EVALUATION OF LYMPHATIC AND GLYMPHATIC ASSOCIATED EXTRACELLULAR VESICLE BIOMARKERS FOR SPORT-RELATED CONCUSSION

Abstract

Purpose: Interdisciplinary research in epidemiology, neurology, neuroscience, and sports medicine commonly highlight the dangerous short- and long-term sequelae of sport-related concussions (SRC). Despite advancements in clinical evaluation and recognition, many SRCs are not properly diagnosed and managed, leaving many athletes in danger of acute and chronic neurological deficits. Epidemiological studies suggest the prevalence of chronic traumatic encephalopathy (CTE) is three times, and Alzheimer's disease is four times greater in former athletes with a history of SRC than non-athletes. The underlying mechanisms linking SRC and contact-sport participation to neurodegeneration are not fully understood. Herein, I hypothesized that transient insufficiency of the lymphatic and glymphatic clearance systems in the central nervous system (CNS) could play a crucial role in the SRC-mediated neurological conditions. Therefore, this study aimed to examine the differences in plasma levels of extracellular vesicles (EV) that are associated with the lymphatic and glymphatic clearance systems of the CNS among athletes following sport-related head impacts. Participants: Plasma EV concentrations were analyzed in collegiate athletes (controls n=29, SRC n=19) with and without SRC. In a parallel study, fourteen college-aged soccer players participated in a laboratory-based, repetitive subconcussion paradigm. All participants provided written informed consent, and the study was approved by institutional review board at Temple University. Methods: We evaluated EVs containing markers associated with the CNS lymphatic and glymphatic systems, including lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), astrocyte-specific glial fibrillary acidic protein (GFAP), aquaporin 4 (AQP4), and the platelet and endothelial cell adhesion molecule 1(PECAM-1 or CD31). Tetraspanin-28 (CD81) was used as an EV-specific marker. Blood samples from athlete controls were collected once during preseason baseline assessments. Samples from athletes with SRC were collected within 72 hours of injury. Whole blood was double-centrifuged to obtain platelet-poor plasma, snap-frozen in liquid nitrogen, and stored at -80°C until analyzed. Quantification of plasma EVs was performed using spectral flow cytometry. Mann-Whitney U tests were used for group comparisons of single and double-positive EV concentrations, and receiver-operating characteristic curve (ROC) and area under the curve (AUC) analyses assessed diagnostic efficacy. Within-group changes in plasma EVs following repetitive, subconcussive head impacts were assessed with Friedman's test using Dunn's correction for multiple comparisons. Results: Among athletes with SRC, plasma concentrations of LYVE1+EVs and CD31+EVs were significantly elevated within 72 hours of injury compared to controls (LYVE1+EVs, p < 0.0001; CD31+EVs, p = 0.005). ROC analysis revealed plasma concentrations of LYVE1+EVs demonstrated significant diagnostic accuracy to differentiate athletes with SRC from athlete controls (AUC: 0.971, 95% C.I. = 0.933-1.000, p < 0.0001). Notably, concentrations of LYVE1+/CD81+ double-positive EVs, CD31+/CD81+ double-positive EVs, and GFAP+/CD81+ double-positive EVs were significantly higher among athletes with SRC within 72 hours of injury compared to control athletes (p < 0.0001; p = 0.0002; p < 0.0001, respectively). Plasma AQP4+/GFAP+ double-positive EVs and AQP4+/CD81+ double-positive EVs were not. However, plasma concentrations of GFAP+/CD81+ double-positive EVs and AQP4+/GFAP+ double-positive EVs were significantly elevated after repetitive, subconcussive head impacts (p < 0.0001 and p = 0.004, respectively). Conclusion: Plasma concentrations of double-positive EVs, including LYVE1+/CD81+EVs, CD31+/CD81+EVs, and GFAP+/CD81+EVs, may be promising biomarkers for acute SRC. EVs associated with the glymphatic system, GFAP+/CD81+EVs and AQP4+/GFAP+EVs, were significantly elevated after repetitive subconcussive head impacts. The differences observed in EV responses to SRC and subconcussion may provide novel mechanistic insights about sport-associated neurodegeneration for current and future athletes.