使用双光子显微镜对闭合颅骨创伤性脑损伤和颅窗小鼠荧光蛋白表达进行活体成像
Summary
这项研究表明，向小鼠提供重复性创伤性脑损伤，并同时植入颅窗，以便随后使用双光子显微镜对神经元表达的 EGFP 进行活体成像。
Abstract
该协议的目标是演示在暴露于外源性刺激后，如何纵向可视化动物大脑特定细胞类型中目标蛋白的表达和定位。在这里，显示了闭合颅骨创伤性脑损伤（TBI）的施用和同时植入颅窗以进行随后的小鼠纵向活体成像。向小鼠颅内注射在神经元特异性启动子下表达增强绿色荧光蛋白（EGFP）的腺相关病毒（AAV）。2至4周后，在AAV注射位置使用体重下降装置对小鼠进行重复TBI。在同一手术过程中，将小鼠植入金属头柱，然后在TBI撞击部位植入玻璃颅窗。使用双光子显微镜在暴露于创伤的同一大脑区域检查EGFP的表达和细胞定位，历时数月。
Introduction
创伤性脑损伤 （TBI） 可由运动损伤、车辆碰撞和军事战斗引起，是一个全球性的健康问题。TBI 可导致生理、认知和行为缺陷，以及终身残疾或死亡 1,2。TBI 的严重程度可分为轻度、中度和重度，绝大多数为轻度 TBI （75%-90%）3。人们越来越认识到，TBI，特别是TBI的重复发生，可以促进神经元变性，并成为多种神经退行性疾病的危险因素，包括阿尔茨海默病（AD），肌萎缩侧索硬化症（ALS），额颞叶痴呆（FTD）和慢性创伤性脑病（CTE）4,5,6.然而，TBI诱导的神经退行性变的分子机制仍不清楚，因此是一个活跃的研究领域。为了深入了解神经元如何对 TBI 做出反应并从中恢复，本文描述了一种在 TBI 后通过纵向活体成像在小鼠中监测荧光标记的目标蛋白的方法，特别是在神经元内。
为此，本研究展示了如何将用于闭合颅骨 TBI 的外科手术（类似于之前报道的7,8）与植入颅内窗口的外科手术相结合，用于下游活体成像，如 Goldey 等人所述9.值得注意的是，先植入颅窗，然后在同一区域进行TBI是不可行的，因为诱发TBI的体重下降的影响可能会损坏颅窗并对小鼠造成无法弥补的伤害。因此，该方案旨在在同一手术过程中进行 TBI，然后将颅窗直接植入撞击部位。在一次手术中结合 TBI 和颅窗植入的一个优点是减少了小鼠接受手术的次数。此外，它允许人们监测对 TBI 的即时反应（即，在小时的时间尺度上），而不是在以后的手术过程中植入窗口（即，在 TBI 后几天的时间尺度上开始初始成像）。与通过固定组织免疫染色等传统方法监测神经元蛋白相比，颅窗和活体成像平台也具有优势。例如，活体成像需要较少的小鼠，因为同一只小鼠可以在多个时间点进行研究，而不是在离散时间点需要单独的小鼠队列。此外，可以随着时间的推移监测相同的神经元，从而允许跟踪同一细胞内的特定生物学或病理事件。
作为概念证明，这里证明了突触蛋白启动子下增强的绿色荧光蛋白 （EGFP） 的神经元特异性表达10。这种方法可以扩展到 1） 通过利用其他细胞类型特异性启动子来扩展不同的脑细胞类型，例如少突胶质细胞的髓鞘碱性蛋白 （MBP） 启动子和星形胶质细胞的神经胶质纤维酸性蛋白 （GFAP） 启动子11 ，2） 通过将其基因与 EGFP 基因融合不同的目标蛋白，以及 3） 共表达融合到不同荧光团的多种蛋白质。在这里，EGFP 通过颅内注射通过腺相关病毒 （AAV） 递送进行包装和表达。使用减重装置进行闭合性颅骨 TBI，然后植入颅窗。神经元EGFP的可视化是通过颅窗实现的，使用双光子显微镜检测体内的EGFP荧光。使用双光子激光，可以以最小的光损伤更深入地穿透皮质组织，从而可以对单个小鼠内的相同皮质区域进行数天至数月的重复纵向成像12,13,14,15。总之，这种将 TBI 手术与活体成像相结合的方法旨在促进对导致 TBI 诱发疾病病理学的分子事件的理解16,17。
Protocol
所有与动物相关的方案均按照美国国家研究委员会（US）委员会发布的《实验动物护理和使用指南》进行。该协议已获得马萨诸塞大学陈医学院（UMMS）机构动物护理和使用委员会的批准（许可证编号202100057）。简而言之，如研究示意图（图1）所示，动物接受病毒注射、TBI、窗口植入，然后按时间顺序进行活体成像。 注意：商业条款已被删除。有关使用?…
Representative Results
作为该协议的概念证明，在3个月大时将表达AAV-Syn1-EGFP的病毒颗粒注射到雄性TDP-43Q331K / Q331K小鼠（C57BL / 6J背景）19的大脑皮层中。值得注意的是，也可以使用野生型 C57BL/6J 动物，但这项研究是在 TDP-43 Q331K/Q331K 小鼠中进行的，因为该实验室专注于神经退行性疾病研究。注射 AAV 后 4 周进行 TBI 手术。在相同的手术环境中，植入了头柱和颅窗。在TBI手术后第0天，1周…
Discussion
在这项研究中，将 AAV 注射、TBI 给药和颅窗植入头柱相结合，对小鼠大脑皮层（IV 层和 V 层）内 EGFP 标记的神经元进行纵向成像分析，以观察 TBI 对皮层神经元的影响。这项研究指出，这里选择的 TBI 部位，在海马体上方，为颅窗植入提供了一个相对平坦和宽阔的表面。相反，颅骨在该部位的前方相对较窄，因此很难确保头柱能够有效地接触颅骨表面。虽然本研究中仅使用了TDP-43Q331K / Q331K…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢马萨诸塞大学陈医学院的 Miguel Sena-Esteves 博士赠送了 AAV（PHP.eB）-Syn1-EGFP 病毒，并感谢马萨诸塞大学陈医学院的 Debra Cameron 绘制了小鼠头骨草图。我们还要感谢 Bosco、Schafer 和 Henninger 实验室的现任和前任成员的建议和支持。这项工作由国防部 （W81XWH202071/PRARP） 资助给 DAB、DS 和 NH。
