新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量
Summary
这里演示的是 (1) 新鲜隔离完整的大脑内皮 “管” 和 (2) 同时测量内皮钙和膜电位的协议, 在内皮衍生的超极化过程中。此外, 这些方法允许药理调节内皮细胞钙和电信号作为个人或交互式实验变量。
Abstract
大脑动脉及其各自的微循环通过血液流动调节向大脑输送氧气和营养物质。内皮细胞排列血管腔, 并根据需要控制血管直径的变化, 以满足神经元的代谢需求。膜电位 (vm) 和一氧化氮超极化的初级内皮依赖性信号通路通常平行调节血管扩张, 从而增加血液流动。虽然在几个毫米的血管长度的血管扩张协调不可或缺的一部分, 内皮衍生的超极化 (edh) 的组成部分一直是很难测量的历史。edh 的这些成分包含细胞内 ca2 + [ca2 +]i增加并随后激活小型和中间电导 ca2 +-激活 k + (skca/ik ca)通道.
在这里, 我们提出了一个从小鼠大脑动脉中分离新鲜内皮细胞的简化说明;同时测量内皮 [ca2 +] i 和 v m 分别使用 fura-2 分光光度法和细胞内尖锐电极;以及在生理条件下 (ph 7.4, 37°c) 下盐溶液和药理剂的连续超融合。从威利斯圆环的脑后动脉被移除后部沟通和基底动脉。经清理的脑后动脉段的酶消化和随后的三口治疗有助于切除外膜、血管周围神经和平滑肌细胞。然后, 在显微镜下固定下断陷的后脑动脉内皮 “管”, 并在连续超融合的情况下使用相机、光电倍增管和一到两个静电计进行检查。总之, 这种方法可以同时测量在离散细胞位置的内皮 [ca2 +]i和 vm 的变化, 除了通过间隙连接传播 edh, 直到毫米的距离沿完整内皮。这种方法有望产生一个高通量分析大脑内皮功能的基础机制的血液流动调节在正常和患病的大脑。
Introduction
血管网络1中脑动脉和动脉之间血管舒张的协调调节整个大脑的血液流动.大脑阻力动脉内的内皮细胞根据需要控制血管直径的变化, 以满足神经元1,2,3的代谢需求。特别是在内皮细胞产生的超极化 (俗称 edh) 期间, 细胞内 ca2 + ([ca2 +]i) 和内皮细胞中的电信号键协调内皮细胞之间的血管舒张作用。周围的平滑肌细胞通过缝隙连接动脉放松4。edh 的生理启动顺序意味着刺激 gq偶联受体 (gpcr), 增加 [ca2 +]i, 激活内皮小和中-钙 2 + 激活 k+(skca/ik ca)通道超极化脑血管电位 (vm)5,6,7。因此, 内皮 [ca2 +]i和 vm 之间的亲密关系是血液流动调节的组成部分, 也是心脑血管功能6、8 不可缺少的。在广泛的文献中, 大量的研究报告了血管内皮功能障碍与慢性病的发展 (如高血压, 糖尿病, 心力衰竭, 冠状动脉疾病, 慢性肾功能衰竭,外周动脉疾病)9,10, 表明研究内皮功能在生理和病理条件的意义。
血管内皮是产生超极化、血管扩张和组织灌注的组成部分, 因此, 检查其自身的自身特性至关重要。作为一般的研究模型, 小鼠动脉内皮管模型的制备已发表之前为骨骼肌11,12, 肠 13,肺14, 最近为大脑6。特别是同时进行的 [ca2 +]i和 vm 测量的研究已经发表了骨骼肌动脉内皮 15,16以及淋巴管内皮细胞 17.除了使用内皮管方法的初步研究外, 还可以参考对其优缺点的全面审查, 8 以确定这一实验工具是否适合于特定的研究。简而言之, 一个优点是保留了内皮细胞功能的关键生理成分 (例如, ca2+流入和细胞内释放, vm 的超极化到 nernstk +的潜力通过skca/ikca 激活, 和内皮细胞间耦合通过缝隙连接), 没有混淆因素, 如血管周围神经输入, 平滑肌电压门控通道功能和收缩力,血液循环, 和荷尔蒙的影响8。相反, 常用的细胞培养方法在形态 18和离子通道表达19方面有显著的改变, 这种改变可以极大地模糊与在体内确定的生理观察的比较。在体内。限制包括缺乏与调节血液流动的其他基本成分的整合, 如平滑肌和实验时间表中的灵活性受限, 因为该模型在完整的血管段隔离4小时内进行了最佳测试从动物。
根据 socha 和 segal12编写的前一段视频协议以及在中间6、15、16 的最新实验发展, 我们在此演示了从后脑动脉和同时测量内皮 [ca2 +]i和 v m分别使用 fura-2 分光光度法和细胞内尖锐电极。此外, 这项实验需要在生理条件下 (ph 7.4, 37°c) 持续超融合盐溶液和药理剂。我们选择了大脑后动脉, 因为它产生的是具有结构完整性 (细胞通过缝隙连接结合) 和足够尺寸 (宽度≥50μm, 长度≥300μm) 的孤立内皮, 适合细胞内和细胞间信号。内皮细胞。此外, 啮齿类动物大脑后动脉的研究在文献中得到了大量的体现, 包括对基本内皮信号机制、血管发育和病理20的检查,21,22. 这一实验应用预计将产生对大脑内皮功能 (和功能障碍) 的高通量分析, 从而使整个过程中对血液流动调节的理解有重大进展。衰老和神经退行性疾病的发展。
Protocol
Representative Results
Discussion
