Blood–Brain Barrier Breakdown and Astrocyte Reactivity Evident in the Absence of Behavioral Changes after Repeated Traumatic Brain Injury

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blood–brain barrier breakdown, mild TBI, neuroinflammation, repeated TBI, traumatic brain injury (TBI)


Repeated traumatic brain injuries (TBIs) cause debilitating effects. Without understanding the acute effects of repeated TBIs, treatment options to halt further degeneration and damage cannot be developed. This study sought to examine the acute effects of blood–brain barrier (BBB) dysfunction, edema, inflammation and behavioral changes after either a single or double TBI using a C57BL/6 mouse model. We examined the effects of one or two TBIs, of either a mild or moderate severity. Double injuries were spaced 7 days apart, and all analysis was performed 24 h post-injury. To examine edema and inflammation, protein levels of glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B, interleukin-6, and matrix metallopeptidase 9 (MMP9) were analyzed. Aquaporin-4 (AQP4) and zonula occludens-1 (ZO-1) were analyzed to observe BBB dysfunction. Ionized calcium-binding adapter molecule 1 (IBA1) was analyzed to observe microglial activation. Rotarod, beam walking, and grip strength tests were used to measure changes in physical behavior post-injury. A sample size of ≥5 was used for all analysis. Double injuries led to an increase in BBB breakdown, as indicated by altered MMP-9, AQP4, and ZO-1 protein expression. Single injuries showed an increase in microglial activation, astrocyte activation, and BBB breakdown. Behavioral tasks showed no significant differences between injured and control groups. Based on our findings, we suggest that behavioral studies should not be used as the sole clinical indicator on brain tissue recovery. Analysis of markers such as IBA1, GFAP, MMP-9, AQP4, and ZO-1 provide valuable insight on pathophysiological response to injury.


This article was published with support from the Open Access Publishing Fund administered through the University of Arkansas Libraries.

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.