这项工作是由美国心脏协会（EIA 0740129N）和NIH的T32 HL90610支持。

1。实验前<ol><li>实验一天前，站适当尺寸的玻璃毛细管拉成微电极（无论是水平或垂直的车夫，都可以使用）。尖端直径可以根据被隔离的船只随时调整，虽然我们通常使用直径范围50-150微米之间。这些微血管站了以下丁烷火焰加热到适当的配置，然后弯曲。微量提示身体打破近似直径（用细镊子）微血管中的问题，可以适当的构象，如针对特定应用所需的研磨或抛光。读者对这些程序的广泛和出色的审查，导演戴维斯等。16。两个微电极，然后放置在反对微血管室移液器持有人。这些必须面向这样的提示在相同的水平和垂直平面内的血管腔。 在这个手稿（ 图1和2）微血管腔是一个建先生，David Eick威斯康星医学院生理学教研室（定制<a href="http://www.phys.mcw.edu/">http://www.phys.mcw.edu/</a>和<a href="http://www.eicktech.com">WWW 。eicktech.com</a> ）。然而，微血管室的其他几个配置都是现成市售，与那些生活仪表系统（开发<a href="http://www.livingsys.com/">http://www.livingsys.com/</a> ）和IonOptix（ <a href="http://www.ionoptix.com/">http://www.ionoptix.com/</a> ）是很常见的。使用这些其他系统，它可能必须利用倒置显微镜，而不是一个传统的直立，虽然这是依赖于具体的实验和设备的要求。倒置显微镜的使用将是可取的实验protocOLS需要荧光或共聚焦成像。 </li><li>准备生理盐溶液（PSS的;所述），并确保pH值设置为特定的实验条件（7.38-7.42是典型的，属于生理范围内）到适当的水平。 PSS的解决方案，可事先准备和冷藏，但必须在使用前检查pH值适宜的温度。 PSS可以包含白蛋白，如果实验者或特定的协议要求，虽然这不是普遍强制性。 </li><li>应校准卡钳用来测量血管直径的数字视频，使用血球或显微镜阶段微米。精确的校准，这是至关重要的千分尺放置在血管腔的流入/流出微电极在同一平面上。 </li><li>附加微血管的移液器的领带回路应准备和易于后续使用。这些可以很容易地从8-0或小眼小号准备uture。单领带循环，可以事先准备，并存储在一个小的反转磁带，直到需要（这将保持他们丢失）带周围的显微镜幻灯片盖培养皿中。 （ 图3）。 </li></ol> 2。天实验<ol><li>应打开所有配套设备和适当的功能检查。这包括anti-vibration/floating表和循环恒温水浴（提供船只浴在适当的温度-常用37°C间），所有的压力传感器和船只室排水泵（ 图1A和1B）。打开船室和灌流灌流水库的平衡气体。 </li><li>填写所有的水库，管，商会与PSS的解决方案。灌流线，导致流入移液器必须填写完整，以避免气泡在这条线的存在，这可能驱逐和损坏的VAscular内皮细胞（ 图1C）。如果需要，使用注射器轻轻推动的PSS通过吸管，以确保它是完全充分的，并没有任何堵塞可能阻碍灌流流一路。流入压力应保持在合理的限度内，以避免损坏压力传感器。 </li></ol> 3。微血管收获<ol><li>是要采取微血管的动物从麻醉测定，船只问题，应根据所要研究的血管是最合适的程序分离。在某些情况下，这可能需要切除器官（如脑或冠状动脉微血管），而在其他国家，船只可以从麻醉动物（如肌肉）。例如船只在股薄肌的方向，提出了在图4。估计在体内的血管长度与前移走CEP证书或小卡钳。