Computational analysis reveals increased blood deposition following repeated mild traumatic brain injury

Abstract

Mild traumatic brain injury (mTBI) has become an increasing public health concern as subsequent injuries can exacerbate existing neuropathology and result in neurological deficits. This study investigated the temporal development of cortical lesions using magnetic resonance imaging (MRI) to assess two mTBIs delivered to opposite cortical hemispheres. The controlled cortical impact model was used to produce an initial mTBI on the right cortex followed by a second injury induced on the left cortex at 3 (rmTBI 3d) or 7 (rmTBI 7d) days later. Histogram analysis was combined with a novel semi-automated computational approach to perform a voxel-wise examination of extravascular blood and edema volumes within the lesion. Examination of lesion volume 1d post last injury revealed increased tissue abnormalities within rmTBI 7d animals compared to other groups, particularly at the site of the second impact. Histogram analysis of lesion T2 values suggested increased edematous tissue within the rmTBI 3d group and elevated blood deposition in the rm TBI 7d animals. Further quantification of lesion composition for blood and edema containing voxels supported our histogram findings, with increased edema at the site of second impact in rmTBI 3d animals and elevated blood deposition in the rmTBI 7d group at the site of the first injury. Histological measurements revealed spatial overlap of regions containing blood deposition and microglial activation within the cortices of all animals. In conclusion, our findings suggest that there is a window of tissue vulnerability where a second distant mTBI, induced 7d after an initial injury, exacerbates tissue abnormalities consistent with hemorrhagic progression.

Keywords: DTI, diffusion tensor imaging; Edema; FA, fractional anisotropy; HPC, hemorrhagic progression of the contusion; Hemorrhagic progression; Histogram; MD, mean diffusivity; Quantitative magnetic resonance imaging; T2.

Fig. 1

Experimental design and schematic of computational analysis algorithm. A. Experimental mTBI and neuroimaging timeline. An mTBI was induced to the right cortex on D0 (denoted as an *) in all animals. The second mTBI was induced to the left cortex at either 3 or 7 days later (*). MRI was performed 1d post first (D1), 1d post last (D1 Singles, D4 rmTBI 3d, D8 rmTBI 7d) and 14d post injury (D14: 14d post first, D21: 14d post last injury) (red circles). B. Illustration of the mTBI locations for the first (right) and second (left) injuries. C. Computational schema for T2 MRI analysis to assess lesion volumes and composition for blood (< 69 ms), edema (> 90 ms), normal appearing brain (NAB; 70–89 ms), or noise (> 500 ms) containing voxels. D. Raw T2 weighted MRI after a Single mTBI revealed observable abnormal signal intensities (yellow arrows) that were then subjected to computational analysis for tissue classification (edema, blood, NAB, noise).

Fig. 2

Temporal shifts in T2 value distribution following rmTBI. Histograms of the total lesion (first + second) at 1d post last and 14d post injury (14d post first injury) demonstrate global changes in T2 value distribution between groups. The total lesion was then separated into the first and second lesions at 1d post last injury and 14d post injury (first: 14d post first, second:14d post last injury) to illustrate the differences in T2 distribution between the two injury sites. Color coding in the total lesion histograms illustrates the range of T2 values for tissue classification: blood (green), edema (red), or normal appearing brain (NAB; blue). Single mTBI histograms in the second injury graphs (dotted line) are those taken from the injured cortex (first injury) for comparison.

Fig. 3

rmTBI 7d animals have increased lesion volumes. A. Representative T2WIs illustrating the presence of abnormal tissue after the initial (1d post first; yellow arrows) and second (1d post last; white arrows) mTBI. B. 3D reconstruction of injury volumes illustrates the mild nature of the mTBI injury, where average total lesion volumes from Single (1.4%), rmTBI 3d (0.9%) and rmTBI 7d (2.0%) animals were collected 1d post last injury. C. The temporal evolution of total (first + second) mTBI lesion volumes over the experimental period, demonstrates a significantly increased lesion volume in rmTBI 7d animals 1d post last injury compared to other groups. D. Evaluation of the first and second injuries revealed a transient increase (p = 0.029) in the second lesion volume of rmTBI 7d animals compared to Singles 1d post last injury. Normalized data presented as means ± SEM, where *p < 0.05.

Fig. 4

Voxel-wise analysis reveals increased blood volume in the rmTBI 7d animals. A. MRI taken 1d post last injury reveals abnormal tissue on T2 (yellow arrows) and blood deposition on SWI (yellow arrows) following mTBI. Computationally color-coded images illustrate voxel characterization (green = blood, red = edema, blue = normal appearing brain) in these animals. B. Edema volume analysis within the first and second lesions revealed changes in edematous tissue at 1d post last injury, while no differences were observed between groups at 14d post injury (first: 14d post first, second: 14d post last injury). C. Analysis of blood volume within the first and second lesions demonstrated significantly increased blood deposition within rmTBI 7d animals at 1d post last injury and between the first lesions at 14d post injury (first: 14d post first, second: 14d post last injury). D. Prussian blue staining in the first and second lesions of animals 14d post first injury at the site of maximal lesion shows increased blood deposition in the tissues from rmTBI 7d animals. Normalized graphs presented as means ± SEM where *p < 0.05; cal bar = 100 μm.

Fig. 5

Microglial activation and blood deposition demonstrate spatial overlap within the lesion sites. A. A depiction of the rat cortex illustrates the locations of observable tissue damage where blood and microglial measurements were taken. Depth and width graphs demonstrate a spatial overlap in the blood and activated microglial localization at 14d post first injury. Single data in the second injury (dotted line) is from the injured cortex (first injury) for comparison. B. Representative Prussian blue and IBA1 images, taken from adjacent sections of rmTBI 7d (second injury) and Single (first injury) animals, show the localization of blood and activated microglia within the cortices. cal bar = 100 μm; inset cal bar = 20 μm.