用于清醒、头部固定大鼠神经成像的头部植入物
Summary
描述了清醒的固定头大鼠功能成像的详细新程序。
Abstract
麻醉剂通常用于临床前和基础科学研究，对大脑的代谢、神经元和血管功能有抑制作用，并可能对神经生理学结果产生不利影响。使用清醒的动物进行研究是有利的，但带来了保持动物平静和静止的主要挑战，以最大限度地减少整个数据采集过程中的运动伪影。在较小尺寸的啮齿动物（例如小鼠）中，清醒成像非常常见，但在大鼠中仍然很少，因为大鼠更大，更强壮，并且在成像所需的长时间内更倾向于反对运动限制和头部固定。描述了使用定制的手工缝制吊带，3D打印头部植入物，头帽和头架的清醒，头部固定大鼠的神经成像新模型。单次单须刺激试验后获得的结果表明诱发功能反应的强度增加。从清醒的固定头部大鼠获得诱发的功能反应比从麻醉大鼠获得更快，可靠，可重复，可用于重复的纵向研究。
Introduction
大多数基础、临床前和转化科学神经影像学研究都是从麻醉动物身上获得的1，2。麻醉剂简化了实验，但不断影响大脑和身体的新陈代谢、血压和心率3.麻醉剂的类型以及给药的持续时间和途径为数据解释增加了混杂变量，这可能导致可重复性和转化失败4。清醒，头部固定大鼠神经影像学研究的一个主要瓶颈是需要在整个制备和数据采集过程中保持大鼠静止和平静。小动作会产生不必要的运动伪影，这可能会对数据分析和解释产生不利影响。
已经设计了一种来自清醒的头部固定大鼠的神经成像新模型，使用定制的吊带，三维（3D）打印的头部植入物，头帽和头架，该模型为易于实验提供了多种优势。3D头部植入物很轻，覆盖了转固定所需的一小部分颅骨。3D打印的头部植入物和帽子是使用计算机辅助设计（CAD）软件设计的。晶须刺激，数据采集，数据分析和麻醉大鼠结果的方案已在以前的工作5，6，7中详细描述。
Protocol
所有程序均符合美国国立卫生研究院的指导方针，并得到加州大学欧文分校动物护理和使用委员会的批准。本研究使用了七只雄性和一只雌性大鼠（Sprague-Dawley，体重：185-350克）。研究完成后，使用二氧化碳过量处死大鼠。 1. 不同组件的设计 头部植入物的设计：使用CAD软件（图1C）制作头部植入物，并将其设计为对前膛后方和…
Representative Results
显示了来自麻醉大鼠单次试验的代表性光学成像信号和清醒大鼠的总和反应（40项收集的试验）（图4）。清醒大鼠单须刺激的信号强度可以在比麻醉大鼠更高的阈值下可视化，显示出来自清醒动物的更强信号。以5Hz刺激大鼠的C2胡须1秒，并且功能反应显示为与基线相比的分数变化。较暗的区域（低于负阈值）是神经元活动的主要区域，明亮的白色区域（高于正阈值）显示含?…
Discussion
使用清醒的头部固定大鼠成像在易用性和定制方面具有许多优势。定制设计的吊索允许通过透气的网材料包裹大鼠，无需将动物长时间封闭在封闭的塑料约束室中10，11。在连续成像会话的长时间内，使用非常低剂量的乙酰丙嗪低于大鼠轻度镇静水平（1.0-2.5mg / kg）12，使大鼠保持冷静和无压力。为了保持大鼠稳定并在成像过程中进一步…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢Clara Jones，James Stirwalt，Linh Hoang，Young Joon Ha和Amirsoheil Zareh在训练老鼠和准备吊索期间提供的帮助。资金由美国国立卫生研究院（NIH，批准号：NS119852）和Leducq基金会（批准号：15CVD02）提供。
Materials
| Rats | Charles River | Sprague Dawley | |
| Isoflurane | Pivetal | 21295098 | General anesthetic |
| Lidocaine HCl 2% injection | Phoenix | L-2000-04 | Local anesthetic |
| Atropine sulfate injection | Vedco | 5098907512 | Help in respiration |
| Lactated Ringer's injection solution | Vedco | 50989088317 | |
| Flunixin injection | Vedco | 6064408670 | Pain management |
| Enrosite injection (Enrofloxacin 2.27%) | VetOne | 501084 | Avoid infection |
| PromAce injection (Acepromazine maleate) | Beohringer Ingelheim | 136059 | |
| Animax ointment | Dechra Veterinary Products | 122-75 | active ingredients of nystatin 1000units per gram, neomycin sulfate 2.5mg per gram, thiostrepton 2500 units per gram, and triamcinolone acetonide 1mg per gram |
| Puralube ophthalmic ointment | Dechra Veterinary Products | 211-38 | |
| Povidone-iodine PVP prep pads | Medline | MDS093917 | Betadine generic |
| Isopropyl alcohol swabs | BD | 326895 | |
| Vetbond tissue adhesive | 3M | 1469SB | |
| Bur (drill bit), standard operatory carbide | SS White Burs | 14829 | #3 bur |
| Screws, 00-90 x 1/8 flat head stainless steel | J.I. Morris | F0090CE125 | Anchor screws |
| Stereotaxic system | Kopf Instruments | 1430 | |
| Homeothermic heating blanket | Harvard Apparatus | 50-7220-F | |
| Pulse oximeter & heart rate monitor | Kent Scientific | MouseStat Jr. | |
| Petrolatum | Fisher Scientific | P66-1LB | Vaseline generic |
| Wire, bare copper | Fisher Scientific | 15-545-2C | 20 gauge |
| Teets Cold Cure powder | Pearson Dental | C73-0054 | active ingredient: Methyl Methacrylate |
| Teets Cold Cure liquid | Pearson Dental | C73-0078 | active ingredient: Methyl Methacrylate |
| Silicone mold rubber | Smooth-On | Body Double Fast | silicon polymer |
| Metricide 28 (Germicide) | Metrex | Oct-05 | |
| India ink, black | Pelikan | 301051 | |
| Dental drill | NSK Dental | Ultimate XL-F | |
| 3D printer | Prusa Research | i3 MK3S+ | |
| Sew on fasteners | Velcro | 90030 | |
| Pet screening utility fabric | Joann | 10173334 | Netting material |
| Bur (drill bit), standard operatory carbide | SS White Burs | 14829 | #1 bur |
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Citar este artigo
Bhatti, M., Malone, H., Hui, G., Frostig, R. D. Head Implants for the Neuroimaging of Awake, Head-Fixed Rats. J. Vis. Exp. (187), e64324, doi:10.3791/64324 (2022).