晚期无脊椎动物中心率微创测量的阻抗肺造影
Summary
在热挑战期间测量心率有助于深入了解由于急性环境变化而对生物体的生理反应。该协议使用美国龙虾（霍马鲁斯美洲）作为模型有机体，将阻抗肺造像描述为一种相对非侵入性和非致命的方法，以测量晚期无脊椎动物的心率。
Abstract
由于世界气候的广泛变化，海洋温度正在迅速上升。由于生物生理学受到环境温度的严重影响，这有可能改变各种海洋生物的热生理性能。该协议使用美国龙虾（霍马鲁斯美洲）作为模型有机体，描述了使用阻抗肺造影来了解晚期无脊椎动物的心脏性能在急性热应力下如何变化。该协议提出了一种微创技术，允许在温度升高实验中实时收集心率。数据很容易纵，以生成一个 Arrhenius 图，用于计算 Arrhenius 破碎温度（ ABT ），心率开始下降的温度随着温度的升高而下降。这种技术可用于各种晚期无脊椎动物（即螃蟹、贝类或虾）。虽然该协议只关注温度对心脏性能的影响，但可以修改，以了解额外的压力因素（例如缺氧或重卡过激）与温度相互作用以影响生理性能的可能性。因此，该方法有可能广泛应用，以进一步了解海洋无脊椎动物如何应对环境的急剧变化。
Introduction
近几十年来，温室气体（即二氧化碳、甲烷和一氧化二氮）对大气的投入增加，导致环境变化模式普遍。世界海洋正在迅速变暖22，3，3这一趋势可能对生物生理学产生严重影响。温度严重影响生理速率，生物体具有最佳温度范围，性能为,4、5、6。,6因此，当温度偏离此范围时，个人在保持适当的氧气输送到组织方面可能会遇到困难。这有可能导致在海洋温度上升5，77时，有氧表演下降。
在实验室环境中，了解环境变化的生理影响的方法是在热应力背景下检查心脏性能。这提供了洞察暴露于预测的变暖条件如何改变性能曲线55，66以及适应可塑性8的可能性。已经成功地实施了多种方法，以测量海洋无脊椎动物的心率。然而，许多这些技术涉及手术切除或主要操纵外骨骼和延长植入测量装置9,9，10，11，,11这给测试对象带来了额外的压力，并增加了在实验前成功恢复所需的时间。此外，侵入性较低的技术（如视觉观察、摄像学）可能仅限于早期生命史阶段，此时生物体可能是完全或半透明的12。此外，对于对技术更先进的方法（例如，通过红外传感器或多普勒灌注88、1111进行观测）的研究人员来说，可能还会面临额外的挑战。
该协议使用美国龙虾（霍马鲁斯美洲）作为晚期海洋无脊椎动物模型，以证明在温度升高实验中使用阻抗肺功能学来评估心率变化。阻抗肺活造术涉及在位于心外两侧的两个电极上传递振荡电流（AC），以测量心脏收缩时电压的变化，并放松13，14。13,该技术具有微创性，因为它使用小电极（即直径为0.10~0.12毫米），这些电极被轻轻地植入外骨骼下方。最后，它使用数据记录器提供在斜坡期间心率和水温的实时评估。
该协议还提供了计算Arrhenius破碎温度（ABT）的说明，即心率随着温度升高13、15,15开始下降的温度开始下降。ABT作为测试受试者容量热极限的非致命指标，可能比测量临界热最大值（CT最大值，心脏功能55，66的上限）更受青睐，因为致命极限往往是极端的，在自然环境中很少遇到。
Protocol
1. 设备设置 环绕清晰、可塑性管绕自身，形成直径约8~10厘米、长40~70厘米的热交换线圈。使用电气胶带固定线圈。 将热交换线圈连接到冷却/加热循环水槽的外部电源和回管。使用软管夹确保连接安全。 用反渗透 （RO） 水填充冷却/加热循环水槽的井，并将电源线插入出口。打开水浴，确保与热交换线圈连接时没有泄漏。 通过将黑色 BNC 电缆插入装置上的交流<…
Representative Results
该协议描述了在温度升高实验中使用阻抗肺造影来获取心率（电压）和温度的实时数据。当穿孔此技术时，记录的电压和温度的振幅将因实验设计和焦点物种而异。但是，当协议正确实现时，实时显示的电压输出遵循一般正则分布（图 1A）。随着竞技场温度的升高，电压的实时分布变化，以反映电压峰值（即心跳）的频率增加;图1B。随着竞技场温度继?…
Discussion
该协议描述了在温度升高实验中使用阻抗肺功能学测量晚期无脊椎动物心率变化。与其他基于实验室的方法99、10、1110,11相比，该技术的主要优点是，它具有微创性，不涉及外骨骼的重大手术操作，从而减少了实验前所需的恢复时间。此外，设备易于使用，结果数据可以在建议的软件程序中简单操作和解释。虽然美国龙虾在这?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢保罗·罗森的实验室援助，国家科学基金会向缅因大学缅因州EPSCoR颁发了IIA-1355457，用于购买设备的资金。该项目得到了美国农业部国家粮食和农业研究所的支持，哈奇项目编号MEO-21811，通过缅因州农业和森林实验站，以及NOAA国家海洋渔业服务Saltonstall肯尼迪赠款#18GAR039-136。作者还感谢三位匿名评论者对本手稿的先前版本的评论。缅因州农业和森林实验站出版物编号3733。
Materials
| 1.6 mm (1/16 in) drill bit | Milwaukee Tool at Home Depot | 1001294900 | This is for a 1.6 mm (1/16 in) diameter drill bit. This item can be found at most home-improvement stores. |
| 38 AWG Copper Magnet Wire | TEMCo | MW0093 | This wire is used to make the wire electrode leads that are implanted into the test subjects. This listing is for a 4 oz coil of 38-gauge magnetic wire. TemCo also has 36-gauge magnetic wire that is also suitable for use in constructing wire electrodes. |
| Cyanoacrylate glue | Loctite | 852882 | This item includes a brush tip, which makes it easier to control the amount of glue used to secure electrodes to the carapace. |
| Ethanol, 70% Solution, Molecular Biology Grade | Fisher BioReagents | BP82931GAL | This reagent is used in combination with the sterile cotton balls to disinfect the carapace prior to electrode implantation. |
| Excel | Microsoft | N/A | This program is used in the protocol for organizing, manipulating, and analyzing data. It is compatible with both PC and Mac operating systems. |
| Fisherbrand 8-Piece Dissection Kit | Fisher Scientific | 08-855 | This kit includes the forceps, scissors, dissecting knife (and blades), and dissecting needle needed to accomplish the electrode implantation steps in the protocol. |
| Fisherbrand Isotemp Refrigerated/Heated Bath Circulators: 5.4-6.5L, 115V/60Hz | Fisher Scientific | 13-874-180 | This is a complete system that consists of an immersion circulator and a bath. It can be used as a temperature controlled bath or to circulate fluid externally to an application. Temperature range of this water bath is -20 to +100 °C, and the unit heats/cools rapidly and is easy to drain upon conclusion of use. |
| Fisherbrand Sterile Cotton Balls | Fisher Scientific | 22-456-885 | These swabs should be soaked in 70% ethanol before being used to disinfect the carapace prior to electrode implantation. |
| Fork Terminal, Red Vinyl, Butted Seam, 22 to 16 AWG, 100 PK | Grainger | 5WHE6 | Terminals are soldered to the magnetic wire to construct the wire electrodes. These can be purchased from a variety of home-improvement vendors. |
