获取大鼠的静态功能磁共振成像数据
Summary
本协议描述了一种从使用低剂量异黄酮与低剂量脱氧多米丁结合的大鼠获得稳定静息状态功能磁共振成像 （rs-fMRI） 数据的方法。
Abstract
静态功能磁共振成像 （rs-fMRI） 已成为一种越来越流行的方法，以研究处于休息状态、非任务状态的大脑功能。此协议描述了获取 rs-fMRI 数据的临床前生存方法。将低剂量异黄酮与连续输注α 2 肾上腺素受体激动剂脱氧核糖核酸相结合，为稳定、高质量的数据采集提供了强有力的选择，同时保留了大脑网络功能。此外，这个程序允许自发呼吸和接近正常的生理在大鼠。使用此方法，其他成像序列可与静态采集相结合，创建麻醉稳定性高达 5 h 的实验协议。本协议描述了设备的设置、麻醉四个不同阶段对大鼠生理学的监测、获得休息状态扫描、数据质量评估、动物恢复以及处理后数据分析的简短讨论。此协议可用于多种临床前啮齿动物模型，以帮助揭示在休息时发生的大脑网络变化。
Introduction
静止状态功能磁共振成像 （rs-fMRI） 是衡量大脑处于静止状态且不从事任何特定任务时血氧水平依赖 （BOLD） 信号的指标。这些信号可用于测量大脑区域之间的相关性，以确定神经网络中的功能连接。rs-fMRI 广泛应用于临床研究，因为它具有非侵入性，并且患者所需的努力量较低（与基于任务的 fMRI 相比），因此最适合不同的患者群体1。
技术进步使rs-fMRI能够适应啮齿动物模型，以揭示疾病状态背后的机制（参见参考2 供审查）。临床前动物模型，包括疾病或淘汰模型，允许广泛的实验操作不适用于人类，研究还可以利用验尸样本，以进一步加强实验2。然而，由于限制运动和减轻压力的困难，啮齿动物的MRI采集传统上是在麻醉下进行的。麻醉剂，取决于他们的药理动力学，药理动力学和分子靶点，影响脑血流，大脑新陈代谢，并可能影响神经血管耦合通路。
有许多努力，以开发麻醉方案，以保持神经血管耦合和大脑网络功能3，4，5，6，7，8。我们之前报告了麻醉系统，应用低剂量异黄酮以及低剂量的α 2肾上腺素受体激动剂脱氧丙酮9。根据这种麻醉方法，大鼠在与既定投影通路（腹腔和腹腔胸腔核、原发和二级胸膜感官皮层）相一致的区域对胡须刺激表现出强大的BOLD反应：大规模静态脑网络，包括默认模式网络10、11和显着网络12也一直被检测到。此外，这种麻醉方案允许在同一种动物上重复成像，这对纵向监测疾病进展和实验操作的效果非常重要。
在本研究中，我们详细介绍了实验设置、动物准备和生理监测程序。我们特别描述了每个阶段的特定麻醉阶段和扫描采集。每次休息状态扫描后都会评估数据质量。讨论中还包括扫描后分析的简要摘要。有兴趣发现在老鼠身上使用rs-fMRI潜力的实验室会发现这个协议很有用。
Protocol
所有实验均使用 9.4 T MRI 扫描仪进行，并得到了达特茅斯学院机构动物护理和使用委员会的批准。获得额外批准，以记录和显示视频中使用的动物和下面的数字。 1. 扫描前的准备工作 皮下输液管 部分从包装中取出 23 G 针头，使针点保持无菌。 牢固地握住针头的轮毂，并使用剃须刀刀片在针轴与针头接合的地方得分。 将针架夹在得分正下方的轴?…
Representative Results
每次休息状态扫描后，使用独立的组件分析（ICA; 补充文件中包含的示例脚本）评估稳定性。 图 6 显示了休息状态扫描的组件输出示例。 图 6a 显示了高稳定性扫描中的信号组件。请注意，从空间上讲，该组件具有很高的区域性。在空间组件下方的时间过程中，信号是稳定的，不可预测的，表明大脑活动的真实性。底部的功率频谱显示的频…
Discussion
动物的稳定性，无论是身体还是生理，都是获得高质量的休息状态数据的关键。本协议通过通过四个不同的麻醉阶段实现稳定性。在进入麻醉的下一阶段之前，动物必须达到设定的生理阈值：由于这种方法依赖于生理自调节机制，个别动物在每个麻醉阶段可能需要的时间略有不同。我们的经验是，在每个阶段花更多的时间比匆忙通过早期阶段，而不给老鼠的生理学足够的时间来解决更有效。允许?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究所国家药物滥用研究所（NIDA）的资金支持。 EDKS 和 EMB 得到了授予艾伦 I. 格林和 DJW 的格兰特 R21DA044501 的支持，格兰特 T32DA037202 授予艾伦 J. Budney] 和国家酒精滥用和酒精中毒研究所 （NIAAA） [授予艾米丽 D. K. 沙利文 F31AA028413] 。作为达特茅斯精神病学教授雷蒙德·索贝尔教授，艾伦·格林的基金提供了额外的支持。
陆汉兵得到了国家药物滥用研究所耳内研究项目国家卫生研究院的支持。
作者要感谢和感谢已故的艾伦·格林。他对共同发生的疾病领域的坚定不移的奉献有助于建立作者之间的协作。我们感谢他的指导和指导，这将非常怀念他。
Materials
| 9.4T MRI | Varian/Bruker | Varian upgraded with Bruker console running Paravision 6.0.1 software | |
| Air-Oxygen Mixer | Sechrist | Model 3500CP-G | |
| Analysis of Functional NeuroImages (AFNI) | NIMH/NIH | Version AFNI_18.3.03 | Freely available at: https://afni.nimh.nih.gov/ |
| Animal Cradle | RAPID Biomedical | LHRXGS-00563 | rat holder with bite bar, nose cone and ear bars |
