Repeated mild traumatic brain injury results in long-term white-matter disruption

Abstract

Mild traumatic brain injury (mTBI) is an increasing public health concern as repetitive injuries can exacerbate existing neuropathology and result in increased neurologic deficits. In contrast to other models of repeated mTBI (rmTBI), our study focused on long-term white-matter abnormalities after bilateral mTBIs induced 7 days apart. A controlled cortical impact (CCI) was used to induce an initial mTBI to the right cortex of Single and rmTBI Sprague Dawley rats, followed by a second injury to the left cortex of rmTBI animals. Shams received only a craniectomy. Ex vivo diffusion tensor imaging (DTI), transmission electron microscopy (TEM), and histology were performed on the anterior corpus callosum at 60 days after injury. The rmTBI animals showed a significant bilateral increase in radial diffusivity (myelin), while only modest changes in axial diffusivity (axonal) were seen between the groups. Further, the rmTBI group showed an increased g-ratio and axon caliber in addition to myelin sheath abnormalities using TEM. Our DTI results indicate ongoing myelin changes, while the TEM data show continuing axonal changes at 60 days after rmTBI. These data suggest that bilateral rmTBI induced 7 days apart leads to progressive alterations in white matter that are not observed after a single mTBI.

Figure 1

Experimental design and region of interest analysis. (A) A mild traumatic brain injury (mTBI) (denoted as *) was induced to the right cortex of all injured animals, while Sham controls only received a craniectomy (n=4 per group). A second mTBI was then induced to the left cortex of rmTBI animals 7 days later (*). All animals were killed at 60 days after initial injury and ex vivo diffusion tensor imaging (DTI) was performed. (B) Schematic of the first (right) and second (left) mTBI locations. (C) DTI images were analyzed using the whole right and left corpus callosum (CC; left, yellow regions of interest (ROIs)). The CC was then examined using a total of 12 ROIs (6 each bilaterally) as shown on the relative anisotropy color maps, which depict the directionality of water diffusion along the CC (red: medial-lateral; green: dorsal-ventral; blue: anterior-posterior). (D) Changes in CC integrity were further examined using Luxol fast blue (LFB; n=3 per group) staining and transmission electron microscopy (TEM; n=3 per group). The area of the whole right and left CC (yellow ROI), cingulum (cingulum+CC) width, and midline CC width (red boxes) was measured in addition to LFB staining intensity. TEM analysis was performed on the right CC of Single and Sham animals and the left CC of rmTBI animals (black box denotes CC region). MRI, magnetic resonance imaging.

Figure 2

Radial diffusivity is increased in repeated mild traumatic brain injury (rmTBI) animals. (A) Representative diffusion tensor imaging (DTI) images illustrate changes in axial (AD) and radial (RD) diffusivities between Sham and rmTBI animals. The relative anisotropy (RA) color map reflects overall differences in water diffusion between the groups (red: medial-lateral; green: dorsal-ventral; blue: anterior-posterior). (B) Evaluation of AD revealed no significant differences between the injured groups within the right and left corpus callosum (CC). However, a significant increase (*P<0.001) in RD was observed bilaterally within the rmTBI animals compared with the Single and Sham groups.

Figure 3

Radial diffusivity is elevated throughout the entire corpus callosum (CC) in repeated mild traumatic brain injury (rmTBI) animals. (A) Schematic depicting the right and left regions of interest (ROIs) used to further examine regional diffusion tensor imaging (DTI) changes within the CC (red: medial-lateral; green: dorsal-ventral; blue: anterior-posterior). (B) Axial diffusivity (AD) measurements revealed no significant changes between the groups within the right (first mTBI) or left (second mTBI) CC. (C) Radial diffusivity (RD) measurements taken from the segmented regions showed no significant differences between the experimental groups. However, the rmTBI animals showed elevated RD in every segment compared with the Sham and Single groups.

Figure 4

Ongoing white-matter damage is evident in the corpus callosum (CC). (A) Representative transmission electron microscopy (TEM) images illustrate increased white-matter abnormalities (arrows) within repeated mild traumatic brain injury (rmTBI) animals (A3) at the site of the second injury (left) compared with the site of first injury (right) within the Sham (A1) and Single (A2) groups; cal bar=1 μm. (B) Normal myelinated axons appear with a tightly wrapped sheath located near the axon (B1). Typical abnormalities (arrows) within the CC included large separations of the myelin sheath from the axon (B2), breaking of the sheath layers (B3), and deterioration of the myelin (B4 and B5); cal bar=0.2 μm. (C) The rmTBI group revealed a significant increase (*P<0.001) in g-ratio (axon/fiber diameter) compared with other groups. Similarly, the axon caliber of rmTBI animals was also increased (*P<0.001) compared with Sham and Single groups. The Single group also exhibited a significant decrease (*P<0.001) in axon caliber compared with Shams. Both the Single and rmTBI groups showed a significant (*P<0.001) decrease in myelin thickness compared with Sham animals.