><li>Harris, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+responses+between+rat+strains+and+colonies.">Differences in responses between rat strains and colonies.</a> Food and cosmetics toxicology. 3, 199-202 (1965).</li></ol>

系统监控看什么东西的时候试验运行良好，如: 一旦插管已经置于电路和肺部换气，有多种方法，以确保正常工作的系统。应该有整个管道无渗漏灌流液。肺血管阻力（PVR）应保持相对恒定（假设恒定的流量）。氧气交换应增加一次呼吸机工作正常，扩大肺部招募更多的肺泡进行气体交换。 图12a显示适当通风和灌注连接到EVLP电路人工胸廓内肺。 看什么东西的时候实验没有运行良好，如: 有迹象表明，曾发生过程中EVLP实验的开始阶段率最高的一些常见问题。第一个和最简单吨Ø补救措施是在该行从肺部排出泄漏。此下电路，并在贮存器的水平不断下降的部分是由灌流池的池引人注目。检查并拧紧周围的泄漏区域的任何管接头，检查管本身的泄漏。如果肺之前发生这种泄漏，它也可能会引入气泡进入肺部。这应尽可能快地补救作为气泡在灌注液会导致组织损伤，并导致在PVR一个显著增加。还可以有一个泄漏从肺或套管中的一个的到来。这可能是由一个套管的任一滑动或阻塞在出射线引起压力增加而引起的。检查两个套管，保证既不已经滑落或扭曲的位置。在此过程中，因为在PA压力瞬时增加是一个明显的迹象表明，最近已发生某种障碍物PA的压力也应加以监测。 图12B示出了破裂肺即破裂，由于高的压力。从肺本身甲泄漏也可以由在组织中的撕裂引起的。这个问题可能是也可能不是可修复而是重新定位和重新拧紧套管是在这种情况下的最佳选择。 学习要点/机会: 审判和前体肺灌注系统的错误的发展使我们能够确定我们在这里勾勒，以促进有效实施EVLP系统的几个关键问题。首先，对于采购，但重要的是标准麻醉技术遵循适当麻醉动物（足以麻醉剂，注射入腹膜）和遵守需要所有IACUC策略。插管（ 图13中的A，B和C示出），应反复冲洗以除去肺vasculat内的任何凝块和/或碎屑URE。对于动物的选择，我们建议您使用只SD或路易斯大鼠，体重250-350克。应特别注意cannulating大鼠，体重接近至250g由于船只将会更小，因此更难以不损伤脉管到导管插入时可采取。如果小鼠或小鼠模型中，将被使用的，更小的套管可能需要被使用。 气管插管是不是通常的缝合时，首先通过一个丝线缝合后气管解剖周围筋膜和前插管后妥善固定，只要挑战。按照这个与前切口1-2气管环缝合上述通过套管。为了更好的安全性（ 图4C）的凹槽内固定配合方形结在气管环之间。与气管插管相比肺动脉（PA）的插管是更具挑战性。下面的步骤在本研究中使用对于此过程。首先，把握心脏先端具一对镊子。通过另一对镊子在横窦和线程的缝合线固定在近端的PA套管。紧接在右心室流出道（RVOT）（ 图14A）之前切开右心室。该切口进入RVOT后，套管将朝向肺动脉流出道被引导。具有在肺动脉/主动脉后面位置缝合右ventriculotomy前提高了效率（ 图5C）。插管应该固定到位与缝线，以防止脱出。一个主要的并发症可能发生，如果在PA插管是不是在正确的解剖方向。插管可以被插入太远，仅灌注一个分支或成为失当定位在除去从胸腔的心脏 - 肺标本的扭转。这可以很容易地被定向回原来位置，以保持anatomi的适当的角度CAL位置。最后，左心房（LA）插管的过程中最具挑战性的部分。对LA插管需要被放置在左心房内。与组织是非常易碎的，心不使用显著力或扭转以防止肺静脉与左心房这会再进行实验无法挽救内的撕裂。该PA套管的LA插管前，最好放置。阿左ventriculotomy与去除的顶点的已被证明扰乱cordae tendinae并允许通过二尖瓣小叶更容易获得。另外，ventriculotomy可以更容易地扩张和可视化的二尖瓣和通过二尖瓣喂插管。二尖瓣环带一对小钝端皮卡的扩张可以以可视化的道成LA（ 图14B）来完成。缝线应插管前，放在背后的心脏。