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Medicine

微电极阵列记录中天节点发射率，以识别小鼠内在心脏起搏缺陷

Published: July 05, 2021
doi:
10.3791/62735
, , ,
1Department of Biological Sciences,Southern Methodist University

Summary

该协议旨在描述一种新的方法，测量内在心脏发射率使用微电极阵列记录整个中天节点组织，以确定小鼠的制动缺陷。药理制剂也可以在这种方法中引入，以研究它们对内在起搏的影响。

Abstract

位于右中庭的中天节点（SAN）包含心脏的心脏起搏器细胞，该区域功能障碍可导致心动过速或心动过速。心脏起搏缺陷的可靠识别需要通过在很大程度上防止自主神经系统的影响来测量内在心率，而自主神经系统可以掩盖心率缺陷。分析内在心脏起搏器功能的传统方法包括药物诱导的自主封锁，以测量 体内 心率，分离心脏记录测量内在心率，以及中性心脏起搏器细胞的中性条带或单细胞贴片夹记录，以测量自发行动的潜在发射率。然而，这些更传统的技术在技术上可能具有挑战性，而且难以执行。在这里，我们提出了一种新的方法，通过执行微电极阵列（MEA）记录从小鼠的全安装中天节点制剂来测量内在心脏发射速率。MEA 由多个微电极组成，这些微电极以网格状模式排列，用于记录 体外 细胞外场电位。与以往记录内在心率的方法相比，此处描述的方法具有相对更快、更简单、更精确的综合优势，同时允许简单的药理学审讯。

Introduction

心脏是一个复杂的器官，由心脏内在和外在的影响，如那些起源于大脑的影响。中性节点（SAN）是心脏中一个定义区域，容纳心脏起搏器细胞（也称为中腹细胞，或SA细胞），负责启动和延续哺乳动物心跳1，2。内在心率是由心脏起搏器细胞驱动的，不受其他心脏或神经幽默的影响，但传统的心率测量在人类和活的动物，如心电图，反映了心脏起搏器和神经对心脏的影响。对SA细胞最显著的神经影响来自自主神经系统，它不断调节发射模式，以满足身体的生理要求3。支持这个想法，同情和寄生虫预测可以在SAN4附近找到。内在心脏神经系统（ICNS）是另一个重要的神经影响，其中结石丛，特别是在右侧，内侧和调节SAN5，6的活动。

了解制步缺陷在临床上很重要，因为功能障碍可能是许多心脏疾病的基础，并导致其他并发症的风险。病窦综合征（SSS）是一类疾病，其特征是鼻节点功能障碍，妨碍了适当的步调7，8。SSS可以呈现鼻窦心动过速，鼻窦暂停，鼻窦逮捕，鼻窦退出块，交替的胸膜和心动过速9，并可能导致并发症，包括增加脑卒中和突然死亡的风险8，10。那些患有布鲁加达综合征，一种以心室颤动为特征的心脏疾病，心脏骤死的风险增加，如果他们也有合并性SA功能障碍11，12，则患心律失常事件的风险更大。心房功能障碍也可能有超出心脏的生理后果。例如，由于脑溢血13，SSS被观察到会引发患者的癫痫发作。

为了识别中性起搏缺陷，需要通过测量 SAN 的活动来确定内在心率，而不受自主神经系统或幽默因素的影响。临床上，这可以通过药理自主封锁14来近似，但同样的技术也可以应用于哺乳动物模型研究内在心脏功能15，16。虽然这种方法可以阻断大部分促成的神经影响，并允许进行体内心脏检查，但它并不能完全消除对心脏的所有外在影响。另一种用于研究动物模型内在心脏功能的研究技术是使用Langendorff香灌心脏的孤立心脏记录，这通常涉及使用电图、起搏或史诗多电极阵列17、18、19、20进行测量。虽然这项技术更具体于心脏功能，因为它涉及从身体去除心脏，测量可能仍然受机械-电力自动调节机制的影响，可能会影响内在心率测量21。孤立的心脏记录也可能仍然受到自主调节的影响，通过ICNS5，6，22，23。此外，在孤立的心脏接近20时，维持与生理相关的心脏温度（对心脏功能测量至关重要）可能很困难。研究 SAN 功能的更直接的方法是专门分离 SAN 组织并测量其活性。这可以通过SAN条（孤立的SAN组织）或孤立的SAN心脏起搏器细胞24，25完成。两者都需要高度的技术培训，因为 SAN 是一个非常小且定义高度明确的区域，并且细胞隔离构成更大的挑战，因为分离如果执行不当，可能会损害细胞的整体健康。此外，这些技术需要专家的电生理技能，以便使用单个记录微电极从组织或细胞成功记录。

在此协议中，我们描述了一种使用微电极阵列 （MEA） 进行体外记录 SAN 的技术，以获得内在心率测量。这种方法的优点是使缺乏密集电生理技能的研究人员能够获得高度特定的电生理记录。MEA以前曾用于研究心肌细胞主要培养物中心肌细胞功能26、27、28、29、30、31、32、心表33、34、35、36、37、38、39和组织整体坐骑40， 41，42，43，44，45，46，47。先前的工作也已完成，以检查现场潜力在SAN组织41，42。在这里，我们提供了一种方法，使用MEA记录和分析穆林内在SAN发射率。我们还描述了如何利用这项技术来测试药物对SAN内在燃烧率的药理学影响，提供一个样本实验，显示4-氨基苯丙胺（4-AP）的效果，一种电压门K+通道阻滞剂。使用定义的解剖地标，我们可以准确地记录 SAN，而无需执行其他方法所需的广泛组织解剖或细胞隔离。虽然 MEA 的成本高得令人望而却步，但录音提供了非常具体和可靠的制进度度，可用于大量的临床和生理研究应用。

