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Bioingegneria

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

Published: April 09, 2018
doi:
10.3791/57451
, , ,
1Department of Cardiovascular and Renal Research, Institute of Molecular Medicine,University of Southern Denmark, 2Department of Biochemistry and Molecular Biology,University of Southern Denmark, 3Department of Cardiac, Thoracic and Vascular Surgery,Odense University Hospital

Summary

我们描述同时进行的机械测试和 3 d 成像的动脉壁的孤立, 活着的人的抵抗动脉, 斐济和 Ilastik 图像分析, 以量化的弹性蛋白和胶原的空间组织和体积密度。我们讨论了这些数据在动脉壁力学数学模型中的应用。

Abstract

在原发性高血压、糖尿病和代谢综合征中, 抗血管重塑的致病作用有记录。microstructurally 动机的研究与发展了解人体阻力动脉力学性能的数学模型在健康和疾病方面有可能帮助理解疾病和医疗治疗影响人体微循环。为了开发这些数学模型, 必须对微血管壁的力学和 microarchitectural 特性之间的关系进行解密。在这项工作中, 我们描述了一个前体方法进行被动的机械测试和同时无标签的三维成像的弹性体和胶原质的微结构的动脉壁上的孤立的人的阻力动脉。成像协议可应用于任何感兴趣物种的抗性动脉。图像分析描述量化 i) 压力诱发变化的内部弹性板分枝角和血管外膜胶原直线度使用斐济和 ii) 胶原蛋白和弹力蛋白体积密度确定使用 Ilastik 软件。最好所有的机械和影像测量都是在活的, 灌注的动脉, 但是, 一个替代方法使用标准的视频显微镜压力 myography 结合后固定成像的再加压血管是讨论。此替代方法为用户提供了不同的分析方法选项。讨论了机械和成像数据在动脉壁力学数学模型中的包含, 并提出了该协议的未来发展和补充。

Introduction

原发性高血压、糖尿病和代谢综合征的致病作用和抗性动脉重塑的作用记录在1,2,3,4,5。破译微血管壁的力学和 microarchitectural 特性之间的关系对于发展该协会的数学模型是必不可少的。这些模型将改善理解的重塑过程, 并将支持在硅模型的发展, 有助于测试的药理策略针对与疾病相关的重建的动脉壁。

先前的研究集中于了解动脉壁微结构与动脉壁力学的关系, 包括机械措施和细胞外基质 (ECM) 的微体系结构, 几乎完全是在大, 从小鼠或猪的弹性导管动脉6,7,8,9,10,11。通常采用非线性光学技术, 利用弹性蛋白的荧光性和胶原蛋白的二次谐波产生, 对墙体的显微组织进行成像。这允许时空成像的两个主要组成部分的细胞外基质, 弹性蛋白和胶原蛋白, 不需要染色。在厚膜介质中, 由于光的散射, 全厚度动脉壁的成像是大导管动脉的一个挑战。然而, 为了确定动脉壁结构成分的微体系与观测到的力学性能有关, 必须在机械测试过程中获得三维信息。对于像人主动脉这样的大动脉, 这需要双轴安装, 机械测试和成像区域的兴趣在 1-2 cm2块的动脉壁7,9,10,12. 只有部分墙可以进行成像和机械测试。

对于任何种类的小动脉 (例如, 人心包13, 肺14和皮下15动脉, 大鼠肠系膜动脉16,17,18 ,19, 20, 小鼠提睾肌, 肠系膜, 脑, 股骨和颈动脉21, 22, 23, 24, 25, 26,27) 整个壁厚的成像是可能的, 可以与机械测试相结合。这允许同时记录的机械性能和结构安排在墙内。然而, 一个直接的数学模型的关系, 观察改变的三维结构的 ECM 和改变的力学性质的阻力动脉壁, 有最好的知识, 只有报告后,最近在人体阻力动脉13,15。

在这项工作中, 介绍了一种用于被动机械测试的前体方法, 以及在隔离的人体阻力动脉的动脉壁上, 弹性蛋白和胶原质微结构的同时三维成像。成像协议可应用于任何感兴趣物种的抗性动脉。本文介绍了利用斐济28对内弹性板分枝角和血管外膜胶原直线度13 进行图像分析的方法。用 Ilastik 软件29确定胶原蛋白和弹力蛋白的体积密度, 最后讨论了在动脉壁力学数学模型中包含的机械和成像数据。