这可以简单地通过使用一对SMAL起吊的​​心脏向上进行升钝端皮卡和放置下方缝合和整个心脏。洛杉矶是现在可以插管。饲料通过皮卡的LA套管，以正确的可视化安置的套管进入左心房。请特别注意不要打跑套管回左心室。缝线应该然后沿左心室的心肌被紧密地固定。固定缝合到左心房可能阻塞整个或插管的一部分。 在手术过程中，至关重要的是，没有空气留在装置的流入部分。任何显著空气可以产生空气栓塞增加的PVR（有效"空气锁定"），这将导致低得多的灌液流对于给定的压力。的各个点可以用来在系统内除去空气。流出部内的空气被预期并且不应该对肺的任何有害作用。肺动脉高压猪模型已经示重新创建从在8周的时间内连续少量空气的病理。该增加的空气减少的灌注的存在量而引起炎症到周围组织19。 灌注的开始可以发生，一旦插管已完成，但距离LA前的管来连接到EVLP线。灌流应通过运行，以清除任何血块，这灌流可以清空到胸壁没有任何问题。切换灌流泵为手动模式，并缓慢增加流量〜2毫升/分钟允许密切监测PA的压力。压力在20-30 CMH 2 O可以指示障碍物，看的灌流退出LA也是一个指标，但是这可能是很难看到的。如果压力不增加至超过20-30 CMH 2 O，停泵，并重新检查均插管。一旦压力恒定10-20 CMH 2 O允许日Ë灌流液，以2分钟贯穿而进入胸腔。此时距离LA的线可以连接到EVLP电路。灌流泵速度可提高到5-10毫升/分钟。当流体头前进通过电路，将有增加PA的压力，由于增加的流体头部的高度，因此，该静压力。如果流体不能流过的最高点的线，就可能有必要要么施加抽吸力就行的相对端或试图降低线的最高部分。一旦这个问题得到解决，灌流液循环应该没有任何问题。 几个问题应相对于所述呼吸监测。第一，可能会发生支气管/气管和心脏 - 肺位置的扭转如肺部变得更加水肿和重量增大。它对于套管保持在相对紧密的解剖位置，因此改变是重要的一个或两个套管E可以是必要的。压力或体积控制通气机以及正或负通气可用于本EVLP系统。对于大鼠模型中，我们用积极的压力，体积控制的通风效果很好，在4-10之间毫升/千克，并在2-8 CMH 2 O.之间的呼气末压力（PEEP）潮气量发现然而，8 水柱一个PEEP可以在气管的分岔导致可能的破裂。每个实验后（或一组实验，如果进行背到背），通风线通向气管应清洗任何支气管肺泡灌洗（BAL）液，可能已到达了气管。这种液体会变硬，如果保持不变，并可以完全阻塞通风线。 灌注液组合物，是一个成功的EVLP实验的关键。将5％的葡聚糖混合物允许肺灌注即接近生理条件，保持一个稳定的胶体渗透压驱动液BACK插入血管以防止水肿，并防止肺血管内血栓形成。要注意的是一些物种大鼠可以是过敏葡聚糖这可能会导致肺水肿20是很重要的。灌注液中的内容是跨所有实验组一致的在这项研究中，因此，葡聚糖含量不应该是一个混杂因素。的胶体渗透压是具有提高或产生组织水肿的潜在的关键变量。被冷静态存储或常温灌注优化市售灌注液已被用于在本系统中，以增加肺生存能力次。我们注意到，一些这些解决方案包含白蛋白和一个值得关注的是牛血清白蛋白的触发在啮齿动物的肺的炎症反应的可能性。虽然最优灌注液组合物是调查的一个正在进行的课题，灌注液需要考虑的胶体渗透压，渗透压和缓冲能力。 W¯¯Ë建议的溶液可基于修饰的Krebs-Henseleit溶液或细胞培养基。的胶体渗透压应由葡聚糖或白蛋白进行维护，这取决于应用。的灌注压和流量影响器官和超生理灌注参数可以使器官发生机械损伤。 实验过程中视觉指标: 有许多视觉线索，以及从可用于确定是否一个EVLP实验运行良好的实时数据指示。肺将保持在相同的大小和将放气到每次呼吸后相同体积。也将有没有来自肺本身泄漏。的PVR，肺的重量，和遵守将保持相对恒定。氧气产量将保持不变或略有增加。 有些情况下，肺部的实验过程中变得损害了许多视觉上的指标。肺水肿成为一个次在尺寸和重量增长迅速。的肺的变化（从褐色粉红色至白）和液体袋的颜色可以在组织被识别。如果从气压伤或过度扩张气管或肺部破裂，就会有气泡从点伤（ 图12B）。氧的生产将减少，并且PVR和遵守将显着增加。 使用小动物的EVLP模型中，诸如啮齿类动物的电位打开门供未来研究改善肺移植的治疗。然而，在小动物模型需要更好地了解真正模仿肺移植。该模型可能在将来被用来改善药物治疗和为将来的肺移植研究确定基线参数。