从动物/器官的血管被拆除之前，它可以是非常有益的，进行最后的检查，以确定该船只室准备和正常运作（包括全部到位领带循环）。 </li><li>从动物/机关抓船只的外观方面，在一端用细镊子，沿着船只的长度切割，直到它是免费的，格外小心，以避免或船只上的任何揪着拉船。一旦释放，立即将它充满与PSS在1.7 mL离心管，但不放手船只。这是要记住的方向，这样在洗澡的的灌流方向将是在动物的血液流动方向相同。 </li></ol> 4。插管的船只<ol><li>放置在充满浴船只和cannulate近端流入移液器。这样才能最好地完成套管通过了温和灌注率（〜50毫米汞柱）一双细镊子用来存放每一侧近端管腔墙（即每手钳子）​​。在我们的实验室中，我们用细镊子从精细的科学工具（ <a href="http://www.finescience.com/Home.aspx">http://www.finescience.com/Home.aspx</a> ），虽然任何适当的钳将有效地开展工作。滑的船只上的插管技巧和推进尖点，其中两个的领带循环可以确保船只上的船只。 </li><li>提高流入压力〜75毫米汞柱，进一步助长船只和促进远端枪头插管。流通过的船只，应被视为在该船只的远端流出（显微镜下）在灌流水库的失真。 Cannulate船只上流出吸管末端，并确保它有两个并列循环（这些最后的循环必须到位，插管前， 图5）。 </li><li>在XYZ坐标作为neede的调整，直到套管d，直到有失真最小的船只，船在体内的长度接近。提高流入的压力，直到它接近通常经历了由该船只段（我们实验室利用股薄肌8大阻力小动脉平均动脉压的80％）平均动脉压的百分比。 </li><li>提供适当的混合气体进入浴缸和地方的保鲜膜或玻璃盖在室内，以避免飞溅的显微镜镜头放置一个小气泡石。我们使用冒泡，通常是在所有宠物/水族店是直径约1厘米的石头，虽然规模较小，以及有效的工作。冒泡石的存在，确保适当的气体供应船只在任何时候都将延长船舶的可行性。在所有的测量周期（或空气流通过暂时中断的石头），以防止变形，这块石头被删除形象。释放流出吸管钳，并允许通过30分钟的船流。定期流动应夹紧在流出油管，以确定该船只正在开发休息音。在这一点上，所有船只必须检查压力泄漏。泄漏会引入到已知的船只（我们通常使用100-120毫米汞柱）的压力和夹紧流出，流入，线条明显。如果腔内的压力是稳定的，有没有明显的泄漏。然而，如果压力开始下降，一个重要的泄漏是目前必须关闭。流入或流出移液器泄漏一般可以通过增加一个额外的回路并列周围船只上的吸管纠正。另外，如果船只有一个小的分支，是允许的泄漏，这将危及船只的能力，有效地包含压力，可以引入一个额外的错误源。如果发现泄漏，它通常可以被关闭瓦特绑随着一个10-0眼科缝线的单回路。另外，从6-0缝合戏弄单链，使领带循环也是非常有效的。如果泄漏不能被识别，或可以不进行有效的捆绑关闭（例如，有没有的侧分支和简单地在血管壁上的洞主干），这可能在1实验过早终止和使用1替代成为船只必要（这通常是相同​​的船只在另一条腿 - 如有必要，或对侧 - 动物）。 船只应允许至少30分钟没有流量条件下平衡。所有的实验都无流量条件下进行。一旦船只已开发令人满意的音色和响应问题的实验纳入标准，该船是为后续研究做好准备。 这些入选标准可以根据不同的具体的实验方案和研究者。在我们的实验室中，我们经常UTilize了以下内容: <ol class="lalpha"><li> &gt; 30％（计算为ΔD/ D MAX×100，其中ΔD是平衡压力下的值在动脉内壁直径增加曝光的Ca 2 +无PSS和D MAX是最大的积极音直径测量中的Ca 2 +免费PSS的平衡压力）。 </li><li>生肌激活（维修，最好是减少，腔内压力增高的动脉直径）指数作为一个可行的血管平滑肌层。 </li><li>一个可行的内皮衬里，轻快的扩张对乙酰胆碱的反应（10 -6米的浴缸）。 </li><li>可以添加额外的入选标准，以适应具体的实验需要的协议，包括大蟒肾上腺素受体 ​​激动剂，如福林（10-7 M），扩张器，额外的刺激，如腺苷（10-5 </SUP&gt; M）或缺氧（O 2的平衡气体减少0％）。 </li></ol></li><li>下面的部分是"模拟实验"的例子。所有要求的解决方案是准备适当，注意与支付帐户的稀释因子在血管腔的PSS。作为一个例子，我们的一些设备，船只室容积为20毫升。正因为如此，20μL10 -2 M股票有效浓度在10-5中号船室洗澡解决方案的结果。实验是在去除血管，插管，平衡和验证如上所述，准备开始。如治疗随机和有效性的具体是在特定实验，并会消耗过多的空间，这方面的努力。因此，这些不包括细节。 初步控制反应的决心。举一个例子，如生肌激活（压力引起的收缩），intralumenal公关生理刺激是随机改变essure在所需的范围内，允许该船只适当的时间内响应（在这种情况下，我们将允许5-7分钟的时间内）。举一个例子，如与乙酰胆碱的挑战药理刺激，药物被添加到后面从库存解决方案，以随机顺序。在这种情况下，我们通常会使用范围从10-9到10-5米的浴缸，冲洗被定义为恢复平衡的直径。由于乙酰胆碱是一种快速反应剂，可以很容易地实现最大的扩张反应只有几秒钟。其他药物（如肽）可以花费更长的时间，引起极大的反应，这多少帐户，以及一段时间，采取有效冲刷。一旦控制的反应已收集，具体的干预措施可以对系统施加，包括船只孵化受体激动剂/拮抗剂，代理商在特定的离子通道行为，煽动改变腔内的压力，在针对特定的酶系统的平衡气体或药物制剂的差异，仅举几个最常见的干预。 干预后的适当时间是有效的（应适当测试），随后全面的数据收集可以启动。根据实验的具体协议，随后干预可以征收船只或第一，可以删除（通过冲洗 - 在可行的情况 - 或简单的生理挑战去除）。一旦船只已返回到其平衡状态（应核实），随后的干预措施，可应用于需要收集更多的数据。这是一般的程序将继续进行，直到实验终止或直到船只准备的质量低于先前确定的阈值标准（如响应激动剂或能力开发足够的活性音）。在这一点上，实验结束后，研究者可能希望收集的数据是独立的孤立船只的反应（如血管壁的力学，它可以进行使用生肌激活相同的过程，只是船只缺乏所有积极的基调14）。 </li><li>所有设备的定期清洗是必不可少的盐积聚的去除和防止任何有害生物实体的增长。在每个实验日结束，至少，完全刷新所有行和商会的PSS与蒸馏水或去离子水冲洗，并让其充分干燥。每2-3天使用后，用酒精洗净，以及执行和设备被允许完全干燥。在一贯使用的每个星期结束，所有线路和商会都装满0.1 M盐酸，允许以"孵化"30分钟，然后用清水洗净，充分如上。最后，在年底，连续使用2-3周更换新油管的所有部分，并与1.0 M盐酸清洗室和水库其次是一个广泛的蒸馏水/去离子水冲洗。如果有任何油管增长室/水库，这些被更换或适当彻底清洗油管或任何元素或视觉建立的变色。定期清洗和一般维修，设备可以持续很长一段时间。在写作时，我们的船室到8 日或9 年日常使用，我们经历了与他们没有显着的困难。 </li></ol> 5。代表结果<img alt="图1A" src="/files/ftp_upload/3674/3674fig1A.jpg" /> 图1A，这个数字呈现的基本的微血管站设置。