麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化
Summary
测量中枢神经系统 (CNS) 的交感神经和心血管反应的方法对于推进神经科学是很重要的。该协议的制定, 以帮助科学家测量和量化的急性肾脏交感神经活动 (北美放射学会) 在麻醉大鼠 (不存活) 的变化。
Abstract
肾交感神经活动 (北美放射学会) 和平均动脉压是心血管和自主研究的重要参数;然而, 在测量和分析这些变量的技术上, 有有限的资源指导科学家。本协议描述了麻醉大鼠北美放射学会和平均动脉压力的测量方法。该议定书还包括在中枢神经系统 (CNS) 操作北美放射学会录音期间访问大脑的方法。北美放射学会记录技术与中枢神经系统的药理、optogenetic 或电刺激兼容。当研究人员在非生存实验中测量短期 (最小到 h) 自主性反应时, 这种方法是有用的, 以解剖与中枢神经系统核相关。该方法不打算用于获得北美放射学会在大鼠的慢性 (生存) 记录。北美放射学会的放电, 平均矫正北美放射学会, 平均动脉压力可以量化和分析进一步使用参数统计试验。本文还介绍了获得静脉通路、记录平均动脉压 telemetrically 和脑内固定的方法。
Introduction
关于心血管系统自主控制的临床前发现告知管理疾病的策略, 如高血压、心力衰竭和慢性肾脏疾病。交感神经系统的过度活动和减少的迷走神经心脏口气贡献高血压 (BP)1。慢性升高的肾交感神经流出增强儿茶酚胺分泌, 减少肾血流量, 对心血管/肾脏系统2,3有害后果。为了定义导致自主功能障碍的神经生物学途径, 啮齿类动物的研究对于确定中枢神经系统 (CNS) 神经元调节交感神经参数是很重要的。本议定书的目的是提供有关测量肾交感神经活动 ( 北美放射学会 ) 和 BP 的技术信息 , 并概述在麻醉大鼠中枢神经系统操作中对急感神经变化进行量化的方法。
急性 (非生存) 北美放射学会测量 (持久极小到 h) 是有用的, 当科学家将探测中枢神经系统药理, 电, 或 optogenetically 麻醉大鼠, 以确定特定核的功能。利用这些方法, 研究了孤立核、periaqueductal 灰、pedunculopontine tegmentum 和延髓延髓髓质等结构, 确定了调节交感神经参数4的神经生物学通路, 5,6,7。这一方法对于确定在慢性自主功能障碍8、9的慢性病模型中进一步研究中枢神经系统目标是很重要的。为了完成这些实验, 实验室需要一个焊接铁, 外科显微镜, 立体定向框架, 微电极放大器, 和音频显示器。根据实验室中存在的影响电子噪音的因素, 手术/录音区域可能需要法拉第笼/接地带, 以减少北美放射学会记录中的电噪声。如果大脑分析需要组织固定, 需要一个灌注泵和通风罩。数据可以用多种生理学软件/数据采集 (模拟数字转换器) 单元4、5进行数字化和记录, 并采用不同的分析方法和兼容性, 用于合并遥测信号。.
Protocol
所描述的所有方法都是由芝加哥伊利诺伊大学的机构动物护理委员会批准的。 1. 创建双极北美放射学会电极 要制造电极, 切两件不锈钢丝每约18毫米长。切割一条聚乙烯 (PE-50) 油管约15毫米长。将两条导线进油管, 使导线两端突出。 从导线的两端除去绝缘物;修剪电线留下2-3 毫米裸露的电线。一端, 在暴露的导线上卷曲男性针脚。将针脚安全地焊接到导线上, 确?…
Representative Results
图 1显示了一个戊巴比妥麻醉鼠的样本北美放射学会和 BP 记录。静脉注射肾上腺素被用来诱发平均动脉压力的增加, 并唤起反射和瞬态 sympathoinhibition4,6。为量化北美放射学会, 对未重叠的十年代段的原始北美放射学会进行了矫正和平均;从每个段中减去噪声估计值。 <p class="jove_content" fo:keep-together.within-pag…
Discussion
测量北美放射学会的关键步骤包括: (1) 在将肾脏与椎旁肌分离时避免肾动脉和神经的伸展, 以及将神经段放在记录电极上时 (2) 仔细解剖肾神经纤维从周围组织/血管, (3) 确保电极线没有组织, 血液, 或淋巴液, (4) 防止神经干燥, 通过应用矿物油的肾神经和硅胶的神经电极单位。为了进行故障排除, 必须确保记录系统充分接地。为了获得清晰的北美放射学会信号, 在嵌入硅胶之前, 可以在可视化和侦听?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国立护理研究所 (K99/R00NR014369) 的支持。
Materials
| Stainless steel wire | A-M Systems; Sequim, WA | 791000 | RSNA electrode |
| Polyethylene (PE-50) tubing | VWR; Radnor, PA | 63019-048 | RSNA electrode; vessel cannulation |
| Miniature pin connector | A-M Systems; Sequim, WA | 520200 | RSNA electrode |
| Crimping tool | Daniels Manufacturing Corp.; Orlando, FL | M22520 | RSNA electrode |
| Connector strip | Amphenol; Clinton Township, MI | 221-2653 | RSNA electrode |
| J-B Kwik Epoxy | J-B Weld, Sulphur Springs, TX | 8270 | RSNA electrode |
| Silicone | Permatex; Hartford, CT | 2222 | RSNA electrode |
| Heparin sodium; Injectable (10 mL vial, 1000 U/mL) | KV Veterinary Supply; David City, NE | P03466 | Venous line patency |
| Phenylephrine HCl; Injectable (1 mL vial; 10 mg/mL) | ACE Surgical Supply; Brockton, MA | 950-6312 | Testing renal sympathoinhibition |
| Single-hook elastic surgical stays | Harvard Apparatus; Holliston, MA | 72-2595 | Incision |
| Silk surgical tape | 3M, Minneapolis, MN | 1538-0 | Secure surgical stays |
| Needles, 20 G | Sigma-Aldrich; St. Louis, MO | Z192554-100EA | Vessel cannulation |
| Dumont #7 curved forceps | Fine Science Tools; Foster City, CA | 11274-20 | Vessel cannulation |