临床意义目前可供移植的肺合适的，只有19％的肺能够在全国范围内利用导致旷日持久的等待列表的时间或死亡患者等待移植1的缺乏。这种短缺可能是因为年龄大捐助者，外伤，感染，多系统器官功能衰竭和收获2时，有时受伤供体肺。此外，肺是胸腔和标准的运输和保存技术以外的体弱器官可导致劣化和非存活肺部。因此，维持和改善肺活力的前体最近成为肺移植医学的一大重点。 离体肺灌注（EVLP） 离体肺灌注（EVLP）已发展到持续灌注器官正在评估移植，使一个时期的评估，所有OWS肺复苏或再生的潜力。 EVLP可以延长的总出身体器官缺血时间，并允许捐赠器官前往更远的距离3。典型地，肺部通气，在30％50％总的肺容量或20 水柱与吸入氧的一小部分的峰值气道压力（氧合指数2）70％的4。保存溶液灌注在40-60毫升/公斤（100毫升/公斤预测心输出量的大约40％）在人类和大型动物5,6，但灌注在20％左右的心输出量为大鼠7。列入STEEN解决方案使人体肺部在RT环境无肺水肿9开发旅游。这种开拓性的工作已经细化了多伦多大学的肺移植计划10-13，并正在评估边际供体肺移植14,15改善的评估。然而，最优ventilatio以再生边缘和/或低于标准的肺移植需要n和灌注条件是未知的，目前是一个活跃的研究领域。 隔离肺灌注系统已被用于在小动物引起的肺损伤，重新创建呼吸道疾病，并灌注以不同的解决方案的肺部，以防止缺血损伤。研究者已经通过使用分离的肺灌注系统以模仿，可以在人类和较大的动物16-18中使用的EVLP协议创建的肺移植的小动物模型。然而，该实验模型具有关于用于模拟人体生理学的各种技术和参数许多挑战。特别是，有许多微妙之处EVLP过程中保持肺的可行性。这些细微之处可能发生由于在收割技术中，正压通气设置，灌注液的组成和流动条件和肺的插管的差异。疗法安伏，这里的目标是提供研究界与一些，我们已经发现导致了可靠的方法用于在啮齿动物模型实施EVLP故障排除和执行提示。