| Impedance converter | UFI | Model 2991 | Measures impedance changes correlated with very small voltage changes, ranging from 0.2 ohm to over 5 ohms. This model can convert impedance changes that stem from resistance, capacitance, or inductance variations, as well as a combination of all three. |
| LabChart software | ADInstruments | N/A | Purchase of the PowerLab datalogger includes the LabChart software, but a license for the software can also be directly downloaded online. LabChart allows the user to record data, open and read LabChart files, analyze data, as well as save and export files. There is a free version of the software, LabChart Reader, but users can only open and read LabChart files and analyze them (i.e., it cannot be used to record, save, or export data files). One also has the option of selecting LabChart Pro, which includes LabChart teaching modules that can be used for educational purposes. |
| LED Soldering Iron | Grainger | 28EA35 | This is a generic soldering iron that can be used to solder the magnetic wire to the fork terminals to create the wire electrodes. |
| PowerLab datalogger | ADInstruments | ML826 | There are a variety of models of the PowerLab. This catalog number is for the 2/26 model that is a 2 channel, 16 bit resolution recorder with two analog input channels, independently selectable input sensitivities, two independent analog outputs for stimulation or pulse generation and a trigger input. The PowerLab features a wide range of low-pass filters, AC or DC coupling and adaptive mains filter. This unit has a USB interface for connection to Windows or Mac OS computers and a sampling rate of 100,000 samples/s per channel. |
| Prism8 | GraphPad | N/A | This program provides an additional option for calculating the Arrhenius Break Temperature through its “Segmental linear regression” data analysis option. This program does not require any programming and is compatible with both Mac and Windows operating systems. |
| R | R Project | N/A | This is free software for statistical computing that is compatible with UNIX platforms, as well as Windows and Mac operating systems. This program can also be used to calculate the Arrhenius Break Temperature using the “segmented” package. There are a number of tutorials and user guides available online through the r-project.org website. |
| Rosin Core Solder | Grainger | 331856 | This product has a diameter of 0.031 in (0.76 mm) and is ideal for use in soldering speaker wire (similar gauge as magnetic wire used for electrodes). |
| SAS | SAS Institute | N/A | This program provides an additional option for calculating the Arrhenius Break Temperature. However, it does require programming and is not compatible with Mac operating systems. |
| SigmaPlot | Systat Software, Inc. | N/A | This is the authors’ preferred program for statistical determination of the Arrhenius Break Temperature. The “Regression Wizard” is easy to use and does not require any programming. One can obtain a free 30-day trial license before purchase. However, it is compatible only with PC computers. |
| T-type Pod | ADInstruments | ML312 | Suitable for measurement of temperatures from 0-50 °C using T-type thermocouples. |
| T-type Thermocouple Probe | ADInstruments | MLT1401 | Compatible with the T-type Pod for connection. Measures temperature up to 150 °C, and is suitable for immersion in various solutions, semi-solids, and tissue (includes a needle for implantation). This product is a 0.6 mm diameter isolated probe that is sheathed in chemical-resistant Teflon and a lead length of 1.0 m. |
| UV Cable Tie, Black | Home Depot | 295813 | This is for a 100-pack of 8-inch (20.32 cm), black cable ties. However, based on the size of test subjects, smaller or larger cable ties may be needed. This item, and others like it, can be purchased at any home-improvement store. |
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Cite This Article
Harrington, A. M., Haverkamp, H., Hamlin, H. J. Impedance Pneumography for Minimally Invasive Measurement of Heart Rate in Late Stage Invertebrates. J. Vis. Exp. (158), e61096, doi:10.3791/61096 (2020).