一个=电视观看船只，B =数显卡尺，C =注射器推灌流THRough流入移液器必要，D =改变灌流流入压力的水压列，E =显微镜，F =微血管腔，G =灌流水库，H =压力显示器（可以根据需要添加多个）;我=微血管室排水泵为灌流; J =平衡气体混合物的坦克，K =废物中（灌流），L =循环热水器，M = anti-vibration/floating表。 <img alt="图1B" src="/files/ftp_upload/3674/3674fig1B.jpg" /> 图1B。这个数字提出了一个近距离观察的微血管室设置。这个数字:E =显微镜，F =微血管腔;高=压力监视器（当我们显示的简单只有一个，可以添加到多个不同地点，必要）;我=微血管室排水泵N =油管连接到一开始灌流流入移液器（图1A中的"D"的延续）;Ø=管路连接到灌流流出移液器的。 <p类"jove_content"&gt; <img alt="图1C" src="/files/ftp_upload/3674/3674fig1C.jpg" /> 图1C，这个数字呈现微血管室最近的观点。在这个数字中，n =灌流线（即，直接连接到灌流流入移液器管），O =管直接连接到灌流流出吸管;，P =流入移液器的水平和垂直位置控制，Q =灌流漏的水洗澡（图1A和1B连接&#39;我&#39;）; r =来自水库的水洗澡流入（连接到"G" 图1A）。 <img alt="图2" src="/files/ftp_upload/3674/3674fig2.jpg" /> 图2。这个数字提出了系统中的流体和气体的流动示意图。在此图中，= PSS灌流线流入，B = PSS灌流线流入滴管，C = PSS的灌流漏浴废物容器，D =<html> PSS的灌流液流出吸管流出，E =热水输入到室夹克，F =室夹克热水输出。 <img alt="图3" src="/files/ftp_upload/3674/3674fig3.jpg" /> 图3这个数字提出个人领带循环眼科缝合（通过解剖显微镜10X目镜和4.5X可调放大倍率看，A组），在我们准备使用的多个领带循环存储从8-0 10-0缝合扭转磁带上，在有盖培养皿中（以避免失去他们周围的显微镜幻灯片; B组）。 <img alt="图4" src="/files/ftp_upload/3674/3674fig4.jpg" /> 图4。这个数字提供了形象的股薄肌性动脉（通过一个标准的解剖显微镜观察）前手术的隔离和清除，允许适当的空间定位。 ve_content"&gt; <img alt="图5" src="/files/ftp_upload/3674/3674fig5.jpg" /> 图5。这个数字提供了一个空心微血管的形象代表。 A显示面板的整个长度，该船只的流入（A）和流出（二）移液器以及领带循环（c）所示。 B组高倍率图像，清楚地显示动脉内径数显卡尺（在这种情况下，直径为112微米）的决心。 <img alt="图6" src="/files/ftp_upload/3674/3674fig6.jpg" /> 图6。这个数字提出了一个空心的微血管以下平衡的代表图像。最上面的图片是控制的船只，无刺激，平衡压力的条件，中间的图像是同一船只扩张，挑战与乙酰胆碱（10 -6米的浴缸），和底部的图像，该船只是继constrictioN的挑战与福林（10-7米的浴缸）。 <img alt="图7" src="/files/ftp_upload/3674/3674fig7.jpg" /> 图7这个数字总结在响应一个孤立的微血管的挑战与回应:缺氧（面板，在我们的系统，减少在灌流灌流宝2〜135毫米汞柱〜40毫米汞柱），乙酰胆碱的浓度的增加（面板B）在血管内压力的控制条件下的变化和船只用无钙PSS的（C组）以下的孵化。