<table><tbody><tr><td>IPL-2 Basic Lung Perfusion System</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Tweezer #5 stainless steel, curved 11&amp;nbsp;cm</td><td>Kent Scientific Corporation</td><td>IND500232</td><td></td></tr><tr><td>Tweezer #5 Dumostar, 11&amp;nbsp;cm</td><td>Kent Scientific Corporation</td><td>INS500085-A</td><td></td></tr><tr><td>Tweezer #7 Titanium, 12&amp;nbsp;cm tips curved</td><td>Kent Scientific Corporation</td><td>INS600187</td><td></td></tr><tr><td>McPherson-Vannas Scissors 8&amp;nbsp;cm, Str 5&amp;nbsp;mm</td><td>Kent Scientific Corporation</td><td>INS14124</td><td></td></tr><tr><td>Vannas Scissors 8&amp;nbsp;cm Str 5&amp;nbsp;mm</td><td>Kent Scientific Corporation</td><td>INS14003</td><td></td></tr><tr><td>Instrument Sterilization Tray 5&quot; x 7&quot;</td><td>Kent Scientific Corporation</td><td>INS800101</td><td></td></tr><tr><td>Heparin 30,000 units per 30 ml</td><td>APP Pharmaceuticals</td><td>Supplied from OSU Pharmacy</td><td></td></tr><tr><td>Ketamine 500&amp;nbsp;mg per 5&amp;nbsp;ml</td><td>JHP Pharmaceuticals</td><td>Supplied from OSU Pharmacy</td><td></td></tr><tr><td>Xylazine 100&amp;nbsp;mg per 1&amp;nbsp;ml</td><td>Akorn</td><td>Supplied from OSU Pharmacy</td><td></td></tr><tr><td>10&amp;nbsp;cc insulin syringe 29&amp;nbsp;G x 1/2&quot; needle</td><td>B-D</td><td>309301</td><td></td></tr><tr><td>Hyflex NBR</td><td>Ansell</td><td>S-17310M</td><td>Bite proof gloves</td></tr><tr><td>BL1500</td><td>Sartarius</td><td>Practum 1102-1S</td><td>Scale</td></tr><tr><td>Large Flat Bottom Restrainer</td><td>Braintree Scientific Inc</td><td>FB L 3.375 dia x 8.5, 250-500gm rat&amp;nbsp;</td><td>Rat tunnel for injection</td></tr><tr><td>Sterling Nitrile Powder-free Exam Gloves, Large</td><td>Kiberly-Clark</td><td>50708</td><td></td></tr><tr><td>Rapidpoint 405</td><td>Siemens</td><td></td><td>blood gas analyzer</td></tr><tr><td>Fiberoxygenator D150</td><td>Hugo Sachs Elektronik</td><td>PY2 73-3762</td><td></td></tr><tr><td>LabChart v7.3.7</td><td>ADInstruments</td><td></td><td></td></tr><tr><td>Tracheal cannula</td><td>Harvard Apparatus</td><td>733557</td><td></td></tr><tr><td>Pulmonary Artery cannula</td><td>Harvard Apparatus</td><td>730710</td><td></td></tr><tr><td>Left Atrium cannula</td><td>Harvard Apparatus</td><td>730712</td><td></td></tr><tr><td>Peristaltic Pump&amp;nbsp;</td><td>Ismatec</td><td>ISM 827B</td><td></td></tr><tr><td>Small Animal Ventilator model 683</td><td>Harvard Apparatus</td><td>55-000</td><td></td></tr><tr><td>Ecoline Star Edition 003, E100</td><td>Lauda</td><td>LCK 1879</td><td>Water Heater</td></tr><tr><td>Tubing Cassette</td><td>Cole-Parmer</td><td>IS 0649</td><td></td></tr><tr><td>Connect kit D150</td><td>Cole-Parmer</td><td>VK 73-3763</td><td></td></tr><tr><td>PowerLab 8/35</td><td>ADInstruments</td><td>730045</td><td></td></tr><tr><td>TAM-A transducer amplifier module type 705/1</td><td>Hugo Sachs - Harvard Apparatus</td><td>73-0065</td><td></td></tr><tr><td>TAM-D transducer amplifier type 705/2</td><td>Hugo Sachs - Harvard Apparatus</td><td>73-1793</td><td></td></tr><tr><td>SCP Servo controller for perfusion type 704</td><td>Hugo Sachs - Harvard Apparatus</td><td>732806</td><td></td></tr><tr><td>CFBA carrier frequency bridge amplifier type 672</td><td>Hugo Sachs - Harvard Apparatus</td><td>731747</td><td></td></tr><tr><td>VCM ventilator control module type 681</td><td>Hugo Sachs - Harvard Apparatus</td><td>731741</td><td></td></tr><tr><td>TCM time control module type 686</td><td>Hugo Sachs - Harvard Apparatus</td><td>731750</td><td></td></tr><tr><td>IL2 Tube set for perfusate</td><td>Harvard Apparatus</td><td>733842</td><td></td></tr><tr><td>Tube set for moist chamber</td><td>Harvard Apparatus</td><td>73V83157</td><td></td></tr><tr><td>Tygon E-3603 Tubing 2.4&amp;nbsp;mm ID</td><td>Harvard Apparatus</td><td>721017</td><td>perfusate line entering lung</td></tr><tr><td>Tygon E-3603 Tubing 3.2&amp;nbsp;mm ID</td><td>Harvard Apparatus</td><td>721019</td><td>perfusate line leaving lung</td></tr><tr><td>low potassium dextran glucose solution</td><td></td><td></td><td>flushing the lung</td></tr></tbody></table>

