用于微CT成像的成人和早期产后小鼠肺的血管铸造
1Translational Vascular Medicine Branch, National Heart Lung and Blood Institute,National Institutes of Health, 2Mouse Imaging Facility, National Institute of Neurological Disorders and Stroke,National Institutes of Health, 3Division of Pediatric Surgery, Department of Surgery,University of North Carolina at Chapel Hill
Summary
该技术的目的是通过肺膨胀和通过肺动脉注射放射性不透明聚合物化合物，对早期产后小鼠和成年小鼠的肺动脉网络进行活体可视化。还讨论了铸造组织的潜在应用。
Abstract
血管在三维空间中形成复杂的网络。因此，很难通过观察组织表面来直观地理解血管网络是如何相互作用和表现的。该方法提供了一种可视化肺部复杂三维血管结构的方法。
为此，导管插入肺动脉，血管同时冲洗血液和化学扩张，以限制阻力。然后，肺以标准压力通过气管膨胀，聚合物化合物以标准流速注入血管床。一旦整个动脉网络被填充并允许治愈，肺血管可以直接可视化或在微CT （μCT） 扫描仪上成像。
当手术成功时，可以欣赏从产后早期到成人的小鼠肺动脉网络。此外，在肺动脉床中演示时，此方法可应用于任何具有优化导管放置和端点的血管床。
Introduction
该技术的重点是使用小鼠聚合物化合物的肺动脉结构可视化。虽然在脑、心脏,和肾脏,1、2、3、4、5,2,3等全身血管病床上进行了广泛的工作，但有关肺动脉网络的准备和填充的信息较少。4因此，本研究的目的是扩展先前的工作6，7，8，7,8并提供详细的书面和视觉参考，调查人员可以很容易地遵循，以产生肺动脉树的高分辨率图像。
虽然有许多方法存在标记和成像肺血管，如磁共振成像，超声心动图，或CT血管造影9,9，10，其中许多模式未能充分填充和/或捕获小血管，限制了可以研究的范围。串行分段和重建等方法提供高分辨率，但时间/劳动密集型11，12，13。,1311,在传统的腐蚀铸造10、13、14、15、16中10,13,14，周围的软组织,完整性受到损害。甚至动物的年龄和大小也成为因素，当试图引入导管或，分辨率是缺乏的。另一方面，聚合物注射技术将动脉填充到毛细管水平，当与 +CT 结合使用时，可实现无与伦比的分辨率 5。从小到产后第14天的小鼠肺部样本已成功施用8个，并在几个小时内得到处理。这些可以无限期地重新扫描，甚至发送组织学准备/电子显微镜（EM），而不损害现有的软组织17。此方法的主要限制是 CT 设备/软件的前期成本、准确监测血管内压力方面的挑战，以及无法在同一动物中纵向获取数据。
本文以现有工作为根据，进一步优化肺动脉注射技术，将年龄/尺寸相关边界推至产后第1天（P1），取得显著效果。对于想要研究动脉血管网络的团队来说，这是最有用的。因此，我们为导管放置/稳定提供新的指导，加强对填充率/体积的控制，并突出提高铸造成功率的显著缺陷。然后，结果铸件可用于未来的表征和形态分析。也许更重要的是，据我们所知，这是引导用户完成这个复杂过程的第一个视觉演示。
Protocol
此处描述的所有方法都已获得国家心肺和血液研究所的机构动物护理和使用委员会 （ACUC） 的批准。 1. 准备 将小鼠内向注入肝素（1单位/克小鼠体重），并允许其调节2分钟。 在CO2室中对动物实施安乐死 。 将鼠标放在手术板上的超前位置，用胶带将四肢固定到手术板上。使用放大镜进行精细解剖。 2. 暴露肺部和气管 …
Representative Results
一个成功的演员将展示整个肺动脉网络的均匀填充。我们在C57Bl/6J小鼠中演示了这一点：产后一天P90（图4A）、P30（图4B）、P7（图4C）和P1（图4D）。通过控制流量和目视监测填充的实时，最远程血管的可靠终点实现了（图5A）。 <p class="jove_content" fo:keep-together…
Discussion
正确执行，这种方法产生肺动脉网络的惊人图像，允许在啮齿动物模型中进行比较和实验。沿途的几个关键步骤确保了成功。首先，调查人员必须在准备阶段对动物进行肝素化，以防止血块在肺血管和心脏腔室中形成。这允许聚合物化合物的完全动脉传递。其次，当刺穿隔膜和去除肋骨时，注意保护肺部免受意外伤害、割伤或伤害。气道中的任何泄漏都会防止完全膨胀，使样品之间的比较不准确?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究部分得到了NHLBI校内研究计划（DIR HL-006247）的支持。我们要感谢NIH鼠标成像设施在图像采集和分析方面的指导。
Materials
| 1cc syringe | Becton Dickinson | 309659 | |
| 20ml Glass Scintillation Vials | Fisher | 03-340-25P | |
| 30G Needle | Becton Dickinson | 305106 | |
| 50mL conical tubes | Cornin | 352098 | For sample Storage and scanning |
