Severe Traumatic Brain Injury Induces Early Changes in the Physical Properties and Protein Composition of Intracranial Extracellular Vesicles

Extracellular vesicles (EVs) are membranous nanostructures that can indicate undergoing processes in organs and thus help in diagnostics and prognostics. They are secreted by all cells, contained in body fluids, and able to transfer proteins, lipids and nucleic acids to distant cells. Intracranial EVs were shown to change their composition after severe traumatic brain injury (TBI) and therefore to have biomarker potential to evaluate brain events. Properties of intracranial EVs early after TBI, however, have not been characterized. Here, we assessed cerebrospinal fluid (CSF) up to seven days after isolated severe TBI for physical properties of EVs and their proteins associated with neuroregeneration. These findings were compared with healthy controls and correlated to patient outcome. The study included 17 patients with TBI and 18 healthy controls. EVs in TBI-CSF were visualized by electron microscopy and confirmed by immunoblotting for membrane associated Flotillin-1 and Flotillin-2. Using nanoparticle tracking analysis, we detected the highest range in EV concentration at day 1 after injury and significantly increased EV size at days 4-7. CSF concentrations of neuroregeneration associated proteins Flotillin-1, ADP-ribosylation Factor 6 (Arf6), and Ras-related protein Rab7a (Rab7a) were monitored by enzyme-linked immunosorbent assays. Flotillin-1 was detected solely in TBI-CSF in about one third of tested patients. Unfavorable outcomes included decreasing Arf6 concentrations and a delayed Rab7a concentration increase in CSF. CSF concentrations of Arf6 and Rab7a were negatively correlated. Our data suggest that the brain response within several days after severe TBI includes shedding of EVs associated with neuroplasticity. Extended studies with a larger number of participants and CSF collected at shorter intervals are necessary to further evaluate neuroregeneration biomarker potential of Rab7a, Arf6, and Flotillin-1.