method of isolated ex vivo lung perfusion in a rat model: lessons learned from developing a rat evlp program

可用于肺移植接受供体肺的数量是非常有限的，由于质量差。 离体肺灌注（EVLP）已获准在人类肺移植变得更容易获得通过使评估机构，扩大捐助池的能力。作为这种技术的扩展和改进，以潜在地评估和改进不合肺移植前的质量的能力是一个重要的需求。为了更严格地评估这些方法中，一个再现的动物模型需要建立将允许对改进的技术测试捐赠肺部和管理，以及对肺移植接受者。此外，相关的病状， 例如一个EVLP动物模型中，通气性肺损伤（VILI），将提供一种新的方法，以评价对这些疾病的治疗方法。在这里，我们描述了一个大鼠肺EVLP程序和改进的发展，这个我的ThOD允许用于将来扩展的可重现模型。我们还描述了本EVLP系统的应用建模VILI大鼠肺部。我们的目标是提供研究界提供关键信息和"珍珠智慧"/技术，出现了从试验和错误，并建立一个EVLP系统，强大的和可重复性的关键。

通过数据采集程序收集的实时机械数据可以很容易地进行分析，以测试任何数量的假设。例如， 图10A示出了通过60分钟从10大鼠实验中动物换气以4毫升/千克和2的低潮气量/低PEEP CMH 2 O的平均肺重量虽然在整个实验一非常轻微的增加在肺的重量，这增加不是统计学上显著（ANOVA，P = 0.92）。 图10B示出了从12大鼠实验的平均肺动脉压（PAP）通过60分钟。在0分钟时间点较低的PAP是在所有实验和PAP的开始使用低流量和通风设置，结果仍然没有统计学显著改变这个时间点之后常数t = 10分钟（ANOVA的行列后，P = 0.89）。 图10C示出了肺血管阻力（PVR），通过60分钟从12鼠实验虽然有小幅度下降PVR吨= 20分钟后，出现了这个实验过程中PVR无统计学差异显著（ANOVA秩，P = 0.65）。相较于此处示出的PVR的数据，野田等人 。已示出的PVR的稍微增加随时间4小时。然而，这些作者报告数据中的PVR开始在1小时，代替在实验的开始和被提供7没有标准偏差的值。野田等人 。也没有显示肺水肿的数据为4小时实验所以没有比较可以与这里在图10A中呈现的数据作出。在野田等人的主要区别。过程相比，现在示出在本文中包括:一个1小时冷保存在EVLP之前LPS溶液，将大鼠最初通气，气体混合物包括异氟烷，以使其失去知觉，灌流溶液补充有50毫克甲泼尼龙和50毫克头孢菌素，总FL流被定义为所计算出的心输出量的20％，灌注液取样之后才肺已通风的100％的O 2，以便事先5分钟，并在实验运行4小时。 从灌注液在实验过程中所采取的样品也可用于多种目的分析。作为一个例子，在图11中，我们展示了如何高的潮气量/高PEEP换气可诱导在60分钟的促炎性响应。对于这些实验，从4只大鼠灌流损害性的条件下， 即 10毫升/公斤，8高PEEP 水柱高的潮气量下通风，均使用标准亲和抗炎细胞因子IL1β，TNFα和IL-4分析ELISA技术。 如图11，相比于细胞因子水平通气前（0分钟），损害通气60分钟，导致了IL-1β和TNFα（促炎性细胞因子）一个具有统计学显著增加<html>次中的IL-4无变化（抗炎细胞因子）的浓度。因此，这种EVLP系统能够产生肺损伤型材机械通气时通常观察到的。 <img alt="图1" src="/files/ftp_upload/52309/52309fig1highres.jpg" /> 图1.图和照片，小动物离体肺灌注（EVLP）电路。英皇图中匹配与照片的信件。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52309/52309fig2highres.jpg" /> 图2.所有的传感器都牢固地连接到控制箱。"&gt;请点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52309/52309fig3highres.jpg" /> 图3.大鼠手术台牢固地建立了相邻的体外灌注肺（EVLP）电路。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52309/52309fig4highres.jpg" /> 图4（A）的一个切口，颅暴露气管。胸腔不露出。 （B）用丝线缝合放置气管后面。 （C）的气管被部分地切割以用于插管准备。 （D）中的气管插管放入positio<html> n和固定用丝线缝合。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52309/52309fig5highres.jpg" /> 图5.（A）中的胸腔被拉回，以允许进入心脏和肺。 （B）准备将缝合肺动脉后面。 （ 三）肺动脉插管，用绑在前面放置丝线缝合。（D）低K +电解质溶液是通过肺动脉和出左心房冲洗，以去除任何血块。