JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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의학

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

Published: December 05, 2017
doi:
10.3791/56133
, , , , , ,
1Department of Physical Therapy,Marquette University, 2Medical College of Wisconsin, 3Department of Physiology,Medical College of Wisconsin, 4Graduate Programs of Nurse Anesthesia,Texas Wesleyan University, 5Office of Research,Medical College of Wisconsin

Summary

这篇手稿描述了体外大鼠脑阻力动脉血管功能的视频显微术。该手稿还描述了用激光多普勒血荧光标记凝集素和组织灌注评价微血管密度的技术。

Abstract

该协议描述了使用在体外电视显微镜来评估孤立的脑阻力动脉 (和其他血管) 的血管功能, 并描述了使用激光多普勒血组织灌注的评价技术 () 和微血管密度利用荧光标记的加纳单叶(GS1) 凝集素。目前的方法研究孤立阻力动脉在壁压力遇到在体内和在缺乏实质细胞的影响提供了一个关键的联系之间的体内研究和信息获得的分子简化的方法, 提供有限的洞察力的整体反应在整个动物的水平。荧光标记的 GS1 凝集素有选择地识别动脉和毛细血管的方法, 提供了切实可行的解决方案, 使调查人员能够扩大从孤立阻力动脉的研究中获得的知识。本文介绍了这些技术在大鼠血管生理学和病理学基础知识中的应用, 作为一个一般的实验模型, 并在各种专门的基因工程 “设计” 大鼠菌株, 可以提供重要的洞察力特定基因对重要血管表型的影响。利用这些有价值的实验方法, 在大鼠的选育策略和新技术的培育下, 建立基因敲除模型, 将扩大在挖空小鼠模型中开发的科学前提的严谨性, 并将这一知识扩展到一个更相关的动物模型中, 因为它的尺寸较大, 具有很好的生理背景和生理研究的适用性。

Introduction

动脉血管功能的最早研究利用导管动脉, 在许多情况下是主动脉。通过将动脉环段附着在组织浴中的力传感器上, 对大动脉的力产生进行了研究;在主动脉的情况下, 通过切割血管的螺旋条, 使平滑肌纤维在附着点和力传感器之间的纵向方向上定向, 以提供由收缩产生的力的最佳估计沿其纵轴的平滑肌肉。切割脉螺旋条的标准技术是在容器的流明处放置一根玻璃棒, 在容器壁上以所需的角度进行切割, 并在容器壁的外露边缘的一端保持, 因为切口被延伸以产生整个螺旋带的船只。在这一点上, 血管的内皮侧通常被涂抹, 以去除碎片之前, 连接到力传感器的船只带和淹没准备在一个氧和温度控制组织浴。最终, 这种方法导致了在生理学史上最著名和最重要的发现之一, Furchgott 和 Zawadski1, 即内皮衍生舒张因子 (EDRF) 的作用, 后来被确定为一氧化氮, 在调节血管功能。导致这一发现的关键事件是, 在这种情况下, 研究者通过避免动脉与外表面的内皮侧接触来维持完整的内皮, 并注意到主动脉带没有表现出预期收缩到乙酰胆碱, 但在反应中松弛。根据这一观察, 调查人员研制了一种 “三明治” 制剂, 在其中将主动脉段与一个完整的内皮细胞 (但不能产生收缩力) 连接到主动脉的标准螺旋带, 并转化为收缩成一个放松。

今天广泛使用的两个主要进展是在小阻力动脉中测量主动收缩力的准备工作的发展2,3 (如肠道肠系膜上的那些)3) 和空心阻力动脉准备4,5,6。在最早的一份报告中, Mulvany 和 Halpern3描述了使用金属丝 myograph 的制备方法, 研究自发性高血压大鼠 (SHR) 肠肠系膜上孤立阻力动脉的主动收缩力, 并正常 WKY 控制。在导线 myograph 系统的发展之后, 空心抵抗动脉准备被开发了允许研究更接近在体内情况4,5,6。 虽然这两种方法都提供了有价值的结果, 但空心动脉的制备具有更有效地保持动脉内固有活动张力的优点;并允许研究人员对壁压力和血管反应对流速和内皮剪切力变化的影响进行活性的肌源反应 (参见 Halpern 和凯利6的回顾)。

本论文的主要目的是描述如何使用孤立的, 空心阻力动脉的视频显微术的时间, 以获得准确的信息, 有关的机制, 调节主动语气在这些关键血管, 独立于神经、体液或实质细胞的影响。这一基本信息, 使用一个标准的大鼠模型和我们的研究新的基因工程大鼠菌株的例子, 将提供给读者一个关于血管功能的见解类型的想法, 可以获得与电视显微镜方法, 可用于研究涉及任何控制和实验组的研究者的选择, 包括强大的新的实验大鼠模型的选择性近亲繁殖和新发展的遗传工程技术。

