Evaluating spatiotemporal microstructural alterations following diffuse traumatic brain injury

Abstract

Background: Diffuse traumatic brain injury (TBI) is known to lead to microstructural changes within both white and grey matter detected in vivo with diffusion tensor imaging (DTI). Numerous studies have shown alterations in fractional anisotropy (FA) and mean diffusivity (MD) within prominent white matter tracts, but few have linked these to changes within the grey matter with confirmation via histological assessment. This is especially important as alterations in the grey matter may be predictive of long-term functional deficits.

Methods: A total of 33 male Sprague Dawley rats underwent severe closed-head TBI. Eight animals underwent tensor-based morphometry (TBM) and DTI at baseline (pre-TBI), 24 hours (24 h), 7, 14, and 30 days post-TBI. Immunohistochemical analysis for the detection of ionised calcium-binding adaptor molecule 1 (IBA1) to assess microglia number and percentage of activated cells, β-amyloid precursor protein (APP) as a marker of axonal injury, and myelin basic protein (MBP) to investigate myelination was performed at each time-point.

Results: DTI showed significant alterations in FA and RD in numerous white matter tracts including the corpus callosum, internal and external capsule, and optic tract and in the grey-matter in the cortex, thalamus, and hippocampus, with the most significant effects observed at 14 D post-TBI. TBM confirmed volumetric changes within the hippocampus and thalamus. Changes in DTI were in line with significant axonal injury noted at 24 h post-injury via immunohistochemical analysis of APP, with widespread microglial activation seen within prominent white matter tracts and the grey matter, which persisted to 30 D within the hippocampus and thalamus. Microstructural alterations in MBP+ve fibres were also noted within the hippocampus and thalamus, as well as the cortex.

Conclusion: This study confirms the widespread effects of diffuse TBI on white matter tracts which could be detected via DTI and extends these findings to key grey matter regions, with a comprehensive investigation of the whole brain. In particular, the hippocampus and thalamus appear to be vulnerable to ongoing pathology post-TBI, with DTI able to detect these alterations supporting the clinical utility in evaluating these regions post-TBI.

Keywords: Axonal injury; Diffusion tensor Imaging; Neuroinflammation; Structural magnetic resonance imaging; Traumatic brain injury.

Fig. 1

Brain volume changes following TBI. It shows the different results of (A) the tensor-based morphometry, (B) representing group average of the T2 images showing increased ventricle size at 24 h, and 7days with recovery at 14 D and 30 D post-TBI, (C) ROI schematic showing regions used to calculate the box-plots in (D-F) including (D) posterior-dorsal hippocampus (in pink), (E) subiculum hippocampus (in red), and (F) thalamus (in brown).

Fig. 2

Longitudinal diffusion tensor imaging (DTI) reveals altered white- and grey-matter microstructures over one-month following TBI. (A) Voxel-wise comparison of FA values and (B) RD values between TBI time points (i.e. 24 h, 7 D, 14 D, and 30 D post-TBI) and the baseline time point (each row) at different slice positions (columns). Yellow-red indicates TBI > Baseline, and blue-light blue indicates TBI < Baseline. (C) shows the FA values in four ROIs and (D) shows the RD values at the same ROIs. OP, optic tract; CCb, the body of the corpus callosum; CCl, lateral corpus callosum; CCg, genu of corpus callosum; CCs, splenium of corpus callosum; IC, internal capsule; EC, external capsule, pF, piriform cortex; Thl, thalamus; Amg, amygdala; HP, hippocampus; * P < 0.05.

Fig. 3

Representative images of APP immunohistochemistry from our regions of interest. Scale bar = 50 µm (A), with counts of the total number of APP+ve lengths and bulbs (B), * indicates significantly different compared to sham (* = p < 0.05; ** = p < 0.01: *** = p < 0.001).

Fig. 4

Heat map representation of the number of APP+ve immunoreactivity bulbs and lengths counted in each of the white matter tracts of interest. Value calculated as the average of n = 5.

Fig. 5

Representative images of IBA1 immunohistochemistry from our regions of interest. Scale bar = 50 µm. Image representative of n = 5 per group (A), with counts of the number of IBA1+ve cells/mm2 (B) and% activated microglia (C). * indicates significantly different compared to sham (* = p < 0.05; ** = p < 0.01: *** = p < 0.001); # indicates significantly different compared to 24 h (# = p < 0.05; ## = p < 0.01; ### = p < 0.001).

Fig. 6

Heat map representation of the% activated microglia within our areas of interest. Values calculated as the average of n = 5 per group.

Fig. 7

Representative images of MBP immunohistochemistry from our regions of interest following removal of background, thresholding and creation of a binary image (A), with evaluation of the effects of TBI on expression of MBP as examined via% area stained (B), coherency (C), total myelin length (D) and number of intersections (E). Scale bar = 50 µm. Image representative of n = 5 per group. (* = p<0.05; ** = p < 0.01: *** = p < 0.001); # indicates significantly different compared to 24 h (# = p<0.05; ## = p<0.01; ### = p<0.001) ^p<0.05, ^^p<0.01 indicates significantly different compared to 7 D animals.