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

没有利益冲突的声明。

提出的协议描述的骨骼肌微血管隔离，清除和双插管，虽然这一般技术可以很容易地适用于大多数组织。对于当前的手稿，"动脉"一词已被用于由作者来形容一个阻力血管直径在70-120微米休息积极的基调下，这也是一个主要因素调节器官灌注阻力或之间不等组织。 一些修改，这个系统可以安装多个应用程序的具体调查，并随时可以提供更深入的实验（例如，17膜电位?...

<table border="1"><tbody><tr><td> 试剂和设备 </td><td> 公司 </td><td> 评论/目录＃ </td></tr><tr><td>船厅</td><td>习俗</td><td>戴夫Eick（都城） </td></tr><tr><td>加热循环水浴</td><td> PolyScience和哈克</td><td>哈克直流10 </td></tr><tr><td>滴管</td><td>冯检基Haer公司</td><td>毛细管2.0 mm外径×1.0 mm内径（27-33-1） </td></tr><tr><td>血压计</td><td>世界精密仪器</td><td></td></tr><tr><td>水夹套水库的</td><td>习俗</td><td></td></tr><tr><td>外部光源</td><td>世界精密仪器</td><td> NovaFlex经股</td></tr><tr><td>吸取普勒</td><td>微观仪器</td><td> PMP102 Micropipet普勒</td></tr><tr><td>全部完井MENT的手术工具</td><td>罚款科学工具</td><td>杜蒙</td></tr><tr><td>超精细镊子</td><td>罚款科学工具</td><td> INOX＃5 </td></tr><tr><td>丝绸缝合线</td><td> ethilon </td><td> ＃10-0或9-0 </td></tr><tr><td>立体显微镜</td><td>奥林巴斯</td><td>奥林巴斯SZ-11 </td></tr><tr><td>模拟视频卡钳</td><td> boeckeler </td><td>通过控制器（VIA-100） </td></tr><tr><td>高分辨率模拟摄像机</td><td>松下</td><td> GP - MF 602 </td></tr><tr><td>氧气罐</td><td>区域性</td><td>余额21％的氮和5％CO 2的平衡氮</td></tr><tr><td>管道</td><td>聚乙烯</td><td></td></tr><tr><td>排水泵</td><td>科尔帕默仪器有限公司</td><td></td></tr><tr><td>修饰大鼠的PSS </td><td>看到配方如下</td><td></TD&gt; </tr><tr><td>凡Breemen松弛的PSS </td><td>看到配方如下</td><td></td></tr></tbody></table> 表1。隔离的微血管站提出的数字设置的主要组成部分的清单。 <table border="1"><tbody><tr><td> 修改大鼠的PSS食谱 </td><td> 为了使两公升的PSS </td><td> 20X盐库存（2L） </td><td> 20X缓冲库存（2L） </td></tr><tr><td>氯化钠</td><td></td><td> 278.0Ğ </td><td></td></tr><tr><td>氯化钾</td><td></td><td> 14.0Ğ </td><td></td></tr><tr><td>硫酸镁4-7H 2 O的 </td><td></td><td> 11.5Ğ </td><td></td></tr><tr><td>氯化钙2-H 2 O </td><td></td><td> 9.4Ğ </td><td></td></tr><tr> &lt;TD&gt;碳酸氢钠3 </td><td></td><td></td><td> 80.8Ğ </td></tr><tr><td> EDTA的</td><td></td><td></td><td> 0.4Ğ </td></tr><tr><td>的NaH 2 PO 4计 </td><td> 0.28Ğ </td><td></td><td></td></tr><tr><td>葡萄糖</td><td> 1.98Ğ </td><td></td><td></td></tr><tr><td> 20倍的盐库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td> 20X缓冲库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td>蒸馏水</td><td> 1800毫升</td><td></td><td></td></tr></tbody></table> 表2。配方为标准生理盐溶液中用于孤立微血管协议"（PSS）。 食谱上的评论:2公升盐库存和缓冲库存的大号2。这些C冷藏不使用时，但动摇他们之前往往准备的PSS。额外的成分，添加在最终的PSS的准备时间。 <table border="1"><tbody><tr><td> 凡Breemen松弛的PSS </td><td> 为了使2升的PSS </td><td> 20X盐库存（1L） </td><td> 20X缓冲库存（1L） </td></tr><tr><td>氯化钠</td><td></td><td> 107.4Ğ </td><td></td></tr><tr><td>氯化钾</td><td></td><td> 7.0Ğ </td><td></td></tr><tr><td>硫酸镁4-7H 2 O的 </td><td></td><td> 5.76Ğ </td><td></td></tr><tr><td>氯化镁2-6H 2 </td><td></td><td> 81.32Ğ </td><td></td></tr><tr><td> 碳酸氢钠 </td><td></td><td></td><td> 40.4Ğ </td></tr><tr><td>美东时间一</td><td></td><td></td><td> 0.2Ğ </td></tr><tr><td> EGTA </td><td></td><td></td><td> 15.22 </td></tr><tr><td>的NaH 2 PO 4计 </td><td> 0.28Ğ </td><td></td><td></td></tr><tr><td>葡萄糖</td><td> 1.98Ğ </td><td></td><td></td></tr><tr><td> 20倍的盐库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td> 20X缓冲库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td>蒸馏水</td><td> 1800毫升</td><td></td><td></td></tr></tbody></table> 表3。配方范Breemen松弛生理盐溶液（PSS），在孤立的微血管协议的零积极音的条件下使用。 食谱上的评论:1 L一盐库存ND 1升的缓冲库存。不使用时，可以冷藏，但动摇他们之前往往准备的PSS。额外的成分，添加在最后松弛的PSS的准备时间。

