Monitoring of Post-Brain Injuries By Measuring Plasma Levels of Neuron-Derived Extracellular Vesicles

Abstract

Background: Extracellular vesicles (EV) released from neurons into the blood can reflect the state of nervous tissue. Measurement of neuron derived EV (NDE) may serve as an indicator of brain injury. Methods: A sandwich immunoassay was established to measure plasma NDE using anti-neuron CD171 and anti-EV CD9 ([CD171 + CD9+]). Plasma samples were obtained from commercial sources, cross-country (n = 9), football (n = 22), soccer (n = 19), and rugby (n = 18) athletes over time. Plasma was also collected from patients undergoing total aortic arch replacement (TAR) with selective cerebral perfusion during cardiopulmonary bypass before and after surgery (n = 36). Results: The specificity, linearity, and reproducibility of NDE assay (measurement of [CD171 + CD9+]) were confirmed. By scanning electron microscopy and nanoparticle tracking, spherical vesicles ranging in size from 150 to 300 nm were confirmed. Plasma levels of NDE were widely spread over 2 to 3 logs in different individuals with a significant age-dependent decrease. However, NDE were very stable in each individual within a ± 50% change over time (cross-country, football, soccer), whereas rugby players were more variable over 4 years. In patients undergoing TAR, NDE increased rapidly in days post-surgery and were significantly ( P = .0004) higher in those developing postoperative delirium (POD) (n = 13) than non-delirium patients (n = 23). Conclusions: The blood test to determine plasma levels of NDE was established by a sandwich immunoassay using 2 antibodies against neuron (CD171) and exosomes (CD9). NDE levels varied widely in different individuals and decreased with age, indicating that NDE levels should be considered as a normalizer of NDE biomarker studies. However, NDE levels were stable over time in each individual, and increased rapidly after TAR with greater increases associated with patients developing POD. This assay may serve as a surrogate for evaluating and monitoring brain injuries.

Elevation of astrocyte-derived extracellular vesicles over the first month post-stroke in humans

“…The protocol for the double label sandwich ELISA immunoassay in this study is similar to a previous publication 21 which demonstrated the size (100–200 nm, consistent with small EVs) and electron micrographic characterization of NDEs using this method. In the current study, EV data represent the double label of the CD171 NDEs or EAAT1 ADEs or MOG ODEs with the CD9 common EV marker.…”

“…A prior study used control plasma samples applied to anti-L1CAM (neuronal marker) and anti-CD81 (common EV marker 35 ) immobilized ELISA wells. Subsequently the ratio of various other anti-neuronal and anti-non-neuronal antibodies were used to compare the two well types 21 . The ratio of these antibodies in anti-L1CAM versus anti-CD81 wells was high for neuronal markers (S100B, AchE, NRGN, CTSD, SV2A, SYNPO, SNAP25) and low for non-neuronal markers (CD9, GAPDH, ALDO, SFTPB, PSEN, BACE).…”

“…Patients with hemorrhagic transformation have significantly greater morbidity and mortality 19 , 20 . As it pertains to BDEs, a prior study showed increased NDE levels immediately after aortic arch replacement surgery that subsided over time and correlated with post-operative delirium 21 . No prior studies have directly assessed multiple BDE types (neural, astrocytic, oligodendrocytic) following human ischemic stroke, nor have they assessed BDEs in the setting of hemorrhagic transformation.…”

An approach for the analysis of axonal neuroinflammation by measuring dual biomarkers of oligodendrocytes and inflammatory cytokine in human plasma

“…When we tested 63 control plasma samples, values were widely distributed over 2 logs (Supplemental Fig. 1), similar to our previous studies on neuron-derived EV (10). Thus, as shown in the next section, we focused on the longitudinal studies.…”

“…Plasma samples from high school cross-country and American football players (Indiana University, Bloomington, IN), ice hockey (University of Louisiana at Lafayette, Lafayette, LA), and college rugby (Keio University, Kanagawa, Japan) were the same as our previous studies (10)(11) with additional samples of cross-country and football from different high schools. Detail of these samples were described in the Results of those studies (10)(11). For capillary blood collection, we used Tasso device (Seattle, WA) in Fig.…”