Increased Behavioral Deficits and Inflammation in a Mouse Model of Co-Morbid Traumatic Brain Injury and Post-Traumatic Stress Disorder

Abstract

Comorbid post-traumatic stress disorder with traumatic brain injury (TBI) produce more severe affective and cognitive deficits than PTSD or TBI alone. Both PTSD and TBI produce long-lasting neuroinflammation, which may be a key underlying mechanism of the deficits observed in co-morbid TBI/PTSD. We developed a model of co-morbid TBI/PTSD by combining the closed head (CHI) model of TBI with the chronic variable stress (CVS) model of PTSD and examined multiple behavioral and neuroinflammatory outcomes. Male C57/Bl6 mice received sham treatment, CHI, CVS, CHI then CVS (CHI → CVS) or CVS then CHI (CVS → CHI). The CVS → CHI group had deficits in Barnes maze or active place avoidance not seen in the other groups. The CVS → CHI, CVS and CHI → CVS groups displayed increased basal anxiety level, based on performance on elevated plus maze. The CVS → CHI had impaired performance on Barnes Maze, and Active Place Avoidance. These performance deficits were strongly correlated with increased hippocampal Iba-1 level an indication of activated MP/MG. These data suggest that greater cognitive deficits in the CVS → CHI group were due to increased inflammation. The increased deficits and neuroinflammation in the CVS → CHI group suggest that the order by which a subject experiences TBI and PTSD is a major determinant of the outcome of brain injury in co-morbid TBI/PTSD.

Keywords: astrocytes; behavior; cognition; macrophage/microglia; memory.

Figure 1.

CVS → CHI Impairs Performance on Barnes Maze and Active Place Avoidance. (a) On the 3rd and 4th day of Barnes testing, the CVS → CHI group had a significantly longer latency to find the escape hole than the other groups (*p < 0.01). (b) On the 4th, 6th, 7th, and 8th trials of active place avoidance testing, the CVS → CHI group CHI had significantly more entrances than the other groups (*p < 0.05). (c) The CVS → CHI group had a significantly shorter time to 1st entrance into the shock zone than the other groups (*p < 0.005). These data suggest that CVS → CHI produces behavioral deficits not seen in the other groups.

Figure 2.

CHI → CVS and CVS → CHI Produces Impairments on Elevated Plus Maze and CHI Impairs Acoustic Startle Response. A: The CVS, CHI → CVS and CVS → CHI groups had spent significantly more time in the open arm compared to the sham-treated group (CVS or CHI → CVS, **p < 0.001; CVS → CHI, *p < 0.05). The CVS, CHI → CVS and CVS → CHI groups also had significantly fewer entrances into open (Panel B) or closed (Panel C) arms (*p < 0.05). D: The CHI group had significantly more trials with behavioral freezing at 105 dB than the other groups (*p < 0.05).

Figure 3.

CHI Activates Astrocytes in CA3. (a) Representative images of GFAP immunoreactivity in the CA3 region of the hippocampus. (b) The number of GFAP⁺ CA3 cells was significantly increased in the CHI group as compared to Sham-group (*p < 0.05). Scale bar, 100 μm.

Figure 4.

CVS → CHI Increases Iba-1⁺ Cell Number in Multiple Hippocampal Regions. (a) Representative images of Iba-1 immunoreactivity in the dentate gyrus (top), CA3 (middle) or CA1 (bottom). Scale bar 100 μm. (b) Summary of Iba-1⁺ cell number. In all three hippocampal regions, the CVS → CHI group had significantly more Iba-1⁺ cells than the other groups (*p < 0.05). Scale bar, 100 μm.

Figure 5.

Increased Hippocampal Arginase-1 Expression Following CHI, CVS, CHI → CVS, CVS → CHI. (a) Representative images of hippocampal arginase-1 immunofluorescence. (b) Summary of the changes in arginase-1 expression. Arginase-1 expression in the CHI, CVS, CHI → CVS, and CVS → CHI groups were significantly greater than the sham-treatment group (*p < 0.005, **p < 0.0005). Scale Bar, 100 μm.

Figure 6.

Neuroinflammation in the Amygdala. (a) Representative images of Iba-1⁺ cells. (b) Summary of differences in Iba-1⁺ cell number. There were significantly more Iba-1⁺ cells in the CHI group than in the sham-treated group (*p < 0.05). (c) Representative images of GFAP⁺ cells in the amygdala. (d) There were significantly more GFAP⁺ cells in all experimental groups as compared to the sham-treated group (stats). Scale bar, 100 μm.

Figure 7.

Inflammation in the Dentate Gyrus Correlates With Impaired Performance on Hippocampal-Dependent Tasks. The number of Iba-1⁺ cells in the dentate gyrus significantly correlated with average latency of the 4 trials on the first day of Barnes maze training (Panel A) (r₂ = 0.77, p < 0.005) and with the number of entrances on trial 6 on active place avoidance (Panel B) (r₂ = 0.64, p < 0.005).

Figure 8.

CHI Increases Hippocampal BDNF Expression. A: Representative images of hippocampal BDNF immunofluorescence in the CA3 regions. B: Summary of differences in BDNF expression. The CHI group had significantly more BDNF expression than the sham-treated group (*p < 0.01).