由于电视显微镜方法的精确度, 测量空心动脉的直径变化可为血管内皮依赖性和内皮独立机制提供极有价值的信息。放松, 以及在高血压、高盐饮食和其他实验干预的血管控制机制中发生的重要 (有时是意想不到的) 改变。此外, 测量的压力-直径关系的孤立和空心阻力的动脉, 最大限度地放宽了治疗的 Ca2 +免费的解决方案或药理血管扩张药物, 使调查员评估血管重塑和计算被动应力-应变关系的动脉结构变化7可以提供重要的洞察力改变动脉的被动机械特性, 从而影响动脉功能独立于 (或除了) 主动控制机制的变化。还必须指出的是, 从隔离的阻力动脉的研究中获得的信息可以补充利用获得的信息, 这是一种评估整个动物级别组织灌注的实用方法8,9 ,10, 以及通过使用荧光标记的 GS1 凝集素来评估微血管密度所获得的信息, 这特别结合了小动脉和毛细血管基底膜的糖蛋白基11,12. 后一种方法提供了一个高度准确的微血管密度的估计, 不受传统的困难, 在估计微血管密度通过计数血管的在体内, 例如失踪非灌注由于动脉主动闭合而导致血流停止的血管。如果一起使用, 这些方法可以提供重要的洞察力相关的功能改变的孤立阻力动脉的变化, 组织灌注在微循环水平;在本手稿中还将提供一些使用这些有价值的方法与空心动脉技术结合的例子。

本文主要研究利用视频显微镜技术对 outbred 大大鼠动脉血管的变化进行评价。然而, 重要的是要注意, 这些技术已证明是非常宝贵的, 以阐明表型变化的高度专业化的基因工程大鼠选育或基因编辑使用的技术。在这篇手稿中, 我们提供的例子, 视频显微技术如何提供了重要信息的血管功能, 在许多有价值的大鼠模型, 包括达尔盐敏感 (SS) 大鼠-一个近交系大鼠菌株, 是最广泛的用实验模型研究了盐敏 hypertenson 的机理18、19、20、21、22、23;和 consomic 大鼠通过选择性选育 SS 大鼠与盐不敏感的棕色挪威 (BN) 大鼠品系。在 consomic 鼠板中, 来自棕挪威鼠的每一个染色体都被单独渗入到达尔 SS24,25,26遗传背景。使用 consomic 鼠板提供了宝贵的线索, 关于特定的染色体, 有助于盐敏感性血压和其他表型, 包括血管反应性24,25,26 ,27,28。

利用 ss 大鼠和携带个别 BN 染色体的 consomic 大鼠的选择性育种策略, 也使同类菌株的小片段渗入到达尔 ss 遗传背景22,29。这些可以提供非常有价值的输入对特定的基因或染色体的狭窄区域, 可以影响关键的生理变量, 如血压, 肾脏损害, 和血管活性22,29。大鼠基因工具箱的另一个强大的补充是利用先进的基因编辑技术, 包括 ZFNs, 转录激活剂类似效应核酸 (TALENS), 最近 CRISPR-Cas913 ,14,15,16,17。这些强大的技术的问世, 使基因被淘汰的老鼠是一个非常重要的发展, 因为基因剔除研究迄今已使用 (并继续使用) 小鼠几乎完全。本论文的另一项实验研究表明, 空心动脉技术和视频显微术对无主抗氧化剂和细胞保护转录的击倒大鼠的生理控制机制有价值因子, 核因子 (红-衍生 2) 样-2 (NRF2)30,31, 这是利用塔伦技术在大遗传背景17中开发的。在这些实验中,体外视频显微镜技术用于提供 NRF2 基因丢失的功能验证, 并在 NRF2-mediated 抗氧化剂直接上调的基础上测试一种潜在的有价值的治疗方法。防御.NRF-2 在对抗血管氧化应激方面具有重要的治疗作用, 因为临床试验的结果令人失望, 例如维生素 C 和 E32直接管理抗氧化剂。

Protocol

威斯康星医学院动物护理和使用委员会 (IACUC) 批准了本文所述的所有协议, 所有程序都符合国家卫生研究院 (NIH) 实验室动物福利办公室 (OLAW)条例. 1. 溶液和容器室的制备 在进行一系列实验之前, 准备 2 l 20x 浓盐溶液, 由278克/L 氯化钠组成;14克/升氯化钾;11.52 克/升 MgSO4.7H2O;和9.4 克/L CaCl2.2H2O。还准备2升的20x 集中缓冲库存, …

