Sultana Hameeda, Neelakanta Girish
Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA.
Methods Mol Biol. 2023;2585:79-95. doi: 10.1007/978-1-0716-2760-0_9.
Several flaviviruses compromise the blood-brain barrier integrity, infect the central nervous system, and elicit neuroinvasion to successfully cause neuropathogenesis in the vertebrate host. Therefore, understanding the pathway(s) and mechanism(s) to block the transmission and/or dissemination of flaviviruses and perhaps other neuroinvasive viruses is considered as an important area of research. Moreover, studies that address mechanism(s) of neuroinvasion by flaviviruses are limited. In this chapter, we discuss detailed methods to isolate exosomes or extracellular vesicles (EVs) from mouse and human N2a cells, primary cultures of murine cortical neurons, and mouse brain tissue. Two different methods including differential ultracentrifugation and density gradient exosome (DG-Exo) isolation are described for the preparation of exosomes/EVs from N2a cells and cortical neurons. In addition, we discuss the detailed DG-Exo method for the isolation of exosomes from murine brain tissue. Studies on neuronal exosomes will perhaps enhance our understanding of the mechanism of neuroinvasion by these deadly viruses.
几种黄病毒会破坏血脑屏障的完整性，感染中枢神经系统，并引发神经侵袭，从而在脊椎动物宿主中成功导致神经病变。因此，了解阻断黄病毒和可能其他神经侵袭性病毒传播和/或扩散的途径和机制被认为是一个重要的研究领域。此外，关于黄病毒神经侵袭机制的研究有限。在本章中，我们将详细讨论从小鼠和人N2a细胞、小鼠皮质神经元原代培养物以及小鼠脑组织中分离外泌体或细胞外囊泡（EVs）的方法。描述了两种不同的方法，包括差速超速离心和密度梯度外泌体（DG-Exo）分离，用于从N2a细胞和皮质神经元中制备外泌体/EVs。此外，我们还讨论了从鼠脑组织中分离外泌体的详细DG-Exo方法。对神经元外泌体的研究可能会增进我们对这些致命病毒神经侵袭机制的理解。