根据最近的发展 6,15,16, 17, 我们现在演示的方法, 分离小鼠脑动脉内皮, 准备同时测量 [ca2 +]i和 vm 在37°c 下始终为 ~ 2小时的 edh 基础。虽然技术上困难, 但我们也可以测量细胞间耦合 (参见参考6, 图 1)。通过这种方式, 我们可以同时测量离散和传导信号事件。有…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢查尔斯·休伊特提供了出色的技术援助, 同时建立了目前协议所需的设备和用品。我们感谢 llu 围产儿生物学中心的 sean m. wilson 博士和 christopher g. wilson 博士分别为我们提供了一个额外的倒置显微镜和静电计。这项研究得到了国家卫生研究院 r00-ag047198 (ejb) 和 loma linda 大学医学院新教师启动资金的支持。内容完全由作者负责, 不一定代表国家卫生研究院的官方观点。
Materials
| Glucose | Sigma-Aldrich (St. Louis, MO, USA) | G7021 | |
| NaCl | Sigma | S7653 | |
| MgCl2 | Sigma | M2670 | |
| CaCl2 | Sigma | 223506 | |
| HEPES | Sigma | H4034 | |
| KCl | Sigma | P9541 | |
| NaOH | Sigma | S8045 | |
| ATP | Sigma | A2383 | |
| HCl | ThermoFisher Scientific (Pittsburgh, PA, USA) | A466250 | |
| Collagenase (Type H Blend) | Sigma | C8051 | |
| Dithioerythritol | Sigma | D8255 | |
| Papain | Sigma | P4762 | |
| Elastase | Sigma | E7885 | |
| BSA | Sigma | A7906 | |
| Propidium iodide | Sigma | P4170 | |
| DMSO | Sigma | D8418 | |
| Fura-2 AM dye | Invitrogen, Carlsbad, CA, USA | F14185 | |
| Recirculating chiller (Isotemp 500LCU) | ThermoFisher Scientific | 13874647 | |
| Plexiglas superfusion chamber | Warner Instruments, Camden, CT, USA | RC-27 | |
| Glass coverslip bottom (2.4 × 5.0 cm) | ThermoFisher Scientific | 12-548-5M | |
| Anodized aluminum platform (diameter: 7.8 cm) | Warner Instruments | PM6 or PH6 | |
| Compact aluminum stage | Siskiyou, Grants Pass, OR, USA | 8090P | |
| Micromanipulator | Siskiyou | MX10 | |
| Stereomicroscopes | Zeiss, NY, USA | Stemi 2000 & 2000-C | |
| Fiber optic light sources | Schott, Mainz, Germany & KL200, Zeiss | Fostec 8375 | |
| Nikon inverted microscope | Nikon Instruments Inc, Melville, NY, USA | Ts2 | |
| Phase contrast objectives | Nikon Instruments Inc | (Ph1 DL; 10X & 20X) | |
| Fluorescent objectives | Nikon Instruments Inc | 20X (S-Fluor), and 40X (Plan Fluor) | |
| Nikon inverted microscope | Nikon Instruments Inc | Eclipse TS100 | |
| Microsyringe pump controller (Micro4 ) | World Precision Instruments (WPI), Sarasota, FL, USA | SYS-MICRO4 | |
| Vibration isolation table | Technical Manufacturing, Peabody, MA, USA | Micro-g | |
| Amplifiers | Molecular Devices, Sunnyvale, CA, USA | Axoclamp 2B & Axoclamp 900A | |
| Headstages | Molecular Devices | HS-2A & HS-9A | |
| Function generator | EZ Digital, Seoul, South Korea | FG-8002 | |
| Data Acquision System | Molecular Devices, Sunnyvale, CA, USA | Digidata 1550A | |
