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Isolation and analysis methods of exosomes in cerebrospinal fluid derived from patients with severe traumatic brain injury

Introduction. Traumatic brain injury (TBI) is a complex clinical condition that commences with primary injury occurring at the moment of trauma, followed by secondary injury characterized by neuroinflammation that leads to neuronal loss, axonal destruction and demyelination. It has recently been proposed that exosomes may mediate immune response after central nervous system injury (1). These small membrane vesicles of endocytic origin are actively secreted from majority of cell types. Depending on cell of origin and its functional status exosomes carry a variety of biologically active molecules such as proteins, mRNAs and microRNAs, including those involved in immune response. Materials and Methods. To investigate whether cerebrospinal fluid (CSF) of patients with severe TBI contains exosomes, we collected samples from ten patients having CSF drainage systems as a standard of care. Information regarding the diagnosis, procedures and outcomes is shown in table 1. CSF was first cleared of cells and debris by sequential centrifugation. The pellet was discarded and the supernatant was passed through a 0.2 μm filter. Exosomes were then isolated using ultracentrifugation procedure or size-exclusion chromatography, detected using western blot analysis for an exosomal biomarker (flotillin-1), or assessing acetylcholinesterase activity. Electron microscopy was used as visualization technique. Results. All samples showed acetylcholinesterase activity and tested positive for flotillin-1 on western blot analysis (figure 1 and 2). Electron microscopy showed cup shaped vesicles smaller than 200 μm in diameter (figure 3). Conclusion. Cerebrospinal fluid derived from patients with severe traumatic brain injury contains exosomes that may have a role in neuroinflammation. References: De Rivero Vaccari JP, Brand III F, Adamczak S, Lee SW, Perez-Barcena J, Wang MY, Bullock MR, Dietrich WD, Keane RW. Exosome-mediated inflammasome signaling after central nervous system injury. Journal of Neurochemistry, 2015, DOI: 10.1111/jnc.13036.

traumatic brain injury; cerebrospinal fluid; exosome isolation and analysis methods; exosomes

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