| 5-0 silk suture ties | Braintree Scientific; Braintree, MA | SUT-S 106 | Vessel cannulation |
| Delicate hemostatic forceps | Roboz Surgical Instrument Co.; Gaithersburg, MD | RS-7117 | Vessel cannulation and RSNA surgery |
| Crile Hemostatic forceps | Fine Science Tools; Foster City, CA | 13004-14 | Needle bending |
| Telemetry transmitter | Data Sciences International; Minneapolis, MN | PA-10 | Mean arterial pressure monitoring (telemetry) |
| Re-gel syringe | Data Sciences International; Minneapolis, MN | 276-0038-001 | Transmitter reuse (telemetry) |
| Disposable pressure transducer | Transpac; San Clemente, CA | MI-1224 | Mean arterial pressure monitoring |
| Clear-Cuff pressure infuser | MILA International Inc.; Florence, KY | 2281339 | Mean arterial pressure monitoring |
| Vessel cannulation forceps | Fine Science Tools; Foster City, CA | 00574-11 | Catheter insertion |
| Black monofilament nylon 4-0 suture on reverse cutting needle | McKesson Medical-Surgical; San Francisco, CA | S661GX | Secure telemetry transmitter |
| Telemetry receiver | Data Sciences International; Minneapolis, MN | RPC-1 | Mean arterial pressure monitoring (telemetry) |
| LabChart Pro (software), PowerLab (acquisition hardware) | AD Instruments; Colorado Springs, CO | ML846, MX2 matrix 2.0 (Compatible with Data Science International telemetry) | 3 options for software/acquisition hardware |
| SciWorks (software), DataWave (acquisition hardware) | DataWave Technologies, Loveland, CO | N/A | |
| Spike 2 (software), Micro1401-3 | Cambridge Electronic Design Ltd., London UK | 1401-3 | |
| Micro-drill | Roboz Surgical Instrument Co.; Gaithersburg, MD | RS-6300 | CNS surgery |
| Stereotaxic surgery frame | Stoelting; Wood Dale, IL | 51600 | CNS surgery |
| Microelectrode amplifier with 10X pre-amplifier | A-M Systems; Sequim, WA | 1800-2 | RSNA recording |
| Retractors | Fine Science Tools; Foster City, CA | 17009-07 | RSNA surgery |
| Micro-dissecting tweezers | Fine Science Tools; Foster City, CA | 11251-10 | RSNA surgery |
| Micro-hook | Fine Science Tools; Foster City, CA | 10064-14 | RSNA surgery |
| Mineral oil | Fisher Scientific; Waltham, MA | 8042-47-5 | RSNA surgery |
| Audio monitor | A-M Systems; Sequim, WA | 3300 | RSNA surgery |
| Silica gel | Wacker, Munchen; Germany | RT601A-B | RSNA surgery |
| Electrical clips | Tyco Electronics; Schaffhausen, Switzerland | EB0283-000 | Grounding or securing perfusion needle |
| Bonn scissors, straight/sharp points | Roboz Surgical Instrument Co; Gaithersburg, MD | RS-5840 | Perfusion |
| Gavage needle | Harvard Apparatus; Holliston, MA | 75-0286 | Perfusion |
| Masterflex perfusion pump | Cole-Parmer; Vernon Hills, IL | 7524-10 | Perfusion |
| Masterflex platinum-cured silicone tubing | Cole-Parmer; Vernon Hills, IL | 96410-15 | Perfusion |
| Formalin (10% buffered solution; 4 L) | Sigma-Aldrich; St. Louis, MO | HT501128 | Perfusion |
| Sucrose | Sigma-Aldrich; St. Louis, MO | S0389 | Cryoprotection |
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Cite This Article
Fink, A. M., Dean, C. Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats. J. Vis. Exp. (139), e58205, doi:10.3791/58205 (2018).