Single Extracellular Vesicle Profiling to Define Brain Specific Traumatic Brain Injury Induced Neuro-Inflammation

Abstract

Traumatic Brain Injury (TBI) triggers secondary molecular processes that contribute to mortality and morbidity. Neuroinflammation is a key factor affecting patient outcomes both acutely and chronically. Traditional diagnostic tools, such as computed tomography imaging and the Glasgow Coma Scale, are limited in detecting molecular changes, particularly related to neuroinflammation. Small extracellular vesicles (sEVs) are cell-specific vesicles that enable cell-to-cell communication and are involved in TBI pathology. In this study, brain-specific sEVs are isolated by targeting brain-associated markers, sodium/potassium-transporting ATPase subunit beta-2 (ATP1B2) and excitatory amino acid transporter 2 (EAAT2), and employed surface-enhanced Raman spectroscopy to profile inflammation-associated cytokine chemokine (C-C motif) ligand 2 (CCL2) bound to single sEV, allowing for blood-based monitoring of neuroinflammation. This approach enabled the direct assessment of neuroinflammation in both human TBI samples and a controlled cortical injury in a rat model. This study found elevated brain-specific sEVs with enhanced CCL2 in TBI samples compared to non-TBI cohorts. The results suggest that the TBI diagnostic platform can detect an increased level of brain-specific sEVs carrying neuroinflammatory signals in TBI clinical samples with high specificity and sensitivity, offering potential as a precise diagnostic tool for TBI diagnosis.

Keywords: TBI; cytokine; extracellular vesicle; liquid biopsy; neuroinflammation.

Figure 1

Single extracellular vesicle profiling to define brain specific TBI induced neuro‐inflammation. A) Astrocyte sEVs bind cytokines present in a neuroinflammatory microenvironment. B) sEVs cross the disrupted blood‐brain barrier (BBB) into peripheral blood and are subsequently isolated from plasma C) The single sEV readout platform captures individual sEVs, phenotypically barcoded with SERS nanobox reporters. The process includes: 1. activation of the gold surface with the crosslinker DSP, 2. immobilization of capture antibodies, 3. binding of sEVs, and 4. incubation with SERS nanobox reporters. D) BBB breakdown and neuroinflammatory changes are quantified in healthy controls and TBI patients by the amount of brain specific sEVs positive for CCL2.

Figure 2

Brain‐specific sEV characterization. A) TEM image of brain organoid sEVs. B) Brain organoid (brain org) sEV characterization by NTA, demonstrating the size range of purified sEVs primarily between 50 and 100 nm. Nano flow cytometry of the sEV marker CD81: C) negative control (antibody only); D) the expression of tetraspanin marker CD81 on brain organoid sEVs. E) SERS signals of canonical tetraspanin markers CD9, CD63, and CD81, indicating the successful capture of brain sEVs using ATP1B2 and EAAT2 on the nanopillar. F) Quantification of nanopillars positive for the tetraspanin makers from sEVs isolated from human brain organoid cultures. Data are represented as mean ± SD. ^** p < 0.01.

Figure 3

Brain sEVs marker validation. A) SERS images of sEVs from human brain organoids and control sEVs from lung cells (H1975). Red and blue dots indicate ATP1B2 and EAAT2 positive sEVs respectively. B) Quantification of nanopillars that are positive for our candidate brain specific markers ATP1B2 and EAAT2. Data are represented as mean ± SD. ^* p < 0.05, ^** p < 0.01.

Figure 4

Specific profiling of brain sEVs in rats after a controlled cortical impact. A) Representative SERS mapping images of the sEVs from rats with a TBI and control sham rats. B) Corresponding sEV molecular profiles from panel A demonstrating elevated ATP1B2, EAAT2, and CCL2 in response to a TBI are sustained for 60 days post‐injury. n = 4 (TBI); n = 5 (Sham), data are represented as mean ± SD. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 5

Analysis of brain sEVs signature in healthy individuals and in patients with TBI. A) Representative SERS mapping images of the brain‐specific sEVs from TBI patients compared to healthy controls. B) Corresponding sEV profiles from panel A demonstrating elevated ATP1B2, EAAT2, and CCL2 in patients with a TBI. n = 15 (healthy) n = 26 (TBI), data are represented as mean ± SD. ^**** p < 0.001. C) ROC curve analysis for each marker between healthy and TBI patients.