在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量
Summary
本文介绍了通过植入遥测装置在整个疾病进展全身体积描记术和肌电自由表现肌萎缩侧索硬化症（ALS）小鼠模型中的通风和呼吸肌肉活动的重复测量的方法。
Abstract
辅助呼吸肌帮助时膈肌功能受损保持通风。以下方案描述了一种用于在数周或辅助呼吸肌肉活动的个月，同时测量在非麻醉，自由地表现小鼠通风重复测量的方法。该技术包括一个无线电发射器的外科植入和电极的插入通向斜角肌和斜方肌测量这些吸气肌肉的肌电活动。通风是通过全身体积描记术测量的，和动物运动由视频评估，并与肌电活动同步。肌萎缩侧索硬化症的小鼠模型中的肌肉活动和通风的测量都显示这个工具如何可以用来研究肌肉活动如何随时间变化的呼吸和评估肌肉活动对通风的影响。所描述的方法可以通过电子邮件asily适合于测量其它肌肉的活动或疾病或损伤的额外的鼠标模型来评估辅助呼吸肌活动。
Introduction
在高需求（ 如运动）的时间辅助呼吸肌（ARMS）加大通风量，并有助于在隔膜功能受损以下损伤或疾病1，2，保持通风。虽然在光圈功能变化在肌萎缩侧索硬化（ALS）的患者和小鼠模型已经很好地描述3，4，5，6，较少被知道关于在ALS臂的活性或功能。然而，一项研究表明，招募ARM的ALS患者比那些具有类似隔膜功能障碍不7预后较好。此外，ARM活性足以在隔膜麻痹8的情况下呼吸。这些研究表明，战略，以增强ARM功能可以改善breathi纳克在从神经肌肉疾病，脊髓损伤，或在其中振动板功能受损的其它条件的患者。然而，控制ARM招聘呼吸的机制在很大程度上是未知的。方法来测量呼吸功能，并研究组是如何招募需要在疾病或损伤的动物模型时间ARM活动的变化，以及评价疗法来改善ARM招聘和通风。此外，手臂与光圈功能的渐进性丧失一致的增加的活性可以是用于神经肌肉疾病如ALS 7，9，10的疾病进展的有用的生物标志物。
该方案描述于非侵入性的方法（以下初始手术）和重复地测量在清醒，表现小鼠呼吸肌和通风的活性。肌电图的同步录音Y（EMG），全身体积描记法（WBP）和视频允许研究者评估ARM活动冲击通风如何变化，并确定当被摄体处于静止或移动的。这种方法的一个主要优点是，它可以在清醒，表现小鼠中执行，而一些可替代的方法来测量的EMG需要麻醉和/或是终端的程序11，12，13。 EMG活动的对清醒小鼠随时间的记录，也可以通过EMG的长期植入引线，其中，所述鼠标通过导线拴到采集系统14，15来完成的。因为栓系小鼠可与正常运动或行为干扰和可能不与一个标准的体积描记法室兼容，所描述的方法使用遥测装置以无线方式传输的EMG信号的采集系统。变送器可以上或关闭用磁铁以节省电池电量，并允许重复EMG活动的测量超过几个月。该协议可以很容易地适应通过将EMG测量的额外呼吸或非呼吸肌活性通向不同的肌肉。可替代地，两个引线中的一个可以被用于测量脑电活动来评估睡眠状态或识别发作活动16。该技术已成功用于测量在休息在整个疾病进展的ALS小鼠模型中ARM活性的变化，并确定关键的神经元驱动臂活动健康小鼠10。
Protocol
实验过程是由辛辛那提儿童医院医学中心的机构动物护理和使用委员会批准，并遵守NIH指南护理和使用实验动物进行。 1.准备遥测装置植入手术穿戴个人防护设备（ 即，磨砂，鞋套，袍，净毛，掩码和手术手套）。 注：此手术需要一个无菌区。 打开培养箱（伺服控制加湿器/婴儿培养箱设定为29℃）并用干燥，白毛巾线它，以允许恢复正确变暖。 在手术之前…
Representative Results
所描述的方案用于植入遥测设备，并记录和斜角肌斜方肌EMG，WBP和SOD1（G93A）ALS模型小鼠的视频。时段，其中所述动物是无活性的（ 例如，不动）用的视频记录确定并通过在微量WBP（ 图3A）缺乏运动相关的活动的确认。不活动时段包括在REM或非REM睡眠所花费的时间，以及时间花费清醒，但仍（ 图3A）。在这个非激活时间EMG活动记录为一个回…
Discussion
这里展示的程序允许呼吸肌肉活动和通风的无创（发射机的初始手术植入后）测量了在相同的动物数月。该技术具有超过在麻醉的小鼠标准EMG技术的若干优点：1）在实验中需要更少的小鼠，并提供在跨越疾病阶段的单个小鼠（以记录的，而不是使用多个小鼠在不同的疾病阶段）来自同一个站点数据的能力; 2）数据分析可以与更强大的统计测试（ 即，使用重复测量的，而不是单独的比较实?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
对这项工作的支持是由辛辛那提儿童医院医疗中心理事奖SAC和美国国立卫生研究院培训资助（T32NS007453）到VNJ提供
Materials
| B6.Cg-Tg (SOD1*G93A)1 Gur/J | Jackson Laboratory | 4435 | |
| Plethysmography Chamber | Buxco Respiratory Products/ Data Sciences International | 601-1425-001 | |
| Telemetry Receivers (Model RPC-1) | Data Sciences International | 272-6001-001 | |
| Bias Flow Pump (Model BFL0500) | Data Sciences International | 601-2201-001 | |
| ACQ-7000 USB | Data Sciences International | PNM-P3P-7002XS | |
| Dataquest A.R.T. Data Exchange Matrix | Data Sciences International | 271-0117-001 | |
| New Ponemah Analysis System | Data Sciences International | PNM-POST-CFG | |
| Ponemah Physiology Platform Acqusition software v5.20 | Data Sciences International | PNM-P3P-520 | |
| Ponemah Unrestrained Whole Breath Plethysmography analysis package v5.20 | Data Sciences International | PNM-URP100W | |
| Configured Ponemah Software System | Data Sciences International | PNM-P3P-CFG | |