描述成像和图像分析技术与数学建模相结合的目标是为研究者提供一个系统的方法来描述和理解观察到的压力引起的电阻动脉 ECM 的变化。所述方法主要是通过比较20、40和 100 mmHg 的 ecm 结构, 在加压过程中对容器中 ecm 的变化进行量化。这些压力的选择, 以确定的动脉壁的结构, 其更符合 (20 mmHg), 僵硬 (100 mmHg) 和中间 (40 mmHg) 状态, 分别。然而, 根据调查者所提出的研究假说, 活动脉血管壁的任何过程, 包括血管活性成分、迟滞和流引起的变化都可以量化。

强调了双光子激发荧光显微术 (TPEM) 与压力 myograph 的结合, 以研究压力 (或其他) 诱发活动脉 ECM 的变化。首先, 因为这允许同时获得的整体三维结构的动脉壁 (直径和壁厚) 连同三维无标签获得高质量的, 详细的胶原蛋白和弹力蛋白图像microarchitectures 如描述的13通过利用弹性蛋白自发荧光和胶原第二谐波产生信号 (SHG)30。第二, TPEM 允许使用低能量近红外激发光, 尽量减少组织的光损伤, 因此, 允许在血管壁内完全相同的位置重复成像, 容许重复测量分析观察变化。

本文讨论了一种利用压力固定动脉共焦成像的替代方法, 使用户没有机会 TPEM 使用所描述的方法。关于 ECM 结构和体积密度的信息也可以从对连续切片的二维组织分析中检索出来, 如31、32所述。然而, 由于缺乏可能检索三维结构信息在动脉的长度刻度以及在变化的情况下使用这种方法, 它不建议使用这种方法来调查压力和治疗引起的三维变化的 ECM。

研究人员对应用本法所述方法的最低要求是, 结合共焦或双光子激发荧光显微镜, 获得动脉插管和加压的设置。在下面的协议中描述的设置是一个定制的压力 myograph 与纵向力传感器, 建立适合于一个定制的倒置双光子励磁荧光显微镜。

Protocol

本工作中使用的人顶叶心包活检的收集是在书面知情同意后进行的, 如前所述的33。对人体组织的研究符合《赫尔辛基宣言》34中概述的原则, 并得到了丹麦南部 (S-20100044 和 S-20140202) 和丹麦数据保护局的卫生研究伦理学区域委员会的批准。 1. 收集组织和隔离 (人) 抵抗动脉 在手术过程中立即收集感兴趣的组织样本。将组织转移到4°c HE…

Representative Results

此工作中使用的用于成像的自定义压力 myograph 显示在图 1中。特别注意为 myograph 的设计被支付了对 i) 房间以小容量 (2 毫升) 和 ii) 可能性安置套管接近, 并且平行与玻璃底部 (图 1B)。房间的底部适合 50 x 24 毫米 #1. 5 玻璃盖玻片 (可更换)。该压力控制器是由标准的1升玻璃瓶和血压计 (袖口删除)。对于具有流量要求的 myograph 设置,…

Discussion

这项工作代表我们建议的标准化, 联合成像和压力 myography 方法, 有价值的同时评估的力学性质的阻力动脉和压力相关变化的动脉结构墙壁在压力范围从0到 100 mmHg。提出的方法是使用定制的设备开发的, 但是, 当两种设备的设计允许动脉壁的成像时, 任何适合于双光子激发荧光显微镜的压力 myograph 都可以使用。应特别注意目标的形状、宽度和工作距离, 以及成像将执行的条件。直立显微镜要求使用水…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者感谢丹麦南部大学自然科学系的丹麦分子生物医学成像中心, 用于实验室和显微镜的使用。kris 专职 Rosenstand 和乌拉梅尔基奥是公认的优秀技术援助与压力 myography 和成像。

