皮内微透析：研究人类微血管功能障碍新机制的方法
Summary
皮内微透析是一种微创技术，用于研究健康和疾病中的微血管功能。该技术可利用剂量反应和局部加热方案来探索皮肤循环中血管舒张和血管收缩的机制。
Abstract
皮肤脉管系统是一种可触及的组织，可用于评估人体的微血管功能。皮内微透析是一种微创技术，用于研究皮肤循环中血管平滑肌和内皮功能的机制。该技术允许对微血管内皮功能障碍的病理生理学进行药理学解剖，其指标是一氧化氮介导的血管舒张减少，这是心血管疾病发展风险的指标。在这种技术中，将微透析探针放置在皮肤的真皮层中，并在探头上放置带有激光多普勒血流探头的局部加热装置以测量红细胞通量。通过直接加热施加夹紧或刺激局部皮肤温度，并通过探针灌注药理学试剂以刺激或抑制细胞内信号通路，以诱导血管舒张或血管收缩或询问感兴趣的机制（辅助因子、抗氧化剂等）。对皮肤血管电导进行量化，并描述疾病状态下内皮功能障碍的机制。
Introduction
心血管疾病 （CVD） 是美国的主要死因1.高血压 （HTN） 是卒中、冠心病和心力衰竭的独立危险因素，估计影响超过 ~50% 的美国人口2。高血压可以发展为独立的心血管疾病（原发性高血压）或由其他疾病引起的，例如多囊肾病和/或内分泌失调（继发性高血压）。HTN 病因的广泛性使得对 HTN 观察到的潜在机制和终末器官损伤的研究变得复杂。需要对与 HTN 相关的终末器官损伤的病理生理学采取多样化和新颖的研究方法。
CVD 最早的病理体征之一是内皮功能障碍，其特征是一氧化氮 （NO） 介导的血管舒张受损 3,4,5。血流介导的扩张是用于量化与 CVD 相关的内皮功能障碍的常用方法，但微血管床中的内皮功能障碍既可以独立于大导管动脉，也可以先于大导管动脉 6,7,8。此外，阻力小动脉比导管动脉更直接地作用于局部组织，并且更直接地控制富氧血液的输送。微血管功能可预测不良心血管无事件生存期 9,10,11。皮肤微血管系统是一种可触及的血管床，可用于检查对生理和药理血管收缩或血管舒张刺激的反应。皮内微透析是一种微创技术，其目的是通过靶向药理学解剖研究皮肤微血管系统中血管平滑肌和内皮功能的机制。这种方法与其他技术形成鲜明对比，例如闭塞后反应性充血，它不允许药理学解剖，离子电渗疗法允许药理学递送，但其作用机制不太精确（详见其他部分12）。
开发和使用这种技术背后的基本原理在别处进行了广泛的审查13.这种方法最初是为啮齿动物的神经学研究而开发的，然后首先应用于人类，从体温调节的角度研究活动性血管舒张的机制。在 1990 年代后期，这种方法被用于检查有关皮肤局部加热的神经和内皮机制。从那时起，该技术已被用于研究皮肤中的许多神经血管信号传导机制。
使用这种技术，我们小组和其他人研究了几种临床人群微血管系统中内皮功能障碍的机制，包括但不限于血脂异常、原发性衰老、糖尿病、慢性肾脏病、多囊卵巢综合征、先兆子痫、重度抑郁症14、15、16、17、18、19 和高血压20,21，22,23,24.例如，先前的一项研究发现，与有正常血压妊娠史的女性相比，有先兆子痫病史的血压正常女性患心血管疾病的风险增加，皮肤循环中 NO 介导的血管舒张减少20。在另一项研究中，与健康对照组相比，诊断为原发性高血压的成人在微血管系统中表现出血管紧张素 II 敏感性增加21，并且原发性高血压患者的慢性巯基供体抗高血压药物治疗已被证明可以降低血压并改善硫化氢和 NO 介导的血管舒张22。Wong 等人 23 在高血压前期成人中发现感觉介导和 NO 介导的血管舒张受损，这与我们发现的内皮功能障碍随着 HTN 分期的增加而进展相吻合，如 2017 年美国心脏协会和美国心脏病学会指南24 所分类。
皮内微透析技术可以对健康和疾病状态下的微血管功能进行严格控制的机制研究。因此，本文旨在描述我们小组和其他人应用的皮内微透析技术。我们详细介绍了用乙酰胆碱 （ACh） 对内皮进行药理刺激的程序，以检查剂量反应关系和使用 39 °C 或 42 °C 局部加热刺激方案对内源性 NO 产生的生理刺激。我们介绍了每种方法的代表性结果，并讨论了该技术产生的发现的临床意义。
Protocol
在参与者招募之前，所有程序均由宾夕法尼亚州立大学机构审查委员会批准。 1. 设备设置 打开局部加热装置和激光多普勒流量计。注意： 两者都应在数据收集之前根据制造商的说明进行校准。激光多普勒流量计应连接到数据采集硬件，以 100 Hz （100 个样本/分钟）采样并在数据采集软件中连续记录。虽然可以使用其他数据采集硬件和软件，但为简单起见，其余指令…
Representative Results
乙酰胆碱剂量反应方案 图1A 描绘了详细描述ACh剂量反应方案的示意图。 图1B 显示了一个受试者的标准化ACh剂量反应方案中红细胞通量值（灌注单位，PU;30 秒平均值）随时间变化的代表性示踪。 图1C 显示了ACh剂量反应方案的原始数据文件。在原始数据文件中保留了额外的基线测量值，但仅使用了 …
Discussion
皮内微透析技术是人体血管研究中的通用工具。研究人员可能会改变协议以进一步多样化其应用。例如，我们描述了 ACh 剂量反应方案，但其他对血管收缩或血管舒缩张力机制的研究，而不是单独的血管舒张力，利用了去甲肾上腺素或硝普钠剂量反应方法 26,27,28,29,30,31。<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
没有。
Materials
| 1 mL syringes | BD Syringes | 302100 | |
