清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。
Summary
麻醉小鼠表现为非生理系统血压, 由于血压和自主神经系统之间的密切关系, 导致对自主语气有意义的评估。为此, 本文概述了在有意识小鼠中, 用静脉输液同时记录肾交感神经活动和血压的新方法。
Abstract
肾脏交感神经对生理和病理生理学都有显著的促进作用。评价肾交感神经活动 (北美放射学会) 对慢性肾脏疾病、高血压、心力衰竭、糖尿病和肥胖等许多研究领域都有很大的兴趣。因此, 明确评估交感神经系统的作用是正确解释实验结果和了解疾病过程的必要条件。传统上, 北美放射学会被麻醉的啮齿类动物所测量, 包括老鼠。然而, 小鼠在麻醉和手术中通常表现出很低的系统性血压和血流动力学不稳定。鉴于交感神经张力与心血管状态的密切关系, 北美放射学会的意义解释被这种非生理状态所迷惑。为了解决这种传统方法的局限性, 我们开发了一种新的方法来测量有意识的、自由移动的小鼠北美放射学会。对小鼠进行长期的 telemeters 检测, 以持续监测血压以及颈静脉输液导管和定制设计的双极性电极直接记录北美放射学会。在48-72 小时的恢复期后, 存活率为 100%, 所有小鼠正常表现。在这个时间点, 北美放射学会成功地记录在80% 只老鼠身上, 在70% 和50% 的小鼠中, 有可行的信号可以获得4和5天的手术后。所有小鼠 (116±2 mmHg; n=10) 均记录生理血压。记录北美放射学会增加与饮食和梳理, 在文献中建立。此外, 北美放射学会的节阻断和调节血压与药理剂。本文介绍了一种有效的、可管理的北美放射学会在有意识、自由移动的小鼠中清晰记录的方法。
Introduction
随着无数基因工程模型的发展, 对在生物医学研究的几个领域使用小鼠的兴趣继续扩大。在大多数情况下, 技术进步与小鼠在生理学中的使用量的增加保持同步, 现在有一个令人印象深刻的选择, 专门用于测量小鼠重要生理参数的微型设备。尽管在有意识的大鼠中直接测量自主神经音的遥测装置已经使用了十年, 但目前尚无用于评估有意识小鼠神经活动的小型化装置。研究者通常通过评估自主神经系统对间接方法的贡献 (如血浆或尿儿茶酚胺、药理自主阻断、血液形态谱分析) 来规避这一局限性。压力/心率)1。
虽然这些方法提供了有价值的信息, 但结果却是整体自主语气的全球图景, 而不是揭示孤立的神经群对正在调查的现象的离散贡献。另外, 在麻醉小鼠中, 直接记录来自特定神经的活动, 这引起了许多关注。在手术后几个小时内, 在麻醉鼠的生理范围内维持稳定的血压是极其困难的。事实上, 在这些类型的实验中, 血压往往未报告或呈现在极低的水平 (即 60-80 mmHg vs > 100mmHg 在有意识的鼠标)2。在麻醉鼠标制剂中显示的心血管系统的脆弱性往往排除了对自主神经活动的有意义的评估, 因为血压和交感神经张力之间的相互依赖关系3, 4。
为了解决这一局限性, 开发了一种新的方法, 直接记录的肾脏交感神经活动 (北美放射学会) 在有意识的, 无拘无束的小鼠, 不受干扰, 在他们的家庭笼子内。本文详细介绍了该技术成功实施的外科和实验方法。该制剂使研究者可以同时记录动脉压力通过 radiotelemetry 除了北美放射学会, 增加的能力, 静脉注射注入的药物的利益, 而不干扰鼠标。
手术后二十四小时, 小鼠表现正常, 不表现出疼痛或窘迫的迹象。实验记录可以在手术后开始48到72小时, 而老鼠在其家庭笼子里舒适地休息, 不受限制地获得食物、水和环境浓缩。明确的北美放射学会痕迹和这种神经种群对动物正常运动的特征反应 (如进食和梳理) 除了对全身血压的药理调节外, 还表现出来。节封锁进一步验证了北美放射学会信号的质量和特异性。这篇手稿包含了对该技术5最初发布的描述的视听补充。
Protocol
所有的实验程序都符合国家卫生研究院关于实验室动物的护理和使用指南, 并得到密西西比大学医学中心机构动物护理和使用委员会的批准。 1. 动物和住房 房屋老鼠 (24-35 克) 抵达后, 机构实验室动物设施。 在温度和湿度控制环境下, 在实验协议的各个阶段提供老鼠标准啮齿动物和自来水广告随意。 2. 植入式北美放射学会电极的定制制作 注: 在预?…
Representative Results
经描述的协议, 生存率为 100%-所有的小鼠在这项研究中幸存下来, 并恢复良好的手术过程后。在24小时的手术准备, 所有的老鼠表现正常, 展示典型的饮食, 梳理和探索行为。在这个时候, 没有动物显示出任何疼痛或痛苦的迹象。术后48小时, 在12只老鼠中, 有一个可核实和清晰的北美放射学会信号记录在10。这个信号在这些老鼠被维护了72小时后手术, 然而一个真正的北美放射学…
Discussion
在这里, 我们概述, 证明和验证了一种新的方法, 目标评估的北美放射学会在有意识的小鼠, 自由移动和休息舒适, 在他们的家庭笼子。术后植入动脉压 radiotelemeter, 留置静脉输液导管和定制设计的双极北美放射学会电极, 小鼠从手术中恢复, 并没有被不受干扰48至72小时。老鼠仍然舒适地定居在他们的笼子里, 在任何时候 (包括试验期), 不受限制地获得食物, 水和环境丰富。所有随后的实验操作由调查?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
S.M.H. 得到了加拿大卫生研究所 (卫生研究院)、加拿大心脏 & 中风基金会 (HSFC) 和艾伯塔省创新保健解决方案 (AiHS) 博士后研究金的支持;J.E.H. 得到国家心脏、肺和血液研究所 PO1HL-51971 的资助。
Materials
| Teflon-coated stainless steel multiple stranded wire | A-M Systems | 793200 | 0.001in diameter bare; 0.0055in diameter coated |
| #11 Scalpel Blade | Fisher Scientific | ALMM9011 | |
| Soldering Iron and solder | Any make or model suitable | ||
| Male miniature pin connectors | A-M Systems | 520200 | Brass with gold plating |
| Female miniature pin connectors | A-M Systems | 520100 | Brass with gold plating |
| Heat Shrink tubing | Radio Shack | Model #: 278-1610 | Catalog #: 2781610 | 1.6 mm diameter |
| Polyethylene 90 (PE90) tubing | VWR | CA-63018-703 | 0.86mm inner diameter; 1.27mm outer diameter |
| Dissecting microscope | Leica Microsystems | Leica M80 | Any make or model also suitable |
| Polyethylene 10 (PE10) tubing | Braintree Scientific | PE10 50 FT | 0.28mm inner diameter; 0.61mm outer diameter |
| Super Glue Liquid | Loctite | n/a | Liquid Formula; any brand suitable |
| Super Glue Gel | Loctite | n/a | Gel Formula; any brand suitable |