Materials

Fine Science Tools 15401-12
Fine Science Tools 11251-23
Nikon SMZ800N
 Sigma-Aldrich, Brøndby, Denmark. 761028 for dissection purpose
Vitrex Medical A/S, Herlev, Denmark 1.63, 2.13, 210mm
Smiths medical Intl, UK
Ethicon Ethilon 11-0
Custom built DK patent number 201200167, University of Southern Denmark, J. Schoubo V. Jensen, F. Jensen. T.R. Uhrenholt
Mettler toledo
 Sigma-Aldrich, Brøndby, Denmark. B3259
 Sigma-Aldrich, Brøndby, Denmark. A7030
 Sigma-Aldrich, Brøndby, Denmark. C5670
 Sigma-Aldrich, Brøndby, Denmark. G7021
 Sigma-Aldrich, Brøndby, Denmark. E3889
Merck Millipore, Hellerup, Denmark 1.00496.9010 Phosphate buffered (pH 6.9) 4% formaldehyde solution 
 Sigma-Aldrich, Brøndby, Denmark. H3784
 Sigma-Aldrich, Brøndby, Denmark. P9666
 Sigma-Aldrich, Brøndby, Denmark. P5655
 Sigma-Aldrich, Brøndby, Denmark. M2643
 Sigma-Aldrich, Brøndby, Denmark. S2002
 Sigma-Aldrich, Brøndby, Denmark. S5886
 Sigma-Aldrich, Brøndby, Denmark. S5761
 Sigma-Aldrich, Brøndby, Denmark. 1.06462
Gibco, ThermoFisher Scientific 10010015
 Sigma-Aldrich, Brøndby, Denmark. PHR1423
 Sigma-Aldrich, Brøndby, Denmark. Z370525
 Tocris Bioscience, Bristol, UK 538944
Nikon Custom built
Spectra Physics, Mountain View, CA
Nikon CFI Plan Apo IR SR 60XWI NA 1.27
Nikon CFI Plan Fluor 20XMI (multi-immersion) NA 0.75
Hamamatsu, Ballerup, Denmark H7422P-40
AHF analysentechnik AG (Tübingen, Germany). ChromaET 460 nm long pass dichroic
AHF analysentechnik AG (Tübingen, Germany). Semrock FF01-520/35-25 BrightLine filter
AHF analysentechnik AG (Tübingen, Germany). Chroma ET402/15X 
Scotch TM
coverslip thickness should match used objective on microscope (#1 or #1.5), alternatively, set adjustment collar to match coverslip
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Briones, A. M., Arribas, S. M., Salaices, M. Role of extracellular matrix in vascular remodeling of hypertension. Curr Opin Nephrol Hy. 19 (2), 187-194 (2010).
  2. Heagerty, A. M., Heerkens, E. H., Izzard, A. S. Small artery structure and function in hypertension. J Cell Mol Med. 14 (5), 1037-1043 (2010).
  3. van den Akker, J., Schoorl, M. J., Bakker, E. N., Vanbavel, E. Small artery remodeling: current concepts and questions. J Vasc Res. 47 (3), 183-202 (2010).
  4. Rizzoni, D., Agabiti-Rosei, E. Structural abnormalities of small resistance arteries in essential hypertension. Intern Emerg Med. 7 (3), 205-212 (2012).
  5. Schiffrin, E. L. Vascular remodeling in hypertension: mechanisms and treatment. Hypertension. 59 (2), 367-374 (2012).
  6. Fonck, E., et al. Effect of elastin degradation on carotid wall mechanics as assessed by a constituent-based biomechanical model. Am J Physiol-Heart C. 292 (6), H2754-H2763 (2007).
  7. Chow, M. J., Turcotte, R., Lin, C. P., Zhang, Y. Arterial extracellular matrix: a mechanobiological study of the contributions and interactions of elastin and collagen. Biophys J. 106 (12), 2684-2692 (2014).
  8. Chen, H., et al. Biaxial deformation of collagen and elastin fibers in coronary adventitia. J Appl Physiol (1985). 115 (11), 1683-1693 (2013).
  9. Schriefl, A. J., Schmidt, T., Balzani, D., Sommer, G., Holzapfel, G. A. Selective enzymatic removal of elastin and collagen from human abdominal aortas: uniaxial mechanical response and constitutive modeling. Acta Biomater. 17, 125-136 (2015).
  10. Zeinali-Davarani, S., Wang, Y., Chow, M. J., Turcotte, R., Zhang, Y. Contribution of collagen fiber undulation to regional biomechanical properties along porcine thoracic aorta. J Biomech Eng. 137 (5), 051001 (2015).
  11. Mattson, J. M., Turcotte, R., Zhang, Y. Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics. Biomech Model Mechan. 16 (1), 213-225 (2017).
