Evans Blue and Fluorescein Isothiocyanate-Dextran Double Labeling Reveals Precise Sequence of Vascular Leakage and Glial Responses After Exposure to Mild-Level Blast-Associated Shock Waves

Abstract

Blast-induced shock waves (BSWs) are responsible for several aspects of psychiatric disorders that are collectively termed mild traumatic brain injury (mTBI). The pathophysiology of mTBI includes vascular leakage resulting from blood–brain barrier (BBB) disruption. In this study, the precise sequence of BBB breakdown was examined using an Evans blue and fluorescein isothiocyanate (FITC)-dextran double labeling technique. Evans blue solution was injected into the tail vein of male C57BL6/J mice just before and 4 h, 1 day, 3 days, and 7 days after a single BSW exposure at as low as 25-kPa peak overpressure. In contrast, the FITC-dextran solution was transcardially injected just before perfusion fixation. Differences in the labeling time-point revealed that BBB breakdown was initiated after approximately 3 h, with significant remodeling by 1 day, and continued until 7 days after BSW exposure. BBB breakdown was upregulated in three distinct regions, namely the brain surface and subsurface areas facing the skull, regions closely associated with capillaries, and the circumventricular organ and choroid plexus. These regions showed distinct responses to BSW; moreover, clusters of reactive astrocytes were closely associated with the sites of BBB breakdown. In severe cases, these reactive astrocytes recruited activated microglia. Our findings provide important insights into the pathogenesis underlying mTBI and indicate that even mild BSW exposure affects the whole brain.