Protocol

这里描述的所有实验程序都是根据国家卫生研究院（NIH）的指导方针进行的，该指南是经南方卫理公会大学机构动物护理和使用委员会（IACUC）批准的。 1. 涂层多电极阵列 （MEA） 用于录制 制作 25 mM 护套缓冲器。 溶解 0.953 g Na2B4O7+10 H2O 在 80 mL 蒸馏水中。 将 pH 值调整到 8.4 与 HCl，然后添加蒸馏水到最终体积 100 mL。 <…

Representative Results

在让组织在盘子中适应15分钟后，记录了10个一分钟的痕迹。我们当前的协议记录了一个多小时的活动，但我们在未在此处未显示的未发布数据中记录了 ≥4 h 的稳定发射模式。如果实验准备对数据收集有好处，则每个录制通道应显示给定通道均匀形状的一致且均匀间隔的重复波形（即尖峰）（图 11D）。这些波形对应于反映内在心脏起搏活动的单个心跳。每个通道的间歇间隔…

Discussion

练习和掌握 SAN 解剖过程势在必行，因为组织是脆弱的，健康组织是成功记录的必要条件。在 SAN 解剖过程中，正确的方向对于获得适当的组织区域至关重要。然而，心脏的原始方向很容易在解剖过程中丢失，这使这一努力复杂化。因此，为了确保正确的左右方向，应对阿丽亚进行目视检查。通常，右中庭往往更透明，而左中庭通常更暗，颜色更红25，48。</s…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作由国家卫生研究院资助，赠款编号为R01NS100954和R01NS099188。

Materials

4-Aminopyridine Sigma A78403-25G
22 gauge syringe needle Fisher Scientific 14-826-5A Used for dissection
23 gauge syringe needle Fisher Scientific 14-826-6C Used for dissection
60mm Petri Dishes Genesee Scientific 32-105G
500mL Pyrex Bottle Fisher Scientific 06-414-1C Used to store solutions
1000 mL Pyrex Bottle Fisher Scientific 06-414-1D Used to store solutions
Bone Forceps Fine Science Tools 16060-11
Calcium chloride dihydrate (CaCl2·2H2O) Sigma-Aldrich C5080-500G
Carbogen (95% O2, 5% CO2)
Castroviejo Scissors, 4" Fine Science Tools 15024-10
D-(+)-Glucose Sigma-Aldrich G7021-1KG
Data Acquisition PC CPU: Intel Xeon or Intel Core i7, Memory: 8GB, HDD: 1TB, Graphic Card: NVIDIA or On-board, Screen: 1920×1080
Dissection Microscope Jenco
Dissecting Pins Fine Science Tools 26002-20
Dumont #2 Laminectomy Forceps Fine Science Tools 11223-20
Dumont #55 Forceps Fine Science Tools 11295-51
 Extra Fine Graefe Forceps Fine Science Tools 11152-10
Glass Chamber Grainger 49WF30 Used for mouse euthanization
Harp Anchor Kit Warner Instruments  SHD-22CL/15 WI 64-0247
HCl Fisher Chemicals SA48-4 Used for pH balancing
Hemostat Fine Science Tools 13013-14
Heparin Aurobindo Pharma Limited IDA, Pashamylaram NDC 63739-953-25
HEPES Sigma-Aldrich H3375-250G
Inverted Microscope Motic AE2000
Isoflurane Patterson Veterinary 07-893-1389
Lab Tape Fisher Scientific 15-950
Light for Dissection Microscope Dolan-Jenner MI150DG 660000391014
Magesium chloride (MgCl2) Sigma-Aldrich 208337-100G
MED64 Head Amplifier MED64 MED-A64HE1S
MED64 Main Amplifier MED64 MED-A64MD1A
MED64 Perfusion Cap MED64 MED-KCAP01
MED64 Perfusion Pipe Holder Kit MED64 MED-KPK02
MED64 ThermoConnector MED64 MED-CP04
Mesh  Warner Instruments 640246
Microelectrode array (MEA) Alpha Med Scientific MED-R515A
Mobius Software WitWerx Inc. Specific software for the MED64
NaOH Fisher Chemicals S320-500 Used for pH balancing
Normal Saline Ultigiene NDC 50989-885-17
Paint Brush Fisher Scientific NC1751733
Parafilm Genesee Scientific PM-996
Peristaltic Pump Gilson F155009
Peristaltic Pump Tubing Fisher Scientific 14-171-298 1/8'' Interior Diameter
Polyethyleneimine Sigma P3143
Potassium chloride (KCl) Sigma-Aldrich P9333-500G
Potassium phosphate monobasic (KH2PO4) Sigma-Aldrich P5655-500G
Sodium Bicarbonate Sigma S6297
Sodium chloride (NaCl) Fisher Scientific S671-3
Sylgruard Elastomer Kit Dow Corning 184 SIL ELAST KIT 0.5KG
Sodium Phosphate Monobasic Sigma S6566
Sodium tetraborate Sigma S9640
Surgical Scissors Fine Science Tools 14074-09
Transfer Pipets (3mL graduated) Samco Scientific 225
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Tags

Keywords: Microelectrode ArraySinoatrial NodeHeart RateCardiac PacemakingMouseDissectionTyrode’s SolutionHeart RemovalAtriumSinoatrial Node Location
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Kumar, P., Si, M., Paulhus, K., Glasscock, E. Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice. J. Vis. Exp. (173), e62735, doi:10.3791/62735 (2021).
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