| Polyethylene tubing | Scientific Commodities | BB31695-PE/13 | For pedestal 2.7mm inner diameter; 4.0mm outer diameter |
| Hospital Sterilization Services & Ozone Sterilization packets | Contact local hospital sterilization services | ||
| Isoflurane anesthesia | Abbott | 05260-05 | |
| Deltaphase isothermal heat pads & surgical table | Braintree Scientific | 39OP | Keep heat pads warm in a 37°C water bath; Corresponding surgical table essential |
| Glycopyrrolate | Amdipharm Mercury Company Limited | n/a | |
| Isoflurane vaporizer system & flow gauge | Braintree Scientific | VP I | Include medical grade oxygen supply |
| Tissue scissors | Fine Science Tools | 14173-12 | |
| Fine spring scissors | Fine Science Tools | 15006-09 | |
| Small cotton-tipped applicators | Fisher Scientific | 23400100 | |
| Fine Straight Forceps | Fine Science Tools | 11254-20 | #5, FST by Dumont Biologie Tip |
| Angled Forceps | Fine Science Tools | 11251-35 | #5/45 FST by Dumont |
| Small Absorbent Spears | Fine Science Tools | 18105-03 | |
| Parafilm | Sigma Aldrich | BR701605 ALDRICH | |
| Kwik-Sil 2 component Silicone Polymer | World Precision Instruments (WPI) | KWIK-SIL | Purchase extra specialized tips from WPI |
| 5-0 Polysorb Suture | Tyco Healthcare | n/a | |
| 6-0 Silk Suture | Braintree Scientific | SUT-S 104 | Deknatel brand, spool |
| Radiotelemetry Probe | Data Sciences International (DSI) | TA11-PAC10 | |
| Radiotelemetry Receiver | Data Sciences International (DSI) | PhysioTel RPC-1 | |
| Ambient Pressure Reference | Data Sciences International (DSI) | Apr-01 | |
| Pressure Output Adapter | Data Sciences International (DSI) | R11CPA | |
| Rena Pulse Tubing | Braintree Scientific | RPT-040 | |
| Infusion Swivel | Instech Solomon | 375/D/22 | |
| Swivel Support Arm & Mount | Instech Solomon | SMCLA | |
| Polysulfone button | Instech Solomon | LW62S/6 | |
| Stainless steel spring | Instech Solomon | PS62 | |
| Vetbond surgical adhesive | 3M | n/a | |
| Triple Antibiotic Ointment | Fougera | n/a | |
| PowerLab 8 Channel Data Acquisition System & Software | ADInstruments | PowerLab 8/35 | |
| PVC Insulated Cable | Belden | PVC Audio Connection Cable 32 AWG | |
| Preamplification Headstage | Dagan Corporation | Model 4002 | |
| Differential Amplifier | Dagan Corporation | EX4-400 | |
| Sodium Nitroprusside | Sigma Aldrich | 71778-25G | |
| Phenylephrine | Sigma Aldrich | P6126-5G | |
| Sterile Physiological Saline 0.9% NaCl | Beckton Dickinson | Contact local hospital supplier | |
| hexamethonium | Sigma Aldrich | H0879-5G | |
| Stainless Steel top anti vibration table | n/a | n/a | Custom designed in-house; Solid steel plate on a benchtop is also suitable |
| Faraday cage | n/a | n/a | Custom designed and constructed in-house |
| Small animal hair trimmer | n/a | n/a | Drugstore, men's beard trimmer suitable |
| Dipilatory Cream | n/a | n/a | Veet brand, sensitive skin formula |
| 10% Povidone Iodine | Purdue Products | Betadiene | |
| 70% Ethanol | n/a | n/a | |
| Steel microretractors | n/a | n/a | Made in-house. Bend a steel paper clip & loop 4-0 silk to form a retractor |
| Hemostats | Fine Science Tools | 13011-12 | |
| Heat Gun | Fisher Scientific | 09-201-27 |
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Cite This Article
Hamza, S. M., Hall, J. E. Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.. J. Vis. Exp. (132), e54120, doi:10.3791/54120 (2018).