| 60cc syringe | Becton Dickinson | 309653 | |
| 7-0 silk suture | Teleflex | 103-S | |
| Analyze 12.0 Software | AnalyzeDirect Inc. | N/A | Primary Software |
| Amira 6.7 Software | Thermo Scientific | N/A | Alternative Sofware |
| CeramaCut Scissors 9cm | Fine Science tools | 14958-09 | |
| Ceramic Coated Curved Forceps | Fine Science tools | 11272-50 | |
| CO2 Tank | Robert's Oxygen Co. | n/a | |
| Dual syringe pump | Cole Parmer | EW-74900-10 | |
| Dumont Mini-Forceps | Fine Science tools | 11200-14 | |
| Ethanol | Pharmco | 111000200 | |
| Formalin | Sigma – Life Sciences | HT501128 | |
| Gauze | Covidien | 441215 | |
| Hemostat | Fine Science tools | 13013-14 | |
| Heparin (1000USP Units/ml) | Hospira | NDC 0409-2720-01 | |
| Horos Software | Horos Project | N/A | Alternative Sofware |
| induction chamber | n/a | n/a | |
| Kimwipe | Fisher | 06-666 | fiber optic cleaning wipe |
| Labelling Tape | Fisher | 15966 | |
| Magnetic Base | Kanetec | N/A | |
| Micro-CT system | SkyScan | 1172 | |
| Microfil (Polymer Compound) | Flowech Inc. | Kit B – MV-122 | 8 oz. of MV compound; 8 oz. of diluent; MV-Curing Agent |
| Micromanipulator | Stoelting | 56131 | |
| Monoject 1/2 ml Insulin Syringe | Covidien | 1188528012 | |
| Octagon Forceps Straight Teeth | Fine Science tools | 11042-08 | |
| Parafilm | Bemis company, Inc. | #PM999 | |
| PE-10 tubing | Instech | BTPE-10 | |
| Phospahte buffered Saline | BioRad | #161-0780 | |
| Ring Stand | Fisher | S13747 | Height 24in. |
| Sodium Nitroprusside | sigma | 71778-25G | |
| Steel Plate | N/A | N/A | 16 x 16 in. area, 1/16 in thick |
| Straight Spring Scissors | Fine Science tools | 15000-08 | |
| SURFLO 24G Teflon I.V. Catheter | Santa Cruz Biotechnology | 360103 | |
| Surgical Board | Fisher | 12-587-20 | This is a converted slide holder |
| Universal 3-prong clamp | Fisher | S24280 | |
| Winged Inf. Set 25X3/4, 12" Tubing | Nipro | PR25G19 | |
| Zeiss Stemi-508 Dissection Scope | Zeiss | n/a |
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Citer Cet Article
Knutsen, R. H., Gober, L. M., Sukinik, J. R., Donahue, D. R., Kronquist, E. K., Levin, M. D., McLean, S. E., Kozel, B. A. Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging. J. Vis. Exp. (160), e61242, doi:10.3791/61242 (2020).