Department of Neurology, University of Massachusetts Chan Medical School; Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University.
Department of Neurobiology, University of Massachusetts Chan Medical School.
J Vis Exp. 2023 Apr 21(194). doi: 10.3791/64701.
The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.
本方案旨在演示如何在动物大脑的特定细胞类型中，对暴露于外源性刺激后的目标蛋白进行长期的表达和定位可视化。这里，展示了通过闭合性颅骨创伤性脑损伤（TBI）和同时植入颅窗，以便随后对小鼠进行长期活体成像。通过在神经元特异性启动子的控制下，将表达增强型绿色荧光蛋白（EGFP）的腺相关病毒（AAV）颅内注射到小鼠中。2 至 4 周后，使用重物下落装置在 AAV 注射部位对小鼠进行重复 TBI。在同一个手术过程中，将金属头柱和玻璃颅窗植入到 TBI 撞击部位。通过双光子显微镜，在同一脑区中检查 EGFP 的表达和细胞定位，该脑区在数月的时间里持续暴露于创伤。