Materials
| Adjustable Precision Applicator Brushes | Parkell | S379 | |
| BD insulin syringe | BD | NDC/HRI#08290-3284-38 | 5/16" x 31G |
| Betadine | Purdue | NDC67618-151-17 | including 7.5% povidone iodine |
| Buprenorphine | PAR Pharmaceutical | NDC 42023-179-05 | |
| Cefazolin | HIKMA Pharmaceutical | NDC 0143-9924-90 | |
| Ceramic Mixing Dish | Parkell | SKU: S387 | For dental cement preparation |
| Cotton Tipped Applicators | ZORO | catlog #: G9531702 | |
| Catalyst | Parkell | S371 | full name: "C" Universal TBB Catalyst |
| Dental cement powder | Parkell | S396 | Radiopaque L-Powder for C&B Metabond |
| Dental drill | Foredom | H.MH-130 | |
| Dental drill controller | Foredom | HP4-310 | |
| Dexamethasone | Phoenix | NDC 57319-519-05 | |
| EF4 carbide bit | Microcopy | Lot# C150113 | Head Dia/Lgth/mm 1.0/4.2 |
| Ethonal | Fisher Scientific | 04355223EA | 75% |
| FG1/4 carbide bit | Microcopy | Lot# C150413 | Head Dia/Lgth/mm 0.5/0.4 |
| FG4 carbide bit | Microcopy | Lot# C150309 | Head Dia/Lgth/mm 1.4/1.1 |
| Headpost | N/A | N/A | Custom-manufactured |
| Heating apparatus | CWE | TC-1000 Mouse | equiped with the stereotaxic instrument and be used while operating surgery |
| Heating blanket | CVS pharmacy | E12107 | extra heating device and be used after surgery |
| Isoflurane | Pivetal | NDC 46066-755-03 | |
| Isoflurane induction chamber | Vetequip | 89012-688 | induction chamber for short |
| Isoflurane volatilizing machine | Vetequip | 911103 | |
| Isoflurane volatilizing machine holder | Vetequip | 901801 | |
| Leica surgical microscope | Leica | LEICA 10450243 | |
| Lubricant ophthalmic ointment | Picetal | NDC 46066-753-55 | |
| Marker pen | Delasco | SMP-BK | |
| Meloxicam | Norbrook | NDC 55529-040-10 | |
| Microinjection pump and its controller | World Precision Instruments | micro4 and UMP3 | |
| Microliter syringe | Hamilton | Hamilton 80014 | 1701 RN, 10 μL gauge for syringe and 32 gauge for needle, 2 in, point style 3 |
| Mosquito forceps | CAROLINA | Item #:625314 | Stainless Steel, Curved, 5 in |
| Depilatory agent | McKesson Corporation | N/A | Nair Hair Aloe & Lanolin Hair Removal Lotion |
| Microscope 1 | Nikon | SMZ745 | Nikon microscope for cranial window preparation |
