小鼠轻度创伤性脑损伤的电磁控制闭头模型
Summary
该协议描述了小鼠模型中的轻度创伤性脑损伤。特别是，充分解释了诱导轻度中线闭合性头部损伤的分步方案和动物模型的特征。
Abstract
需要高度可重复的创伤性脑损伤（TBI）动物模型，具有明确的病理，用于测试治疗干预和了解TBI如何改变大脑功能的机制。TBI的多种动物模型的可用性对于模拟人类中TBI的不同方面和严重程度是必要的。本手稿描述了使用中线闭合性头部损伤 （CHI） 开发轻度 TBI 小鼠模型。该模型被认为是轻微的，因为它不会产生基于神经影像学或严重神经元丢失的结构性脑损伤。然而，一次撞击会产生足够的病理，以至于认知障碍在受伤后至少 1 个月是可测量的。论文中定义了使用立体定向引导的电磁撞击器在小鼠中诱导CHI的分步方案。轻度中线CHI模型的优点包括损伤引起的变化的可重复性和低死亡率。该模型在损伤后长达 1 年内对神经影像学、神经化学、神经病理学和行为变化进行了时间表征。该模型与使用相同的撞击装置的受控皮质撞击的开放颅骨模型相辅相成。因此，实验室可以使用相同的撞击器模拟轻度弥漫性 TBI 和局灶性中度至重度 TBI。
Introduction
创伤性脑损伤 （TBI） 是由大脑上的外力引起的，通常与跌倒、运动损伤、身体暴力或交通事故有关。2014 年，美国疾病控制和预防中心确定，有 253 万美国人前往急诊室寻求 TBI 相关事故的医疗帮助1.由于轻度 TBI （mTBI） 占 TBI 病例的大多数，在过去的几十年中，已经采用了多种 mTBI 模型，包括体重下降、活塞驱动的闭合性头部损伤和控制皮质冲击、旋转损伤、轻度液体叩诊损伤和爆炸损伤模型2，3。mTBI模型的异质性有助于解决与人类中观察到的mTBI相关的不同特征，并帮助评估与脑损伤相关的细胞和分子机制。
在常用的闭合性头部损伤模型中，最早也是最广泛使用的模型之一是重量下降法，其中物体从特定高度掉落到动物的头部（麻醉或清醒）2，4。在减重法中，损伤的严重程度取决于几个参数，包括是否进行开颅术，头部固定或自由，以及坠落物体的距离和重量2，4。该模型的一个缺点是损伤严重程度的高度可变性和与呼吸抑制相关的高死亡率5，6。一种常见的替代方案是使用气动或电磁装置进行冲击，这可以直接在暴露的硬脑膜（受控皮质冲击：CCI）或闭合颅骨（闭合性头部损伤：CHI）上进行。活塞驱动损伤的优势之一是其高可重复性和低死亡率。然而，CCI 需要开颅术7，8，而开颅术本身会诱发炎症9。相反，在CHI模型中，不需要开颅手术。如前所述，每个模型都有局限性。本文中描述的CHI模型的局限性之一是使用立体定位框架进行手术，并且动物的头部是固定的。虽然完全头部固定可确保可重复性，但它不考虑撞击后可能导致与mTBI相关的损伤的运动。
该协议描述了在鼠标10中用市售电磁撞击器装置10执行CHI撞击的基本方法。该协议详细说明了实现高度可重复损伤所涉及的确切参数。特别是，研究者可以精确控制参数（损伤深度、停留时间和撞击速度），以精确定义损伤严重程度。如前所述，该CHI模型产生的损伤导致双侧病理，包括弥漫性和微观（即，神经胶质细胞的慢性激活，轴突和血管损伤）和行为表型11，12，13，14，15。此外，所描述的模型被认为是温和的，因为它即使在受伤后 1 年也不会诱导基于 MRI 的结构性脑损伤或病理学上的大体病变16，17。
Protocol
进行的实验得到了肯塔基大学机构动物护理和使用委员会（IACUC）的批准，并且在研究期间遵循了ARRIVE和实验动物护理和使用指南指南。 1. 手术设置 注意：将小鼠饲养在4-5/笼中，房屋室内湿度保持在43%-47%，温度保持在22-23°C。让小鼠 随意 获得食物和水，并暴露于12小时/ 12小时光照/黑暗循环（上午7点/ 下午7点）。 使用指定的?…
Representative Results
这种立体定位电磁冲击器装置用途广泛。它用于开放性颅骨控制皮质冲击 （CCI） 或闭合性头部损伤 （CHI） 手术。此外，可以通过改变损伤参数（如冲击速度、停留时间、撞击深度、撞击器尖端和损伤目标）来调节损伤严重程度。本文描述了使用5.0毫米钢头冲击器的CHI手术。这种损伤被认为是轻微的，因为没有结构性脑损伤。成年小鼠的死亡率低于0.9，14，<sup class="xr…
Discussion
使用所描述的模型重新创建一致的伤害模型涉及几个步骤。首先，将动物正确固定在立体定位框架中至关重要。动物的头部不应能够横向移动，头骨应完全平坦，前膛和λ读取相同的坐标。正确放置耳杆是这种手术中最困难的方面，这只能通过实践来学习。如果颅骨不水平，应在诱发CHI之前调整头部。 未能调整头部位置会导致颅骨骨折。要评估头骨是否扁平，应该从尖端周围的各个角度查看头骨?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院的部分支持，奖励号为R01NS120882，RF1NS119165和R01NS103785以及国防部奖励号AZ190017。内容完全由作者负责，不代表美国国立卫生研究院或国防部的官方观点。
Materials
| 9 mm Autoclip Applier | Braintree scientific | ACS- APL | Surgery |
| 9 mm Autoclip Remover | Braintree scientific | ACS- RMV | Surgery |
| 9 mm Autoclip, Case of 1,000 clips | Braintree scientific | ACS- CS | Surgery (Staples) |
| Aperio ImageScope software | Leica BioSystems | NA | IHC |
| BladeFLASK Blade Remover | Fisher Scientific | 22-444-275 | Surgery |