  12. Schriefl, A. J., Zeindlinger, G., Pierce, D. M., Regitnig, P., Holzapfel, G. A. Determination of the layer-specific distributed collagen fibre orientations in human thoracic and abdominal aortas and common iliac arteries. J R Soc Interface. 9 (71), 1275-1286 (2012).
  13. Bloksgaard, M., et al. Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries. Am J Physiol-Heart C. , (2017).
  14. Dora, K. A., et al. Isolated Human Pulmonary Artery Structure and Function Pre- and Post-Cardiopulmonary Bypass Surgery. J Am Heart Assoc. 5 (2), (2016).
  15. Bell, J. S., et al. Microstructure and mechanics of human resistance arteries. Am J Physiol-Heart C. 311 (6), H1560-H1568 (2016).
  16. Roque, F. R., et al. Aerobic exercise reduces oxidative stress and improves vascular changes of small mesenteric and coronary arteries in hypertension. Brit J Pharmacol. 168 (3), 686-703 (2013).
  17. Briones, A. M., et al. Alterations in structure and mechanics of resistance arteries from ouabain-induced hypertensive rats. Am J Physiol-Heart C. 291 (1), H193-H201 (2006).
  18. Briones, A. M., et al. Role of elastin in spontaneously hypertensive rat small mesenteric artery remodelling. J Physiol. 552 (Pt 1), 185-195 (2003).
  19. Arribas, S. M., et al. Confocal myography for the study of hypertensive vascular remodelling. Clin Hemorheol Micro. 37 (1-2), 205-210 (2007).
  20. Gonzalez, J. M., et al. Postnatal alterations in elastic fiber organization precede resistance artery narrowing in SHR. Am J Physiol-Heart C. 291 (2), H804-H812 (2006).
  21. Spronck, B., Megens, R. T., Reesink, K. D., Delhaas, T. A method for three-dimensional quantification of vascular smooth muscle orientation: application in viable murine carotid arteries. Biomech Model Mechan. 15 (2), 419-432 (2015).
  22. Megens, R. T., et al. In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy. J Biomed Opt. 15 (1), 011108 (2010).
  23. Megens, R. T., oude Egbrink, M. G., Merkx, M., Slaaf, D. W., van Zandvoort, M. A. Two-photon microscopy on vital carotid arteries: imaging the relationship between collagen and inflammatory cells in atherosclerotic plaques. J Biomed Opt. 13 (4), 044022 (2008).
  24. Bender, S. B., et al. Regional variation in arterial stiffening and dysfunction in Western diet-induced obesity. Am J Physiol-Heart C. 309 (4), H574-H582 (2015).
  25. Clifford, P. S., et al. Spatial distribution and mechanical function of elastin in resistance arteries: a role in bearing longitudinal stress. Arterioscler Thromb. 31 (12), 2889-2896 (2011).
  26. Martinez-Revelles, S., et al. Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK. Antioxid Redox Sign. 27 (7), 379-397 (2017).
  27. Foote, C. A., et al. Arterial Stiffening in Western Diet-Fed Mice Is Associated with Increased Vascular Elastin, Transforming Growth Factor-beta, and Plasma Neuraminidase. Front Physiol. 7, 285 (2016).
  28. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9 (7), 676-682 (2012).
  29. Sommer, C., Straehle, C., Kothe, U., Hamprecht, F. A. Ilastik: Interactive Learning and Segmentation Toolkit. , 230-233 (2011).
  30. Campagnola, P. J., et al. Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys J. 82 (1 Pt 1), 493-508 (2002).
  31. Intengan, H. D., Deng, L. Y., Li, J. S., Schiffrin, E. L. Mechanics and composition of human subcutaneous resistance arteries in essential hypertension. Hypertension. 33 (1 Pt 2), 569-574 (1999).
  32. Saatchi, S., et al. Three-dimensional microstructural changes in murine abdominal aortic aneurysms quantified using immunofluorescent array tomography. J Histochem Cytochem. 60 (2), 97-109 (2012).
  33. Bloksgaard, M., et al. Elastin Organization in Pig and Cardiovascular Disease Patients’ Pericardial Resistance Arteries. J Vasc Res. 52 (1), 1-11 (2015).
