JoVE Journal > Developmental Biology
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
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Biologie du développement

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Published: June 20, 2020
doi:
10.3791/61242
, , , , , , ,
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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References

  1. Vasquez, S. X., et al. Optimization of microCT imaging and blood vessel diameter quantitation of preclinical specimen vasculature with radiopaque polymer injection medium. PLoS One. 6 (4), 19099 (2011).
  2. Hong, S. H., et al. Development of barium-based low viscosity contrast agents for micro CT vascular casting: Application to 3D visualization of the adult mouse cerebrovasculature. Journal of Neuroscience Research. 98 (2), 312-324 (2019).
  3. Perrien, D. S., et al. Novel methods for microCT-based analyses of vasculature in the renal cortex reveal a loss of perfusable arterioles and glomeruli in eNOS-/- mice. BMC Nephrology. 17, 24 (2016).
  4. Weyers, J. J., Carlson, D. D., Murry, C. E., Schwartz, S. M., Mahoney, W. M. Retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging. Journal of Visualized Experiments. (60), e3740 (2012).
  5. Zhang, H., Faber, J. E. De-novo collateral formation following acute myocardial infarction: Dependence on CCR2(+) bone marrow cells. Journal of Molecular and Cellular Cardiology. 87, 4-16 (2015).
  6. Kim, B. G., et al. CXCL12-CXCR4 signalling plays an essential role in proper patterning of aortic arch and pulmonary arteries. Cardiovascular Research. 113 (13), 1677-1687 (2017).
  7. Counter, W. B., Wang, I. Q., Farncombe, T. H., Labiris, N. R. Airway and pulmonary vascular measurements using contrast-enhanced micro-CT in rodents. American Journal of Physiology Lung Cellular and Molecular Physiology. 304 (12), 831-843 (2013).
  8. Phillips, M. R., et al. A method for evaluating the murine pulmonary vasculature using micro-computed tomography. Journal of Surgical Research. 207, 115-122 (2017).
  9. Schuster, D. P., Kovacs, A., Garbow, J., Piwnica-Worms, D. Recent advances in imaging the lungs of intact small animals. American Journal of Respiratory Cell and Molecular Biology. 30 (2), 129-138 (2004).
  10. Samarage, C. R., et al. Technical Note: Contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source. Medical Physics. 43 (11), 6017 (2016).
  11. Grothausmann, R., Knudsen, L., Ochs, M., Muhlfeld, C. Digital 3D reconstructions using histological serial sections of lung tissue including the alveolar capillary network. American Journal of Physiology Lung Cellular and Molecular Physiology. 312 (2), 243-257 (2017).
  12. Hayworth, K. J., et al. Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits. Front Neural Circuits. 8, 68 (2014).
  13. Bussolati, G., Marchio, C., Volante, M. Tissue arrays as fiducial markers for section alignment in 3-D reconstruction technology. Journal of Cellular and Molecular Medicine. 9 (2), 438-445 (2005).
  14. Preissner, M., et al. Application of a novel in vivo imaging approach to measure pulmonary vascular responses in mice. Physiological Reports. 6 (19), 13875 (2018).
  15. Junaid, T. O., Bradley, R. S., Lewis, R. M., Aplin, J. D., Johnstone, E. D. Whole organ vascular casting and microCT examination of the human placental vascular tree reveals novel alterations associated with pregnancy disease. Scientific Reports. 7 (1), 4144 (2017).
  16. Bolender, R. P., Hyde, D. M., Dehoff, R. T. Lung morphometry: a new generation of tools and experiments for organ, tissue, cell, and molecular biology. American Journal of Physiology. 265 (6), 521-548 (1993).
  17. Savai, R., et al. Evaluation of angiogenesis using micro-computed tomography in a xenograft mouse model of lung cancer. Neoplasia. 11 (1), 48-56 (2009).
  18. Ehling, J., et al. Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization. American Journal of Pathology. 184 (2), 431-441 (2014).
  19. Sueyoshi, R., Ralls, M. W., Teitelbaum, D. H. Glucagon-like peptide 2 increases efficacy of distraction enterogenesis. Journal of Surgical Research. 184 (1), 365-373 (2013).
  20. Zhang, H., Jin, B., Faber, J. E. Mouse models of Alzheimer’s disease cause rarefaction of pial collaterals and increased severity of ischemic stroke. Angiogenesis. 22 (2), 263-279 (2019).
  21. Faight, E. M., et al. MicroCT analysis of vascular morphometry: a comparison of right lung lobes in the SUGEN/hypoxic rat model of pulmonary arterial hypertension. Pulmonary Circulation. 7 (2), 522-530 (2017).
  22. Fisher, S., Burgess, W. L., Hines, K. D., Mason, G. L., Owiny, J. R. Interstrain Differences in CO2-Induced Pulmonary Hemorrhage in Mice. Journal of the American Association for Laboratory Animal Science. 55 (6), 811-815 (2016).