| Cotton tip applicator | VWR | 89031-270 | Surgery |
| Digitial mouse stereotaxic frame | Stoelting | 51730D | Surgery |
| Dumont #7 Forceps | Roboz | RS-5047 | Surgery |
| Ear bars | Stoelting | 51649 | Surgery |
| EthoVision XT 11.0 | Noldus Information Technology | NA | RAWM |
| Fiber-Lite | Dolan-Jeffer Industries | UN16103-DG | Surgery |
| Fisherbrand Bulb for Small Pipets | Fisher Scientific | 03-448-21 | Head support apparatus |
| Gemini Avoidance System | San Diego Instruments | NA | Active avoidance |
| Heating Pad | Sunbeam | 732500000U | Surgery prep |
| HRP conjugated goat anti-rabbit IgG | Jackson Immuno Research laboratories | 111-065-144 | IHC |
| Induction chamber | Kent Scientific | VetFlo-0530XS | Surgery prep |
| Isoflurane, USP | Covetrus | NDC: 11695-6777-2 | Surgery |
| Mouse gas anesthesia head holder | Stoelting | 51609M | Surgery |
| Neuropactor Stereotaxic Impactor | Neuroscience Tools | n/a | Surgery: Formally distributed by Lecia as impact one |
| NexGen Mouse 500 | Allentown | n/a | Post-surgery, holding cage |
| Parafilm | Bemis | PM992 | Head support apparatus |
| Peanut – Professional Hair Clipper | Whal | 8655-200 | Surgery prep |
| Povidone-Iodine Solution USP, 10% (w/v), 1% (w/v) available Iodine, for laboratory | Ricca | 3955-16 | Surgery |
| Puralube Vet Oinment,petrolatum ophthalmic ointment, Sterile ocular lubricant | Dechra | 17033-211-38 | Surgery |
| Rabbit anti-GFAP | Dako | Z0334 | IHC |
| Rabbit anti-IBA1 | Wako | 019-19741 | IHC |
| 8-arm Radial Arm Water Maze | MazeEngineers | n/a | RAWM |
| Scale | OHAUS CS series | BAL-101 | Surgery prep |
| Scalpel Handle #7 Solid 6.25" | Roboz | RS-9847 | Surgery |
| Sterile Alcohol Prep Pads (isopropyl alcohol 70% v/v) | Fisher Brand | 22-363-750 | Surgery prep |
| SumnoSuite low-flow anesthesia system | Kent Scientific | SS-01 | Surgery |
| 10 mL syringe Luer-Lok Tip | BD Bard-Parker | 302995 | Head support apparatus |
| Timers | Fisher Scientific | 6KED8 | Surgery |
| Topical anesthetic cream | L.M.X 4 | NDC 0496-0882-15 | Surgery prep |
| Triple antibiotic ointment | Major | NDC 0904-0734-31 | Post-surgery |
| Tubing | MasterFlex | 96410-16 | Head support apparatus |
| Vaporizer Single Channel Anesthesia System | Kent Scientific | VetFlo-1210S | Surgery prep |
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Citar este artigo
Macheda, T., Roberts, K., Bachstetter, A. D. Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice. J. Vis. Exp. (187), e64556, doi:10.3791/64556 (2022).