| Animal Physiology Monitoring & Gating System | SAII | Model 1025 | MR-compatible system including oxygen saturation, temperature, respiration and fiber optic pulse oximetry add-on |
| Antisedan (atipamezole hydrochloride) | Patterson Veterinary | 07-867-7097 | Zoetis, Manufacturer Item #10000449 |
| Ceramic MRI-Safe Scissors | MRIequip.com | MT-6003 | |
| Clippers | Patterson Veterinary | 07-882-1032 | Wahl touch-up trimmer combo kit, Manufacturer Item #09990-1201 |
| Dexmedesed (dexmedetomidine hydrochloride) | Patterson Veterinary | 07-893-1801 | Dechra Veterinary Products, Manufacturer Item#17033-005-10 |
| Digital Rectal Thermometer Covers | Medline | MDS9608 | |
| FMRIB Software Library | FMRIB | MELODIC Version 3.15 | Freely available at: https://fsl.fmrib.ox.ac.uk/fsl/fslwiki |
| Heating Pad | Cara Inc. | Model 50 | |
| Hemostat forceps, straight | Kent Scientific | INS750451-2 | |
| Isoflurane | Patterson Veterinary | 07-893-1389 | Patterson Private Label, Manufacturer Item #14043-0704-06 |
| Isoflurane Vaporizer | VetEquip Inc. | 911103 | |
| Lab Tape, 3/4" | VWR International | 89097-990 | |
| Needles, 23 gauge | BD | 305145 | plastic hub removed |
| Parafilm Laboratory Film | Patterson Veterinary | 07-893-0260 | Medline Industries Inc., Manufacturer Item #HSFHS234526A |
| Planar Surface Coil | Bruker | T12609 | 2cm |
| Polyethylene Tubing | Braintree Scientific | PE50 50FT | 0.023" (inner diameter), 0.038" (outer diameter) |
| Puralube Ophthalmic Ointment | Patterson Veterinary | 07-888-2572 | Dechra Veterinary Products, Manufacturer Item #211-38 |
| Sprague Dawley Rats | Charles River | 400 SAS SD | |
| Sterile 0.9% Saline Solution | Patterson Veterinary | 07-892-4348 | Aspen Vet, Manufacturer Item #14208186 |
| Sterile Alcohol Prep Pads | Medline | MDS090735 | |
| Surgical Tape, 1" (3M Durapore) | Medline | MMM15381Z | 3M Healthcare, "wide medical tape" |
| Surgical White Paper Tape, 1/2" (3M Micropore) | Medline | MMM15300 | 3M Healthcare |
| Syringes, 1 mL w/ 25 gauge needle | BD | 309626 | |
| Syringes, 3 mL | BD | 309657 | |
| Vented induction and scavenging system | VetEquip Inc. | 942102 | 2 liter induction chamber with active scavenging |
| 411724 | omega flowmeter | ||
| 931600 | scavenging cube, "vacuum" | ||
| 921616 | nose cone, non-rebreathing |
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Citazione di questo articolo
Wallin, D. J., Sullivan, E. D. K., Bragg, E. M., Khokhar, J. Y., Lu, H., Doucette, W. T. Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat. J. Vis. Exp. (174), e62596, doi:10.3791/62596 (2021).