Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies

Abstract

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Keywords: biomarkers; brain; central nervous system; cerebrospinal fluid; exosome; extracellular vesicle; recommendations and reporting.

FIGURE 1

CSF physiology and function. (a, top left) CSF flows through the ventricular system and into the subarachnoid space. (a, top right) CSF is produced within the choroid plexus, a pia mater‐derived network of fenestrated blood capillaries within each of the ventricles. AQP1 = aquaporin 1; NCBE = sodium bicarbonate transporter; NKCC1 = sodium–potassium‐chloride cotransporter 1; NHE = sodium–hydrogen exchanger. (b, right) The BBB is formed by the endothelial cells and connected by tight junctions, pericytes, and end‐feet processes of specialised astrocytes. (c, right) The blood‐CSF barrier is formed by capillaries with fenestrated endothelium and epithelial cells in the choroid plexus. The apical region of the choroid plexus epithelium and ependymal cells are connected via tight junctions, both in direct contact with CSF. CSF contains lymphocytes and monocytes involved in CNS immune surveillance. (d) CSF solute gradient, from rostral structures (i.e., lateral ventricles) to caudal lumbar CSF. Total protein concentration exhibits a caudal‐rostral gradient (highest levels in the caudal CSF), while there is a rostro‐caudal gradient for some neurotransmitters like Gamma‐aminobutyric acid (greatest levels in the rostral CSF). Created with BioRender.com.

FIGURE 2

EV and Nanoparticle Composition of CSF. (a) Pooled human CSF from neurologically normal male and female living donors was fractionated by SEC and processed for TEM using a uranyl acetate negative staining protocol, according to methods in Li et al. (2017). Pooled CSF SEC fractions (Fxs) 6–9 (left) show particles that are of the expected size range of EVs (∼50–150 nm) and smaller nanoparticles (<50 nm), Fxs 10–13 (middle) and Fxs 14–17 (right) show an abundance of lipoproteins and proteins in CSF. (b). TEM showing immunogold labeling of human CSF EVs isolated with anti‐CD9 antibody conjugated with 10 nm immunogold. CSF‐derived EVs were separated with an affinity capture kit according to methods in Muraoka, Lin et al. (2020). Scale bars 200 nm.

FIGURE 3

Methodological limitations and knowledge gaps for CSF EV studies. The CSF EV field is challenged by methodological limitations and knowledge gaps pertaining to (i) CSF collection, processing, and storage; (ii) EV separation and analysis and (iii) clinical translatability. Created with BioRender.com.