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

隔离的微血管准备是体外制备，允许考试因素的控制血管直径的不同贡献，因此，灌注阻力1-5。这是一个经典的实验准备，在很大程度上，最初是由内田等 15，几十年以前。这最初的描述提供了进行了广泛的修改和提高，主要是在实验室的布赖恩博士杜陵在弗吉尼亚6-8大学，技术的基础上，我们提出了电流的方法，在下面的网页。这种准备，具体是指在大鼠作为首选微血管纤细的动脉，但基本的准备，可以很容易地应用到几乎任何其他组织或器官9-13跨物种隔离的船只。机械（即二维）在孤立的微血管的变化，可以很容易地评估在浩如烟海的生理（例如，缺氧，血管内的压力，或剪切）或药理的挑战，并能提供洞察到机械元素组成一个完整的综合措施，虽然体外 ，组织响应。这种方法的意义是，它允许简便操作的影响微血管直径的综合调控，同时也让许多从其他来源，包括血管内压力（生肌），自主支配，血流动力学的贡献控制（例如，剪切应力），内皮依赖或独立的刺激，激素，实质影响，提供了部分清单。在适当的实验条件和适当的目标，这可以作为一个优势在体内或原位组织/器官的筹备工作，不轻易让浅显的控制更广泛的系统性变量。 马约旦基本上是该制剂的限制其优势的结果。根据定义，这些船只的行为被许多最重要的贡献者调节血管阻力已被删除，包括神经，体液免疫，代谢，因此等的条件下，研究，研究者应注意避免过度所收集的数据利用这个准备的解释和推断。该制剂方面关心的其他重要领域，它可以很容易损坏，如内皮衬里或血管平滑肌细胞成分，如变量的错误源可以被引入。强烈建议，个别研究者利用，以确保该制剂的质量，无论是在实验开始，并定期在整个协议的过程中适当的测量。

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

一个<em&gt;体外</em&gt;编制说明两个血管反应性血管活性物质的刺激和通过被动墙力学的基本结构性能评估的审讯股薄肌的最大阻力小动脉的隔离。

"体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

the-ex-vivo-isolated-skeletal-microvessel-preparation-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

10.6K 次观看.  West Virginia University. 一个体外编制说明两个血管反应性血管活性物质的刺激和通过被动墙力学的基本结构性能评估的审讯股薄肌的最大阻力小动脉的隔离。

视频: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

科研

生物工程

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

步骤人类隐静脉<em&gt;前体内</em&gt;灌注和外部强化

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

医学

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

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 ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

神经科学

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

在五个脊椎动物群中可靠隔离中枢神经系统微血管

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

生物学

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

"体外骨骼肌微血管制备离体血管反应性的调查

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