| Audible Baseline Monitors | Ampol US LLC, Sarasota, FL, USA | BM-A-TM | |
| Digital Storage Oscilloscope | Tektronix, Beaverton, Oregon, USA | TDS 2024B | |
| Fluorescence System Interface, ARC Lamp + Power Supply, Hyperswitch, PMT | Molecular Devices, Sunnyvale, CA, USA | IonOptix Systems | |
| Temperature Controller | Warner Instruments | TC-344B or C | |
| Inline Heater | Warner Instruments | SH- 27B | |
| Valve Controller | Warner Instruments | VC-6 | |
| Inline Flow Control Valve | Warner Instruments | FR-50 | |
| Electronic Puller | Sutter Instruments, Novato, CA, USA | P-97 or P-1000 | |
| Microforge | Narishige, East Meadow, NY, USA | MF-900 | |
| Borosilicate Glass Tubes (Trituration) | World Precision Instruments (WPI), Sarasota, FL, USA | 1B100-4 | |
| Borosilicate Glass Tubes (Pinning) | Warner Instruments | G150T-6 | |
| Borosilicate Glass Tubes (Sharp Electrodes) | Warner Instruments | GC100F-10 | |
| Syringe Filter (0.22 µm) | ThermoFisher Scientific | 722-2520 | |
| Glass Petri Dish + Charcoal Sylgard | Living Systems Instrumentation, St. Albans City, VT, USA | DD-90-S-BLK | |
| Vannas Style Scissors (3 mm & 9.5 mm) | World Precision Instruments | 555640S, 14364 | |
| Scissors 3 & 7 mm blades | Fine Science Tools (or FST), Foster City, CA, USA | Moria MC52 & 15000-00 | |
| Sharpened fine-tipped forceps | FST | Dumont #5 & Dumont #55 |
References
- Longden, T. A., Hill-Eubanks, D. C., Nelson, M. T. Ion channel networks in the control of cerebral blood flow. Journal of Cerebral Blood Flow & Metabolism. 36 (3), 492-512 (2016).
- Chen, B. R., Kozberg, M. G., Bouchard, M. B., Shaik, M. A., Hillman, E. M. A critical role for the vascular endothelium in functional neurovascular coupling in the brain. Journal of the American Heart Association. 3 (3), e000787 (2014).
- Iadecola, C., Yang, G., Ebner, T. J., Chen, G. Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex. Journal of Neurophysiology. 78 (2), 651-659 (1997).
- Bagher, P., Segal, S. S. Regulation of blood flow in the microcirculation: role of conducted vasodilation. Acta Physiologica (Oxford, England). 202 (3), 271-284 (2011).
- Garland, C. J., Dora, K. A. EDH: endothelium-dependent hyperpolarization and microvascular signalling. Acta Physiologica (Oxford, England). 219 (1), 152-161 (2017).