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig5large.jpg" target="_blank">请点击此处查看大图版本这个数字。</a> lways"&gt; <img alt="图6" src="/files/ftp_upload/52309/52309fig6highres.jpg" /> 图6.增加肺动脉流速冲洗时肺部可引起肺动脉压力急剧增加。如果被正确执行插管并没有什么大堵塞，压力应该降低。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a> <img alt="图7" src="/files/ftp_upload/52309/52309fig7highres.jpg" /> 图7.丝线缝合放在围绕整个心脏，准备左心房插管。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 正页="总是"&gt; <img alt="图8" src="/files/ftp_upload/52309/52309fig8highres.jpg" /> 图8.左心房插管固定到位用丝线缝合。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/52309/52309fig9highres.jpg" /> 图9.（A）中的肺动脉插管连接到离体肺灌注回路。 （B）中的食道夹紧和结缔组织是钝性分离以除去心脏肺集团。 （C）的心脏-肺集团从胸腔除去并放置到离体肺灌注回路。 （D）中的左心房被连接到离体肺灌注回路。<html> HREF ="htt​​ps://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig9large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图10" src="/files/ftp_upload/52309/52309fig10highres.jpg" /> 图通过离体肺灌注（N = 10），60分钟10（A）重隆雄性SD大鼠。 （B）雄性SD大鼠，通过离体肺灌注（N = 12）的60分钟肺动脉压力。 （C）雄性SD大鼠，通过离体肺灌注（N = 12）的60分钟肺血管阻力，NS表示没有统计学差异显著。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="&lt;/ HTML"><html> "图11"SRC ="/文件/ ftp_upload / 52309 / 52309fig11highres.jpg"/&gt; 图11.影响的大潮卷上亲和抗炎细胞因子浓度1小时通风（10毫升/公斤）和高PEEP（8 CMH 2 O）的灌流液。N = 4，*表示有统计学差异显著对于0小时样品（P &lt;0.05）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig11large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图12" src="/files/ftp_upload/52309/52309fig12highres.jpg" /> 图12.（A）有适当通风和灌注肺连接到EVLP电路。 （B）高正呼气末正压（PEEP）导致撕裂的气管分叉导致泡沫形成的伤害，并填写了人工胸部。<html> ES / ftp_upload / 52309 / 52309fig12large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图13" src="/files/ftp_upload/52309/52309fig13highres.jpg" /> 图13.（A）肺动脉导管。此套管是比左心房套管小。 （B）左心房套管。此套管比肺动脉套管大得多。 （C）套管气管。该套管有肋骨在确保用丝线缝合气管帮助。被插入气管年底也略有指向插入套管进入气管，以帮助。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig13large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 09fig14highres.jpg"/&gt; 图14.（A）中的心脏顶点是由一对钳子的作为右心室是大约以导管插入肺动脉被切开举行。 （B）二尖瓣环带一对小钝端皮卡的扩张使得它更容易可视化道进入左心房。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52309/52309fig14large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program at JoVE.com

