右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获
Özet
一个特异，快速的协议，同时探讨合适的心脏功能，肺部炎症和免疫反应，被描述为一种学习工具。视频和数字，描述的生理和显微切割技术在组织团队的方法，是适用于用于小型到大型研究。
Abstract
正确的心脏的功能是把血液通过肺部，从而将右心脏的生理和肺血管生理。炎症的心脏和肺的功能，是一种常见的改性剂，通过制定生产的细胞因子和生长因子，细胞浸润，并开始重塑过程1。
右心室与左心室相比，是一个低压泵工作在一个相对窄的区域的压力变化。增加与肺动脉压力增加的压力在肺血管床和肺动脉高压2。肺动脉高压往往伴有炎症性肺部疾病，如慢性阻塞性肺疾病，自身免疫性疾病3。由于肺动脉高压赋予预后不良的生活质量和寿命，大量的研究是针对了解的机制，mig的HT药物干预4的目标。有效的管理手段，为肺动脉高压的发展面临的主要挑战仍然是复杂的分子和细胞变化在权利的心，肺和免疫系统的同时了解。
在这里，我们提出了一个程序的工作流程，快速，精确的测量压力变化在正确的小鼠心脏的心脏，肺和免疫组织样本的同时收获。该方法是基于直接导管右心室，通过颈内静脉接近上身的小鼠，最早是在20世纪90年代后期，作为替代措施的压力在肺动脉5-13。组织团队的方式有利于快速右心导管检查技术。这使得能够进行测量的小鼠的自发呼吸室内空气。在不同的工作领域的组织的工作流程减少了时间延迟和打开的可能性，同时进行生理实验和收获的免疫系统，心脏和肺组织。
这里列出的程序的工作流程，可以适应各种各样的实验室设置和研究设计，从小型的，有针对性的实验，到大型药物筛选试验。同时采集心肌的生理数据，可以扩展到包括超声心动图5,14-17和收获的心脏，肺和免疫组织减少了需要获得数据的科学知识为基础向前移动的动物。这里介绍的程序的工作流程也提供了理想的基础免疫，肺和心脏功能的网络链接获取知识。这里概述的相同的原则可以适用于研究根据需要的其它的或附加的器官。
Protocol
1。准备准备以下的解决方案和管（ 表1）如下： Hanks溶液，无钙，镁或指示器，与青霉素（100单位/毫升）/链霉素（100毫克/毫升）。 磷酸盐缓冲盐水（PBS），1个，无钙，无镁。 乙醇，70％，500毫升。 与PBS缓冲甲醛，7％至10％，使500毫升。 麻醉科的解决方案： 圣阿韦坦。小心地加入2 – 甲基-2 – 丁醇的5毫升至5克的2,2,2 – 三溴乙醇。一?…
Representative Results
获得合适的心脏压力曲线的主要结果是通过合适的心脏导管的正确位置。右心室的导管内部的正确放置的压力时间曲线的形状是重要的，因为会导致压力高原（ 图4）。相反，高低不平的曲线，表明由呼吸或心脏跳动的运动靠在墙上的右心室导管移动。为了检测的阶段的动物的生存的潜在的问题，需要计算ΔP，和心脏的速度的标准偏差。预计这两个值都没有显着不同的治疗组之间。此…
Discussion
此处列出的实验流程，允许快速和同时测量右心室收缩压和收获的样品分析的反应在肺部，心脏和免疫系统的小鼠。该过程结合心脏生理学测量，微解剖和随后的组织为活细胞研究，组织学分析，或组学分析的组织收获。的完整过程只需不到20分钟，每鼠标。由于工作区域组织的工作流程，2-3的动物可以同时进行研究。因此，程序是适用于小的，有针对性的实验12和设置在很宽的范围内进?…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家机构的健康1R21HL092370-01（GG），1R01 HL095764-01（GG）; R01HL082694（JW），美国心脏协会的创始人联属公司（0855943D，GG），石溪世界 – 赫伯特基金，纽约（SHP）。
Materials
| Name | Company | Catalogue number | Comments (optional) |
| Reagents | |||
| 2-Methyl-2-butanol | Sigma-Aldrich | 152463 | |
| 2,2,2-Tribromoethanol | Sigma-Aldrich | T48402 | |
| disinfectant soap (Coverage Spray TB plus Steris) | Fisher Scientific | 1629-08 | |
| Ethyl Alcohol, 200 Proof, Absolute, Anhydrous ACS/USP Grade | PHARMCO-AAPER | 111000200 | Dilute to 70 % with distilled water |
| Formaldehyde solution | Sigma-Aldrich | F1635-500ML | Dilute to a 7-10 % formaldehyde concentration at a PBS concentration of 1x using PBS stock solution and water |
| Hanks solution, no calcium, magnesium | Fisher Scientific | 21-022-CV | |
| O.C.T | Tissue-Tek | 4583 | |
| Penicillin (10,000 U/ml) / Streptomycin (10,000 mg/ml) solution | Thermo Scientific | SV30010 | |
| Phosphate buffered saline (PBS), no calcium, no magnesium, 1x and 10x solutions | Fisher Scientific | ||
| Sodium pentobarbital 26% | Fort Dodge Animal Health | NDC 0856-0471-01 | |
| Labware | |||
| Plates 12, 24, 96 well | Falcon | ||
| Transfer Pipet | Fisher Scientific | 13-711-9BM | |
| Tube, EDTA coated | Sarstedt | 2013-08 | |
| Tubes 0.65 ml and 1.7 ml micro-centrifuge | VWR | ||
| Tubes 12 x 75 mm polypropylene | Fisher Scientific | 14-956-1D | |
| Tubes, various sizes, polypropylene | Fisher Scientific | ||
| Instruments | |||
| Forceps, Dumon #5 Fine | Fine Science Tools | 11254-20 | |
| Forceps, extra fine graefe -0.5 mm tips curved | Fine Science Tools | 11152-10 | |
| Forceps, extra fine graefe -0.5 mm tips straight | Fine Science Tools | 11150-10 | |
| Cannula 18 ga, 19 ga | BD | Precision Glide Needles | Cut to optimal length, blunted and outside rasped to create a rough outside surface. |
| Scissors, Dissector scissors-slim blades 9 cm | Fine Science Tools | 14081-09 | |
| Suture for BAL, braided silk suture, 4-0 | Fine Science Tools | SP116 | |
| Suture for right heart catheterization, braided silk suture, 6-0 | Teleflex medical | 18020-60 | |
| Syringe, 1 ml | BD | 309659 | |
| Equipment | |||
| Amplifier, PowerLab 4/30 | ADInstrument | Model ML866 | |
| Catheter, pressure F1.4 | Millar Instruments, Inc | 840-6719 | |
| Dissecting Microscope | Variscope | ||
| Forceps, Vannas spring scissors-2 mm blades | Fine Science Tools | 15000-00 | |
| Halogen Illuminated Desk Magnifier | Fisher Scientific | 11-990-56 | |
| Laptop computer | Asus | Model number A52F i5 processor; 15 inch | |
| Light Source | Amscope | HL-250-A | |
| Pressure Control Unit | Millar Instruments, Inc | PCU-2000 | |
| Software, Labchart-Pro V.7 | AD Instruments |
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Bu Makaleden Alıntı Yapın
Chen, W., Park, S., Hoffman, C., Philip, C., Robinson, L., West, J., Grunig, G. Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice. J. Vis. Exp. (71), e50023, doi:10.3791/50023 (2013).