- Hakim, M. A., Buchholz, J. N., Behringer, E. J. Electrical dynamics of isolated cerebral and skeletal muscle endothelial tubes: Differential roles of G-protein-coupled receptors and K+ channels. Pharmacology Research & Perspectives. 6 (2), e00391 (2018).
- Marrelli, S. P., Eckmann, M. S., Hunte, M. S. Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations. American Journal of Physiology-Heart and Circulatory Physiology. 285 (4), H1590-H1599 (2003).
- Behringer, E. J. Calcium and electrical signaling in arterial endothelial tubes: New insights into cellular physiology and cardiovascular function. Microcirculation. 24 (3), (2017).
- Endemann, D. H., Schiffrin, E. L. Endothelial dysfunction. Journal of the American Society of Nephrology. 15 (8), 1983-1992 (2004).
- Rajendran, P., et al. The vascular endothelium and human diseases. International Journal of Biological Sciences. 9 (10), 1057-1069 (2013).
- Socha, M. J., Hakim, C. H., Jackson, W. F., Segal, S. S. Temperature effects on morphological integrity and Ca2+ signaling in freshly isolated murine feed artery endothelial cell tubes. American Journal of Physiology-Heart and Circulatory Physiology. 301 (3), H773-H783 (2011).
- Socha, M. J., Segal, S. S. Isolation of microvascular endothelial tubes from mouse resistance arteries. Journal of Visualized Experiments. (81), (2013).
- Ye, X., Beckett, T., Bagher, P., Garland, C. J., Dora, K. A. VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells. The Journal of Physiology. , (2018).
- Norton, C. E., Segal, S. S. Calcitonin gene-related peptide hyperpolarizes mouse pulmonary artery endothelial tubes through KATP channel activation. American Journal of Physiology-Lung Cellular and Molecular. , (2018).
- Behringer, E. J., Segal, S. S. Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation. The Journal of Physiology. 593 (20), 4531-4548 (2015).
- Behringer, E. J., Segal, S. S. Impact of Aging on Calcium Signaling and Membrane Potential in Endothelium of Resistance Arteries: A Role for Mitochondria. The Journal of Gerontology, Series A: Biological Sciences and Medical Sciences. 72 (12), 1627-1637 (2017).
- Behringer, E. J., et al. Calcium and electrical dynamics in lymphatic endothelium. The Journal of Physiology. 595 (24), 7347-7368 (2017).
- Simmers, M. B., Pryor, A. W., Blackman, B. R. Arterial shear stress regulates endothelial cell-directed migration, polarity, and morphology in confluent monolayers. American Journal of Physiology-Heart and Circulatory Physiolog. 293 (3), H1937-H1946 (2007).
- Sandow, S. L., Grayson, T. H. Limits of isolation and culture: intact vascular endothelium and BKCa. American Journal of Physiology-Heart and Circulatory Physiology. 297 (1), H1-H7 (2009).
- Diaz-Otero, J. M., Garver, H., Fink, G. D., Jackson, W. F., Dorrance, A. M. Aging is associated with changes to the biomechanical properties of the posterior cerebral artery and parenchymal arterioles. American Journal of Physiology-Heart and Circulatory Physiology. 310 (3), H365-H375 (2016).
- Kochukov, M. Y., Balasubramanian, A., Abramowitz, J., Birnbaumer, L., Marrelli, S. P. Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery. Journal of the American Heart Association. 3 (4), (2014).
- Zhang, L., Papadopoulos, P., Hamel, E. Endothelial TRPV4 channels mediate dilation of cerebral arteries: impairment and recovery in cerebrovascular pathologies related to Alzheimer’s disease. British Journal of Pharmacology. 170 (3), 661-670 (2013).
- Socha, M. J., Domeier, T. L., Behringer, E. J., Segal, S. S. Coordination of intercellular Ca2+ signaling in endothelial cell tubes of mouse resistance arteries. Microcirculation. 19 (8), 757-770 (2012).