| Analysis Module (URP) | Data Sciences International | PNM-URP100W | |
| Universal Amplifier | Data Sciences International | 13-7715-59 | |
| Sync Board | Data Sciences International | 271-0401-001 | |
| Sync Cable | Data Sciences International | 274-0030-001 | |
| Transducer-Pressure Buxco | Data Sciences International | 600-1114-001 | |
| Flow Meter | Data Sciences International | 600-1260-001 | |
| Magnet and Radio included in F20-EET Starter Kit | Data Sciences International | 276-0400-001 | |
| Axis P1363 Video Camera | Data Sciences International | 275-0201-001 | |
| Terg-A-Zyme | Fisher Scientific | 50-821-785 | Enzyme Detergent |
| Actril | Minntech Corporation | 78337-000 | Chemical Sterilant |
| Stereo Dissecting Microscope (Model MEB126) | Leica | 10-450-508 | |
| Servo-Controlled Humidifier/Infant Incubator | OHMEDA Ohio Care Plus | 6600-0506-803 | |
| TL11M2-F20-EET Transmitters | Data Sciences International | 270-0124-001 | |
| Dumont #2 Laminectomy Forceps – Standard Tips/Straight/12cm (x2) | Fine Scientific Instruments | 11223-20 | For handling wires |
| Dumont #2 Laminectomy Forceps – Standard Tips/Straight/12cm (x2) | Fine Scientific Instruments | 11223-20 | For surgery |
| Narrow Pattern Forceps- Serrated/Curved/12cm | Fine Scientific Instruments | 17003-12 | |
| Spring Scissors – Tough Cut/Straight/Sharp/12.5cm/6mm Cutting Edge | Fine Scientific Instruments | 15124-12 | |
| Tissue Separating Scissors – Straight/Blunt-Blunt/11.5cm | Fine Scientific Instruments | 14072-10 | |
| Fine Scissors – Tough Cut/Curved/Sharp-Sharp/9 cm | Fine Scientific Instruments | 14058-11 | For cutting wires and clipping nails |
| Scalpel Handle #3 | World Precision Instruments | 500236 | |
| Scalpel Blade | Fine Scientific Instruments | 10010-00 | For preparing lead caps |
| Polysorb Braided Absorbable suture | Coviden | D4G1532X | For coiling transmitter leads |
| Gluture | Zoetis Inc. | 6606-65-1 | Cyanoacrylate adhesive |
| 3 mL Syring Slip Tip – Soft | Vitality Medical | 118030055 | |
| 25G Needle (X2) | Becton Dickinson and Co. | 305-145 | |
| Cotton Tipped Applicators | Henry Schein Animal Health | 100-9175 | |
| Andis Easy Cut Hair Clipper Set | Andis | 049-06-0271 | Electrical Razor sold at Target |
| Isoflurane | Henry Schein Animal Health | 29404 | Anesthetic |
| Isopropyl Alcohol 70% | Priority Care 1 | MS070PC | |
| Dermachlor 2% Medical Scrub (chlorohexidine 2%) | Butler Schein | 55482 | |
| Artificial Tears | Henry Schein Animal Health | 48272 | Lubricant Opthalmic Ointment |
| Vacuum grease | Dow Corning Corporation | 1597418 | |
| Water Blanket | JorVet | JOR784BN |
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Citer Cet Article
Jensen, V. N., Romer, S. H., Turner, S. M., Crone, S. A. Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease. J. Vis. Exp. (122), e55599, doi:10.3791/55599 (2017).