| Acetlycholine | United States Pharmacopeia | 1424511 | Pilot data collected in our lab indicate drying acetylcholine increases variability of CVC response; do not dry, store in desiccator |
| Alcohol swabs | Mckesson | 191089 | |
| Baby Bee Syringe Drive | Bioanalytical Systems, Incorporated | MD-1001 | In this study the optional 3-syringe bracket (catalg number MD-1002) was utilized |
| CMA 30 Linear Microdialysis Probes | Harvard Apparatus | CMA8010460 | |
| Connex Spot Monitor | WelchAllyn | 74CT-B | automated blood pressure monitor |
| Hive Syringe Pump Controller | Bioanalytical Systems, Incorporated | MD-1020 | Controls up to 4 Baby Bee Syringe Drives |
| LabChart 8 | AD Instruments | **PowerLab hardware and LabChart software must be compatible versions | |
| Lactated Ringer's Solution | Avantor (VWR) | 76313-478 | |
| Laser Doppler Blood FlowMeter | Moor Instruments | MoorVMS-LDF | |
| Laser Doppler probe calibration kit | Moor Instruments | CAL | |
| Laser Doppler VP12 probe | Moor Instruments | VP12 | |
| Linear Microdialysis Probes | Bioanalytical Systems, Inc. | MD-2000 | |
| NG-nitro-l-arginine methyl ester | Sigma Aldrich | 483125-M | L-NAME |
| Povidone-iodine / betadine | Dynarex | 1202 | |
| PowerLab C Data Acquisition Device | AD Instruments | PLC01 | ** |
| PowerLab C Instrument Interface | AD Instruments | PLCI1 | ** |
| Probe adhesive discs | Moor Instruments | attach local heating unit to skin | |
| Skin Heater Controller | Moor Instruments | moorVMS-HEAT 1.3 | |
| Small heating probe | Moor Instruments | VHP2 | |
| Sterile drapes | Halyard | 89731 | |
| Sterile gauze | Dukal Corporation | 2085 | |
| Sterile surgical gloves | Esteem Cardinal Health | 8856N | catalogue number followed by the initials of the glove size, then the letter "B" (e.g., 8856NMB for medium) |
| Surgical scissors | Cole-Parmer | UX-06287-26 |
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Tags
Keywords:
Intradermal MicrodialysisMicrovascular DysfunctionVascular FunctionPharmacological DissectionCardiovascular Disease RiskHypertensionMajor Depressive DisorderArginaseReactive Oxygen SpeciesSalicylateInflammationEndometriosisNon-Hispanic Black WomenNitric OxideEndothelial FunctionVascular Smooth Muscle
Citer Cet Article
Williams, A. C., Content, V. G., Kirby, N. V., Alexander, L. M. Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans. J. Vis. Exp. (197), e65579, doi:10.3791/65579 (2023).