| Microscope 2 | Zeiss | LSM 7 MP | two-photon microscope |
| Multiphoton laser | Coherent | Chameleon Ultra II, Model: MRU X1, VERDI 18W | laser for two-photon microscopy |
| Non-absorbable surgical suture | Harvard Apparatus | catlog# 59-6860 | 6-0, with round needle |
| Norland Optical Adhesive 81 | Norland Products | NOA 81 | |
| No-Snag Needle Holder | CAROLINA | Item #: 567912 | |
| Quick base liquid | Parkell | S398 | "B" Quick Base For C&B Metabond |
| Regular scissor 1 | Eurostat | eurostat es5-300 | |
| Regular scissor 2 | World Precision Instruments | No. 501759-G | |
| Round cover glass 1 | Warner instruments | CS-5R Cat# 64-0700 | for 5 mm of diameter |
| Round cover glass 2 | Warner instruments | CS-3R Cat# 64-0720 | for 3 mm of diameter |
| Rubber rings | Orings-Online | Item # OO-014-70-50 | O-Rings |
| Saline | Bioworld | L19102411PR | |
| Spring scissor 1 | World Precision Instruments | No. 91500-09 | tip straight |
| Spring scissor 2 | World Precision Instruments | No. 91501-09 | tip curved |
| Stereotaxic platform | KOPF | Model 900LS | |
| Super glue | Henkel | Item #: 1647358 | |
| surgical Caliper | World Precision Instruments | No. 501200 | |
| Surgical forceps 1 | ELECTRON MICROSCOPY SCIENCES | Catlog# 0508-5/45-PO | style 5/45, curved |
| Surgical forceps 2 | ELECTRON MICROSCOPY SCIENCES | catlog# 0103-5-PO | style 5, straight |
| Surgical forceps 3 | ELECTRON MICROSCOPY SCIENCES | catlog# 72912 | |
| Surgical forceps 4 | ELECTRON MICROSCOPY SCIENCES | Catlog# 0508-5/45-PO | style 5/45, curved |
| Surgical gauze | ZORO | catlog #: G0593801 | |
| Surgical lamp | Leica | Leica KL300 LED | |
| UV box | Spectrolinker | XL-1000 | also called UV crosslinker |
| Vaporguard | Vetequip | 931401 | |
| Vetbond Tissue Adhesive | 3M Animal Care | Part Number:014006 |
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Tags
Intravital ImagingFluorescent ProteinTraumatic Brain InjuryCranial WindowTwo-photon MicroscopyNeuronal ResponseMechanical StressGene Of InterestVirus PromoterCell TypesOphthalmic OintmentMidline IncisionPeriosteumStereotaxic FrameTBI Impact SiteImpactor TipDental CementLateral MusclesCranial Window Implantation
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Zhong, J., Gunner, G., Henninger, N., Schafer, D. P., Bosco, D. A. Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope. J. Vis. Exp. (194), e64701, doi:10.3791/64701 (2023).