Rapid Activation of Neuroinflammation in Stroke: Plasma and Extracellular Vesicles Obtained on a Mobile Stroke Unit

Abstract

Background: Neuroinflammation is ubiquitous in acute stroke and worsens outcome. However, the precise timing of the inflammatory response is unknown, hindering the design of acute anti-inflammatory therapeutic interventions. We sought to identify the onset of the neuroinflammatory cascade using a mobile stroke unit.

Methods: The study is a proof-of-concept, cohort investigation of ultra-early blood- and extracellular vesicle-derived markers of neuroinflammation and outcome in acute stroke. Blood was obtained, prehospital, on an mobile stroke unit. Outcomes were biomarker concentrations, modified Rankin Scale score, and National Institutes of Health Stroke Scale score.

Results: Forty-one adults were analyzed, including 15 patients treated on the mobile stroke unit between August 2021 and April 2022, and 26 healthy controls to establish biomarker reference levels. Median patient age was 74 (range, 36-97) years, 60% were female, and 80% White. Ten (67%) were diagnosed as stroke, with 8 (53%) confirmed and 2 likely transient ischemic attack or stroke averted by thrombolysis; 5 were stroke mimics. For strokes, median initial National Institutes of Health Stroke Scale score was 11 (range, 4-19) and 6 (75%) received tPA (tissue-type plasminogen activator). Blood was obtained a median of 58 (range, 36-133) minutes after symptom onset. Within 36 minutes after stroke, plasma IL-6 (interleukin-6), neurofilament light chain, UCH-L1 (ubiquitin C-terminal hydrolase L1), and GFAP (glial fibrillary acidic protein) were elevated by as much as 10 times normal. In EVs, MMP-9 (matrix metalloproteinase-9), CXCL4 (chemokine (C-X-C motif) ligand 4), CRP (C-reactive protein), IL-6, OPN (osteopontin), and PECAM1 (platelet and endothelial cell adhesion molecule 1) were elevated. Inflammatory markers increased rapidly in the first 2 hours and continued rising for 24 hours.

Conclusions: The neuroinflammatory cascade was found to be activated within 36 to 133 minutes after stroke and progresses rapidly. This is earlier than observed previously in humans and suggests injury from neuroinflammation occurs faster than had been surmised. These findings could inform development of acute immunomodulatory stroke therapies and lead to new diagnostic tools and improved outcomes.

Keywords: adult; biomarker; female; interventions; neuroinflammatory diseases.

Figure 1.

Ultra-early neuroinflammatory factor levels in acute stroke patients^† ECL, electrochemiluminescence immunoassay; ELISA, enzyme-linked immunosorbent assay; SIMOA, single molecule array; HSP, heat shock protein; MSU, mobile stroke unit; IL-6, interleukin 6; NfL, neurofilament light chain; UCH-L1, ubiquitin C-terminal hydrolase L1; GFAP, glial fibrillary astrocytic protein; CRP, C-reactive protein; MMP-9, matrix metalloproteinase 9; CXCL4, C-X-C motif ligand 4; PECAM-1, platelet endothelial cell adhesion molecule-1; OPN, osteopontin. ^†All values are median plasma neuroinflammatory factor concentrations, for stroke patients and controls. ^‡For ECL, the reference is 1.89 pg/mL (Todd J., Simpson P., Estis J., Torres V., Wub A. H. Reference range and short- and long-term biological variation of interleukin (IL)-6, IL-17A and tissue necrosis factor-alpha using high sensitivity assays. Cytokine. Dec 2013;64(3):660–665.) *Values for SIMOA results are reduced by a factor of 10 for scale. **For extracellular vesicle analyses, levels are expressed as arbitrary units of pixel density.

Figure 2.

Plasma IL-6 levels in first 133 minutes after stroke onset* MCA, middle cerebral artery; ICH, intracerebral hemorrhage. *Plasma IL-6 levels measured by electrochemiluminescence immunoassay. ^†Todd J., Simpson P., Estis J., Torres V., Wub A. H. Reference range and short- and long-term biological variation of interleukin (IL)-6, IL-17A and tissue necrosis factor-alpha using high sensitivity assays. Cytokine. Dec 2013;64(3):660-665.