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

离体肺灌注（EVLP）已获准在人体肺移植变得更容易获得通过使评估机构，扩大捐助池的能力。在这里，我们描述了鼠EVLP方案和改进，允许未来扩展可再现模型的发展。

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型:教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

method-isolated-ex-vivo-lung-perfusion-rat-model-lessons-learned-from

Universidad San Sebastian

Research

JoVE Journal

Medicine

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

27.2K 次观看.  Ohio State University Wexner Medical Center.  离体肺灌注（EVLP）已获准在人体肺移植变得更容易获得通过使评估机构，扩大捐助池的能力。在这里，我们描述了鼠EVLP方案和改进，允许未来扩展可再现模型的发展。 

视频: Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

科研

医学

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

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 ...

生物学

Isolated Lung Perfusion System in the Rabbit Model

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.

The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.

兔模型中的孤立肺灌注系统

The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs&#39; inner workings, both micro and ...

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem of a low number of lungs considered viable for the donation process. In recent decades, however, this scenario has been positively changed, given the development of ex vivo lung perfusion (EVLP) as a strategy for evaluating and reconditioning marginal lungs. The establishment of EVLP in large transplant centers has favored an increase in the number of lung transplants, both by increasing the diagnostic accuracy of lung function and by constituting an effective platform for the reconditioning of lung grafts. In this context, faced with ethical and logistical issues, as well as in the study of immunological factors associated with lung transplantation, the development of rodent EVLP models has become important, given their reliability, the possibility of genetic manipulation, and lower costs. This paper describes a protocol for establishing a rat EVLP model and shows the inflammatory profile associated with the perfused lungs. This will help propagate knowledge about the rat EVLP model, promoting our understanding of the biological responses associated with that revolutionary technique.

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Here, we describe the necessary steps for establishing a rat EVLP model and show the inflammatory profile associated with the perfused lungs. The aim is to propagate knowledge and experiences about the rat EVLP model, enabling the integral understanding of the biological responses associated with that revolutionary technique.

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem ...

<meta charset="utf8"></head><body>在大鼠 EVLP 模型中使用基于 PolyhHb 的灌注推进肺移植

Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. However, new and exciting technology, such as ex vivo lung perfusion (EVLP), offers lung transplant providers extended assessment for marginal donor allografts. This dynamic assessment platform has led to an increase in lung transplantation and has allowed providers to use donors that were previously discarded, thus expanding the donor pool. Current perfusion techniques use cellular or acellular perfusates, and both have distinct advantages and disadvantages. Perfusion composition is critical to maintaining a homeostatic environment, providing adequate metabolic support, decreasing inflammation and cellular death, and ultimately improving organ function. Perfusion solutions must contain sufficient protein concentration to maintain appropriate oncotic pressure. However, current perfusion solutions often lead to fluid extravasation through the pulmonary endothelium, resulting in inadvertent pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as the proper protocols to test them in clinically relevant translational transplant models.