  23. Munce, N. R., et al. Intravascular and extravascular microvessel formation in chronic total occlusions a micro-CT imaging study. JACC Cardiovascular Imaging. 3 (8), 797-805 (2010).
  24. Shifren, A., Durmowicz, A. G., Knutsen, R. H., Faury, G., Mecham, R. P. Elastin insufficiency predisposes to elevated pulmonary circulatory pressures through changes in elastic artery structure. Journal of Applied Physiology. 105 (5), 1610-1619 (2008).
  25. Sonobe, T., et al. Imaging of the closed-chest mouse pulmonary circulation using synchrotron radiation microangiography. Journal of Applied Physiology (1985). 111 (1), 75-80 (2011).
  26. Ritman, E. L. Micro-computed tomography of the lungs and pulmonary-vascular system. Proceedings of the American Thoracic Society. 2 (6), 477-480 (2005).
  27. Dinkel, J., et al. Intrinsic gating for small-animal computed tomography: a robust ECG-less paradigm for deriving cardiac phase information and functional imaging. Circulation: Cardiovascular Imaging. 1 (3), 235-243 (2008).
  28. Ashton, J. R., West, J. L., Badea, C. T. In vivo small animal micro-CT using nanoparticle contrast agents. Frontiers in Pharmacology. 6, 256 (2015).
  29. Ford, N. L., Thornton, M. M., Holdsworth, D. W. Fundamental image quality limits for microcomputed tomography in small animals. Medical Physics. 30 (11), 2869-2877 (2003).
  30. Boone, J. M., Velazquez, O., Cherry, S. R. Small-animal X-ray dose from micro-CT. Molecular Imaging. 3 (3), 149-158 (2004).
  31. Giuvarasteanu, I. Scanning electron microscopy of vascular corrosion casts–standard method for studying microvessels. Romanian Journal of Morphology and Embryology. 48 (3), 257-261 (2007).
  32. Polguj, M., et al. Quality and quantity comparison study of corrosion casts of bovine testis made using two synthetic kits: Plastogen G and Batson no 17. Folia Morphologica (Warsz). 78 (3), 487-493 (2019).
  33. Verli, F. D., Rossi-Schneider, T. R., Schneider, F. L., Yurgel, L. S., de Souza, M. A. Vascular corrosion casting technique steps. Scanning. 29 (3), 128-132 (2007).
  34. Azaripour, A., et al. A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue. Progress in Histochemistry and Cytochemistry. 51 (2), 9-23 (2016).
  35. Richardson, D. S., Lichtman, J. W. Clarifying Tissue Clearing. Cell. 162 (2), 246-257 (2015).
  36. Albers, J., Markus, M. A., Alves, F., Dullin, C. X-ray based virtual histology allows guided sectioning of heavy ion stained murine lungs for histological analysis. Scientific Reports. 8 (1), 7712 (2018).
  37. Katsamenis, O. L., et al. X-ray Micro-Computed Tomography for Nondestructive Three-Dimensional (3D) X-ray Histology. American Journal of Pathology. 189 (8), 1608-1620 (2019).
  38. Morales, A. G., et al. Micro-CT scouting for transmission electron microscopy of human tissue specimens. Journal of Microscopy. 263 (1), 113-117 (2016).
  39. Wen, H., et al. Correlative Detection of Isolated Single and Multi-Cellular Calcifications in the Internal Elastic Lamina of Human Coronary Artery Samples. Scientific Reports. 8 (1), 10978 (2018).
  40. Zamir, A., et al. Robust phase retrieval for high resolution edge illumination x-ray phase-contrast computed tomography in non-ideal environments. Scientific Reports. 6, 31197 (2016).
  41. Yu, B., et al. Evaluation of phase retrieval approaches in magnified X-ray phase nano computerized tomography applied to bone tissue. Optics Express. 26 (9), 11110-11124 (2018).
  42. Bidola, P., et al. Application of sensitive, high-resolution imaging at a commercial lab-based X-ray micro-CT system using propagation-based phase retrieval. Journal of Microscopy. 266 (2), 211-220 (2017).
  43. Norvik, C., et al. Synchrotron-based phase-contrast micro-CT as a tool for understanding pulmonary vascular pathobiology and the 3-D microanatomy of alveolar capillary dysplasia. American Journal of Physiology Lung Cellular and Molecular Physiology. 318 (1), 65-75 (2020).
  44. Weibel, E. R. Lung morphometry: the link between structure and function. Cell and Tissue Research. 367 (3), 413-426 (2017).
  45. Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R. An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure. American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).
  46. Sarhaddi, D., et al. Validation of Histologic Bone Analysis Following Microfil Vessel Perfusion. Journal of Histotechnology. 35 (4), 180-183 (2012).
  47. Ehling, J., et al. Quantitative Micro-Computed Tomography Imaging of Vascular Dysfunction in Progressive Kidney Diseases. Journal of the American Society of Nephrology. 27 (2), 520-532 (2016).

Tags

Vascular CastingMicro-CT ImagingPulmonary Arterial VasculatureAdult MousePostnatal MouseVascular MorphologyVascular ArchitecturePE10 TubingSodium NitroprussideSilk SutureRight VentriclePulmonary Artery Trunk
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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).
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