Neutrophil-specific deletion of Syk results in recruitment-independent stabilization of the barrier and a long-term improvement in cognitive function after traumatic injury to the developing brain

Abstract

While traumatic brain injury (TBI) is the leading cause of death and disability in children, we have yet to identify those pathogenic events that determine the extent of recovery. Neutrophils are best known as “first responders” to sites of infection and trauma where they become fully activated, killing pathogens via proteases that are released during degranulation. However, this activational state may generate substantial toxicity in the young brain after TBI that is partially due to developmentally regulated inadequate antioxidant reserves. Neutrophil degranulation is triggered via a downstream signaling pathway that is dependent on spleen tyrosine kinase (Syk). To test the hypothesis that the activational state of neutrophils is a determinant of early pathogenesis and long-term recovery, we compared young, brain-injured conditional knockouts of Syk ( syk ^f/f MRP8-cre ⁺) to congenic littermates ( syk ^f/f ). Based upon flow cytometry, there was an extended recruitment of distinct leukocyte subsets, including Ly6G ⁺/Ly6C ⁻ and Ly6G ⁺/Ly6C ^int, over the first several weeks post-injury which was similar between genotypes. Subsequent assessment of the acutely injured brain revealed a reduction in blood-brain barrier disruption to both high and low molecular weight dextrans and reactive oxygen species in syk ^f/f MRP8-cre ⁺ mice compared to congenic littermates, and this was associated with greater preservation of claudin 5 and neuronal integrity, as determined by Western blot analyses. At adulthood, motor learning was less affected in brain-injured syk ^f/f MRP8-cre ⁺ mice as compared to syk ^f/f mice. Performance in the Morris Water Maze revealed a robust improvement in hippocampal-dependent acquisition and short and long-term spatial memory retention in syk ^f/f MRP8-cre ⁺ mice. Subsequent analyses of swim path lengths during hidden platform training and probe trials showed greater thigmotaxis in brain-injured syk ^f/f mice than sham syk ^f/f mice and injured syk ^f/f MRP8-cre ⁺ mice. Our results establish the first mechanistic link between the activation state of neutrophils and long-term functional recovery after traumatic injury to the developing brain. These results also highlight Syk kinase as a novel therapeutic target that could be further developed for the brain-injured child.