Microstructural and microglial changes after repetitive mild traumatic brain injury in mice

Abstract

Traumatic brain injury (TBI) is a major public health issue, with recently increased awareness of the potential long-term sequelae of repetitive injury. Although TBI is common, objective diagnostic tools with sound neurobiological predictors of outcome are lacking. Indeed, such tools could help to identify those at risk for more severe outcomes after repetitive injury and improve understanding of biological underpinnings to provide important mechanistic insights. We tested the hypothesis that acute and subacute pathological injury, including the microgliosis that results from repeated mild closed head injury (rmCHI), is reflected in susceptibility-weighted magnetic resonance imaging and diffusion-tensor imaging microstructural abnormalities. Using a combination of high-resolution magnetic resonance imaging, stereology, and quantitative PCR, we studied the pathophysiology of male mice that sustained seven consecutive mild traumatic brain injuries over 9 days in acute (24 hr) and subacute (1 week) time periods. rmCHI induced focal cortical microhemorrhages and impaired axial diffusivity at 1 week postinjury. These microstructural abnormalities were associated with a significant increase in microglia. Notably, microgliosis was accompanied by a change in inflammatory microenvironment defined by robust spatiotemporal alterations in tumor necrosis factor-α receptor mRNA. Together these data contribute novel insight into the fundamental biological processes associated with repeated mild brain injury concomitant with subacute imaging abnormalities in a clinically relevant animal model of repeated mild TBI. These findings suggest new diagnostic techniques that can be used as biomarkers to guide the use of future protective or reparative interventions. © 2016 Wiley Periodicals, Inc.

Keywords: DTI; RRID:AB_2144905; RRID:AB_2314667; RRID:AB_323909; SWI; axial diffusion; inflammation; microhemorrhage.

Figure 1

(b&w). Susceptibility-weighted imaging (SWI) 1 week after rmCHI reveals micro-hemorrhages. A. Coronal SWI shows more hypodensities consistent with micro-hemorrhage 1 week after rmCHI (arrows). B. More micro-hemorrhages per brain were observed following rmCHI (n=6, Mann-Whitney U test, p<0.05). C. Representative photomicrograph shown sham animals are negative for Prussian blue in the cortex. D. Representative photomicrograph shown Prussian blue reaction products (arrows) in cortical neuropil 1 week following rmCHI. (Scale bar = 100μm)

Figure 2

(color) Diffusion maps illustrate the directionality of diffusion with colors: red – transverse, green – vertical and blue – orthogonal to the plane. After rmCHI, subtle loss of color and directionality is evident in multiple regions, including white matter, consistent with microstructural injury and abnormal diffusion. Manual drawings of regions of interest on representative T2 image in the coronal view demarcating the corpus callosum (yellow), capsular white matter (orange), fimbria (red), hippocampus (blue) and sensory cortex (green).

Figure 3

(b&w) Mean diffusivity (MD) quantifies the mean of the three eigenvectors, and axial diffusivity (AD) quantifies changes in the primary eigenvector. A. One week after rmCHI, no change in MD is present in white matter corpus callosum. B. Significant reduction of AD is observed one week after rmCHI in white matter corpus callosum. C. Similarly, no change in fimbria MD is evident one week after rmCHI. D. Reduced AD is also present in the fimbria one week after rmCHI. E. Consistent with the other white matter regions, MD is unchanged in the capsular white matter after rmCHI. F. AD is significantly reduced in capsular white matter 1 week following rmCHI (n=10, Student’s two-tailed t test, *p<0.05).

Figure 4

(b&w) Immunolabeling of Iba1+ microglia reveals microgliosis following rmCHI. A. More Iba1+ immunolabeling of microglia is present in the fimbria one week after rmCHI. Bar = 20 μm. B. Stereological estimates of Iba1+ immunolabeling reveal more microglial labeling is present in the fimbria at both early and subacute intervals after rmCHI. C. More CD68+ activated microglia/macrophage immunolabeling is also more prevalent in the fimbria at both acute and subacute intervals following rmCHI. D. In the hippocampal CA1 subfield, more Iba1+ immunolabeling is present one week after rmCHI. E. Stereological estimates confirm markedly more Iba+1 immunolabeled microglia are present in the CA1 one week after rmCHI. F. More CD68-immunolabeled activated microglia/macrophages are also present in CA1 one week after the rmCHI. (n=6–7, two way ANOVA, *p < 0.05, **p < 0.01, ***p ≤ 0.001).

Figure 5

(color) Early and subacute alterations in inflammatory gene expression following rmCHI in cortex and hippocampus. A. Cortical anti-inflammatory marker CD206 mRNA levels are elevated at 24 hours after rmCHI compared to sham B. CD206 (red, punctate) is primarily colocalized (yellow) with Iba1-positive microglia (green, cell bodies) 24h following rmCHI. (Scale bar = 20μm)

Figure 6

(b&w) A. TNFα protein expression is unchanged in the cortex 24 h after rmCHI. B. Similarly, TNFα levels are consistent across groups at 1 week. C. However, loss of TNFR1 mRNA occurs 1 week after rmCHI in the cortex. D. Similarly, cortical TNFR2 expression is also decreased at 1 week following rmCHI. E. Consistent with the cortex, hippocampal TNFα levels are unchanged at 24 h and F. 1 week following rmCHI. G. However, TNFR2 mRNA expression is significantly increased in the hippocampus at 24h. H. This pattern reverses at 1 week with TNFR2 mRNA expression significantly decreased similar to the cortex at this subacute interval. (n=5–8, Student’s two-tailed t test, *p < 0.05, **p < 0.01, ***p ≤ 0.001)