Representative Results

体外空心阻力动脉显微镜允许研究影响小阻力动脉 (和大动脉) 在正常的体内壁压力和缺乏实质细胞的主动音的因素影响.除了评估在正常 pss 中对壁压力升高的各种血管扩张剂和收缩刺激和肌源反应的反应性外, 还可以在液和 superfusate 中添加 Ca2 +-免费的 pss。试验确定了最大容器直径和壁厚。后者的测量是非常有价值的评估血管重塑,即, 改变的?…

Discussion

正如导言中所指出的, 本文介绍了电视显微镜和孤立阻力动脉的方法, 以评估血管功能不仅在标准大鼠模型 (在视频中使用), 而且在高度专门化的基因工程大鼠菌株, 这表明了小说和强大的洞察力, 可以得到利用这些方法。利用这些强大的技术来评估小阻力动脉的主动音和被动力学特性, 可以提供关于多种血管控制机制的重要信息, 包括内皮依赖性调节在正常和病理生理条件下, 血管平滑肌功能与血管…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者衷心感谢凯蒂. 芬克和林恩 Dondlinger 在准备这份手稿方面的宝贵帮助。

补助金支持: NIH #R21-OD018309;#R56 HL065289;还有 #R01 HL128242

Materials

SS Rat Medical College of Wisconsin SS/JHsd/Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
SS.5BN Consomic Rat Medical College of Wisconsin SS-Chr 5BN/Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
SS.13BN Consomic Rat Medical College of Wisconsin SS-Chr 13BN/Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
Ren1-BN Congenic Rat Medical College of Wisconsin SS.BN-(D13hmgc41-D13)hmgc23/Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
Ren1-SSA Congenic Rat Medical College of Wisconsin SS.BN-(D13rat77-D13rat105/Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
Ren1-SSB Congenic Rat Medical College of Wisconsin SS.BN-(D13rat124-D13rat101/Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
Nrf2(-/-) Knockout Rat and Wild Type Littermates Medical College of Wisconsin SD-Nfe212em1Mcwi strain Contact Dr. Aron Geurts (ageurts@mcw.edu)
Low Salt Rat Chow (0.4% NaCl)-AIN-76A Dyets, Inc. 113755
High Salt Rat Chow (4% NaCl)-AIN-76A Dyets, Inc. 113756
Colorado Video Caliper Colorado Video, Inc. Model 308
Video Camera Hitachi KPM1AN
Microscope Olympus Life Science CKX41
Television Monitor Panasonic WVBM1410
Pressure Transducers Stoelting 56360
Blood Pressure Display Unit Stoelting 50115
Cannulated Artery Chamber Living Systems Instrumentation CH-1 Single vessel chamber for general use
Temperature Controller for Single Chamber Living Systems Instrumentation TC-09S
Gas Dispersion Tube, Miniature,Straight Living Systems Instrumentation GD-MS Provides aeration in the vessel bath
Gas Exchange Oxygenator, Miniature Living Systems Instrumentation OX Allows gas exchange with perfusate
Laser-Doppler Flowmeter Perimed PeriFlux 5000 LDPM
GS1 Lectin Vector Labs RL-1102
Glass Capillary Tubes for Micropipettes Fredrich Haer Co. 27-33-1 2 mm ODX1 mm ID
Verticle Pipette Puller David Kopf Instruments Model 700C
Nylon suture material (10/0)-3 PLY Ashaway Line and Twine Manufacturing Co. 114-ANM-10 Single strands of 3 ply nylon suture teased out for use on vessels
Dumont #5 Forceps-Inox Fine Science Tools 11254-20
Vannas Scissors Fine Science Tools 15003-08
Protandim Protandim NRF2 Inducer: Contact Dr. Joe McCord (JOE.MCCORD@UCDENVER.EDU)
Sodium Chloride Fisher Bioreagents BP358-212
Sodium Bicarbonate Fisher Chemical S233-3
Dextrose (d-glucose) anhydrous Fisher Chemical D16-500
Magnesium Sulfate (MgSO4-7H2O) Sigma Aldrich M1880-500 G
Calcium Chloride (CaCl2-2 H2O) Sigma C5080-500G
Sodium Phosphate-Monobasic (NaH2PO4) Sigma S0751-500G
Potassium Chloride (KCl) Fisher Chemical P217-500G
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) Sigma ED255-500G
DOWNLOAD MATERIALS LIST

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Vascular Control MechanismsVideo MicroscopyIsolated Resistance ArteriesRatsVascular BiologyEndothelial ControlVascular Smooth Muscle ControlPeripheral Vascular DiseaseCoronary Artery DiseaseMicrocannulationPhysiological Salt SolutionPH AdjustmentCalcium-free SolutionGas Equilibration
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Lukaszewicz, K. M., Durand, M. J., Priestley, J. R., Schmidt, J. R., Allen, L. A., Geurts, A. M., Lombard, J. H. Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats. J. Vis. Exp. (130), e56133, doi:10.3791/56133 (2017).
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