  34. World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 310 (20), 2191-2194 (2013).
  35. Schneider, C. A., Rasband, W. S., Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9 (7), 671-675 (2012).
  36. Meijering, E., et al. Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A. 58 (2), 167-176 (2004).
  37. Rezakhaniha, R., et al. Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy. Biomech Model Mechan. 11 (3-4), 461-473 (2012).
  38. Green, E. M., Mansfield, J. C., Bell, J. S., Winlove, C. P. The structure and micromechanics of elastic tissue. Interface Focus. 4 (2), 20130058 (2014).
  39. Bell, J. S., et al. Microstructure and mechanics of human resistance arteries. Am J Physiol-Heart C. 311 (6), H1560-H1568 (2016).
  40. Shen, Z., Lu, Z., Chhatbar, P. Y., O’Herron, P., Kara, P. An artery-specific fluorescent dye for studying neurovascular coupling. Nat Methods. 9 (3), 273-276 (2012).
  41. Megens, R. T., et al. Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy. Mol Imaging. 6 (4), 247-260 (2007).
  42. Staiculescu, M. C., et al. Prolonged vasoconstriction of resistance arteries involves vascular smooth muscle actin polymerization leading to inward remodelling. Cardiovasc Res. 98 (3), 428-436 (2013).
  43. Fung, Y. C., Sobin, S. S. The retained elasticity of elastin under fixation agents. J Biomech Eng. 103 (2), 121-122 (1981).
  44. Fung, Y. C. . Biomechanics : mechanical properties of living tissues. , (1993).
  45. Bakker, E. N., et al. Heterogeneity in arterial remodeling among sublines of spontaneously hypertensive rats. PLoS One. 9 (9), e1107998 (2014).
  46. VanBavel, E., Siersma, P., Spaan, J. A. Elasticity of passive blood vessels: a new concept. Am J Physiol-Heart C. 285 (5), H1986-H2000 (2003).
  47. Chen, H., et al. Microstructural constitutive model of active coronary media. Biomaterials. 34 (31), 7575-7583 (2013).
  48. Saez, P., Garcia, A., Pena, E., Gasser, T. C., Martinez, M. A. Microstructural quantification of collagen fiber orientations and its integration in constitutive modeling of the porcine carotid artery. Acta Biomater. 33, 183-193 (2016).
  49. Bellini, C., Ferruzzi, J., Roccabianca, S., Di Martino, E. S., Humphrey, J. D. A microstructurally motivated model of arterial wall mechanics with mechanobiological implications. Ann Biomed Eng. 42 (3), 488-502 (2014).
  50. Schriefl, A. J., Wolinski, H., Regitnig, P., Kohlwein, S. D., Holzapfel, G. A. An automated approach for three-dimensional quantification of fibrillar structures in optically cleared soft biological tissues. J R Soc Interface. 10 (80), 20120760 (2013).
  51. Weisbecker, H., Unterberger, M. J., Holzapfel, G. A. Constitutive modelling of arteries considering fibre recruitment and three-dimensional fibre distribution. J R Soc Interface. 12 (105), 20150111 (2015).
  52. Chen, H., Kassab, G. S. Microstructure-based biomechanics of coronary arteries in health and disease. J Biomech. 49 (12), 2548-2559 (2016).
  53. Chen, H., Kassab, G. S. Microstructure-based constitutive model of coronary artery with active smooth muscle contraction. Sci Rep. 7 (1), 9339 (2017).

Tags

CollagenElastinMicroarchitectureResistance ArteriesLabel-free Fluorescence MicroscopyTwo-photon MicroscopyExtracellular MatrixVascular MechanicsArtery PressurizationSecond Harmonic GenerationAutofluorescenceFiji Image Analysis

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Bloksgaard, M., Thorsted, B., Brewer, J. R., De Mey, J. G. R. Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy. J. Vis. Exp. (134), e57451, doi:10.3791/57451 (2018).
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