Xin-hui Xie,1,* Mian-mian Chen,1,* Shu-xian Xu,1 Junhua Mei,1,2 Qing Yang,2 Chao Wang,1 Honggang Lyu,1 Qian Gong,1 Zhongchun Liu1,3 

1Department of Psychiatry, Renmin Hospital of Wuhan University, Wuhan, Hubei, People’s Republic of China; 2Department of Neurology, Wuhan First Hospital, Wuhan, Hubei, People’s Republic of China; 3Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Xin-hui Xie; Zhongchun Liu, Department of Psychiatry, Renmin Hospital of Wuhan University, No. 99 Jiefang Road, Wuchang District, Wuhan, Hubei, 430060, People’s Republic of China, Email xxh.med@gmail.com; xin-hui.xie@whu.edu.cn; zcliu6@whu.edu.cn.

Introduction: Brain-derived extracellular vesicles (BDEVs) can cross the blood-brain barrier and enter the periphery. Therefore, quantifying and analyzing peripherally circulating BDEVs offer a promising approach to directly obtain a window into central nervous system (CNS) pathobiology in vivo. Rapidly evolving CNS diseases require high-frequency sampling, but daily venipuncture of human subjects is highly invasive and usually unfeasible.
Methods: To address this challenge, here we present a novel method for isolating astrocyte-derived extracellular vesicles from urine (uADEVs), combining urine concentration, ultracentrifugation to isolate total EVs, and then glutamate-aspartate transporter (GLAST) EV isolation using an anti-GLAST antibody.
Results: The identity of these GLAST+EVs as uADEVs was confirmed by transmission electron microscopy, nanoparticle tracking analysis, western blotting, and assessment of astrocyte-related neurotrophins.
Conclusions: Leveraging the convenience and availability of urine samples, the non-invasive uADEV approach provides a novel tool that allows high-frequency sampling to investigate rapidly evolving CNS diseases.

Keywords: urinary astrocyte-derived extracellular vesicles, human in vivo, non-invasive, central nervous system, high-frequency sampling