<meta charset="utf8"></head><body>作者聚焦：利用下一代聚合人血红蛋白改善大鼠供体肺的评估和保存

Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat ex vivo lung perfusion.

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. ...

Evaluation of Apoptosis and Lung Injury in DCD Donor Lungs Preserved by EVLP

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after cardiac death (DCD). However, a critical challenge remains in maximizing the number of viable lungs that meet stringent criteria for clinical use. Ex vivo lung perfusion (EVLP) emerges as a promising technology to evaluate and potentially improve the function of donated lungs. This paper describes the development of a cost-effective and reproducible small animal experimental platform specifically designed for EVLP research. This platform offers a high degree of customization, enabling researchers to tailor experimental parameters to address specific research questions within the field of EVLP. The team established a rat model to simulate DCD and implemented a two-stage preservation approach. Lungs were subjected to static cold storage using a perfusate solution at 4 &#176;C for 4 h, followed by normothermic EVLP for another 4 h. Researchers evaluated various lung function parameters, including pulmonary vascular resistance, pulmonary dynamic compliance, blood gas measurements of the perfusate, and apoptotic cell death in lung tissues. The study demonstrates that EVLP could significantly improve the functional performance of DCD lungs compared to simple cold storage at 4 &#176;C, suggesting that EVLP has the potential to mitigate ischemia-reperfusion injury and enhance graft function following circulatory death, thereby increasing the pool of transplantable lungs.

The objective is to develop a new ex vivo lung perfusion (EVLP) model in rats that can sustain consistent lung function for 4 h throughout EVLP and furnish the industry with implementation specifics that will offer a sturdy method for applying EVLP to rodent models.

The increasing scarcity of donor lungs for transplantation has spurred significant interest in the utilization of organs procured from donors after ...

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro models. Laboratories wishing to use isolated lungs need to be aware of important steps and inherent challenges to establish a setup that is affordable, reliable, and that can be easily adapted to fit the topic of interest. This paper describes a DIY (do it yourself) model for ex vivo rat lung ventilation and perfusion to study drug and gas effects on pulmonary vascular tone, independent of changes in cardiac output. Creating this model includes a) the design and construction of the apparatus, and b) the lung isolation procedure. This model results in a setup that is more cost-effective than commercial alternatives and yet modular enough to adapt to changes in specific research questions. Various obstacles had to be resolved to ensure a consistent model that is capable of being used for a variety of different research topics. Once established, this model has proven to be highly adaptable to different questions and can easily be altered for different fields of study.

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

大鼠 离体 肺灌注模型的设计和实现

Ex vivo lung preparations are a useful model that can be translated to many different fields of research, complementing corresponding in vivo and in vitro ...

的隔离方法

摘要

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此视频中的章节

一个简单的方法小鼠肺插管

青光眼诱导在程序

在离体血液灌注大鼠肺备量化单微血管通透性

兔模型中的孤立肺灌注系统

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

大鼠离体肺灌注模型的设计和实现

的隔离方法

摘要

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此视频中的章节

一个简单的方法小鼠肺插管

青光眼诱导在程序

在离体血液灌注大鼠肺备量化单微血管通透性

兔模型中的孤立肺灌注系统

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

在大鼠离体肺灌注模型中使用下一代聚合基于血红蛋白的氧载体探索替代灌注解决方案

Establishment of a Novel Ex Vivo Lung Perfusion System for Rat Lungs After Circulatory Death

大鼠 离体 肺灌注模型的设计和实现

大鼠离体肺灌注模型的设计和实现