Introduction: Traumatic brain injury (TBI) is a major cause of mortality and disability worldwide. TBI biomarkers secreted from neurons and glial cells after insults have been extensively studied for injury evaluation, management, and prognosis. Despite the advances in TBI biomarker research, the lack of reliable biomarkers that fully corresponded with the pathogenesis and progression of TBI on the molecular level reflects significant unmet healthcare challenges. Extracellular vesicles (EVs) are lipid-bound vesicles secreted by all types of brain cells into body biofluids. Emerging data indicate that EVs are responsible for intercellular communication through specific markers on their surface including DNA, RNA, lipids, protein, and metabolites. Thus alterations of EVs cargo may reflect the state of glial cells and neurons during TBI. Yet, the identification of these markers in the circulating EVs and their role in potentiating secondary injury has been understudied. The present study examined the proteome of plasma EVs in mice using a controlled cortical impact (CCI) model of TBI.
Methods: All animal experiments and surgical procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) from the University of Maryland School of Medicine. Young adult male C57BL/6J mice subjected to CCI surgery were categorized into mild, moderate, and severe injury groups based on the deformation depth of 1.0, 1.5, and 2 mm followed by impact velocity of 3.5 m/s and dwell time 500 ms. After 24h, blood was collected and the EVs were isolated from the platelet-free plasma samples (PFP). First, the larger EVs were isolated from PFP followed by the isolation of smaller EVs by ultracentrifugation method. Smaller EVs were used for all the experiments. The isolated EVs were characterized for Nanoparticle Tracking Analysis (NTA) by ViewSizer and EV markers by western blot analysis. EV proteomics was analyzed using the Olink mouse exploratory panel. Partial Least Squares Discriminant Analysis (PLS-DA) was generated using R and the individual plots of the differentially expressed proteins (DEP) were generated using Graph pad prism. Furthermore, the astrocyte, microglia, and neuron-derived EVs were captured using specific biotinylated antibodies linked to streptavidin magnetic beads. Flow cytometry and western blot analysis were used to confirm the captured EVs. To examine the release of cytokines, primary microglia were plated and direct EVs were added at different concentrations (4 x 109 and 6 x 109 EVs/ml) from the sham and severe TBI groups for 24h, and the supernatant was subjected to CXCL2 ELISA.
Results: NTA showed that there was no significant difference in particle number or the average particle size between the four groups. Western blot analysis confirmed the presence of EV markers in all the groups. PLS-DA plot shows distinct profiles between the sham and severe groups, suggesting the level of differences in their protein cargo. The heatmap of differences in the protein cargo highlighted the alteration between the sham and severe group EV samples. Some of the DEP between the four groups were Eno2, Matn2, Pdgfb, and Tgfb1. Remarkably the level of Eno2 (Enolase-2) was significantly higher in the severe and moderate groups compared to all the other groups suggesting as this can be used as a biomarker in EVs to predict the disease severity. To determine which cell type secrets more Enolase-2, the captured population of the astrocyte (GLAST), microglia (CD11b), and neuron-derived EVs (L1CAM) were stained using the Exo-FITC and the flow cytometry confirmed the staining of EVs on the beads. Western blot analysis showed a higher expression of Enolase-2 in astrocyte-derived EVs along with the presence of EV markers CD9 and CD63, indicating that astrocyte-derived EVs carry elevated levels of Enolase-2 into circulation. In vitro, the direct addition of TBI EVs to the primary microglia induced a significant dose-dependent increase in the CXCL2 levels compared to the sham EVs.
Conclusion: Taken together, this study demonstrates the increased levels of Enolase-2 in the circulating EVs in an injury-severity-dependent manner, and TBI-induced elevation of Enolase-2 was derived from the astrocyte-specific population. Thus, plasma EVs-carried Enolase-2 may act as a potential biomarker involved in pathology and recovery processes during head trauma.