Volumetric and Diffusion Tensor Imaging Abnormalities Are Associated With Behavioral Changes Post-Concussion in a Youth Pig Model of Mild Traumatic Brain Injury

Abstract

Mild traumatic brain injury (mTBI) caused by sports-related incidents in children and youth often leads to prolonged cognitive impairments but remains difficult to diagnose. In order to identify clinically relevant imaging and behavioral biomarkers associated concussion, a closed-head mTBI was induced in adolescent pigs. Twelve (n = 4 male and n = 8 female), 16-week old Yucatan pigs were tested; n = 6 received mTBI and n = 6 received a sham procedure. T1-weighted imaging was used to assess volumetric alterations in different regions of the brain and diffusion tensor imaging (DTI) to examine microstructural damage in white matter. The pigs were imaged at 1- and 3-month post-injury. Neuropsychological screening for executive function and anxiety were performed before and in the months after the injury. The volumetric analysis showed significant longitudinal changes in pigs with mTBI compared with sham, which may be attributed to swelling and neuroinflammation. Fractional anisotropy (FA) values derived from DTI images demonstrated a 21% increase in corpus callosum from 1 to 3 months in mTBI pigs, which is significantly higher than in sham pigs (4.8%). Additionally, comparisons of the left and right internal capsules revealed a decrease in FA in the right internal capsule for mTBI pigs, which may indicate demyelination. The neuroimaging results suggest that the injury had disrupted the maturation of white and gray matter in the developing brain. Behavioral testing showed that compare to sham pigs, mTBI pigs exhibited 23% increased activity in open field tests, 35% incraesed escape attempts, along with a 65% decrease in interaction with the novel object, suggesting possible memory impairments and cognitive deficits. The correlation analysis showed an associations between volumetric features and behavioral metrics. Furthermore, a machine learning model, which integrated FA, volumetric features and behavioral test metrics, achieved 67% accuracy, indicating its potential to differentiate the two groups. Thus, the imaging biomarkers were indicative of long-term behavioral impairments and could be crucial to the clinical management of concussion in youth.

Keywords: MRI; biomarkers; concussion; pig; recovery; sports injuries; trauma; white matter; youth.

FIGURE 1

T1‐weighted MRI scans. (A) T1‐weighted MRI scans of pig brains illustrating manual brain skull stripping across different planes (from left to right, sagittal, coronal, and axial plane, respectively). The planes show the brain with the manually drawn mask overlay used for skull stripping. The mask highlights the brain's boundaries, excluding the surrounding skull and non‐brain tissues. (B) Visualization of ROIs from the registered pig brain atlas (T1‐weighted) after transforming them to our T1 space. A subset of these ROIs is shown overlaid for reference, including the corpus callosum (yellow), cortex (brown), internal capsule (light blue), thalamus (green), hippocampus (dark blue), medulla (yellow), left caudate (red), olfactory bulb (teal), and pons (light blue).

FIGURE 2

Kinematics of head injury. Kinematics of injury for head's rigid motion using the eye as tracking marker. Displacements were calculated with a MATLAB custom‐built code for image processing, applying a polynomial fit and then derived for velocity and acceleration. The graphs depict the results of three tests in one individual (T1, T2, and T3).

FIGURE 3

Volumetric analysis. Displays representative maps of the white and gray matter regions alongside their corresponding group analysis results (mTBI, n = 6; sham, n = 6; avg ± SEM; Student's t‐test, *p < 0.05; **significant at p < 0.003).

FIGURE 4

Volumetric differences in left and right internal capsule post‐mTBI. This volumetric comparison of the left and right internal capsule. The results demonstrate that in the mTBI group there were significant differences between the left and the right internal capsule at both time points after injury (mTBI, n = 6; sham, n = 6; avg ± SEM; Student's t‐test, *p < 0.05).

FIGURE 5

FA maps in grayscale and color‐coded orientations. Left: FA maps (A) in grayscale (sagittal, transverse, and coronal planes, respectively), and FA maps (B) in color code for one mTBI pig. Right: FA maps (C) in grayscale, and FA map (D) in color code for a sham pig. Color codes for orientation are as follows: red indicates left to right, green represents anterior to posterior, and blue denotes superior to inferior.

FIGURE 6

FA changes in corpus callosum and internal capsule post‐mTBI. (A) FA changes in the corpus callosum between 1‐ and 3‐month post‐injury. The graphs depict the mean differences between mTBI and sham groups at the two time points. mTBI group shows a significant increase in FA from 1‐ to 3‐month post‐injury. (B) The comparison of FA values between left and right internal capsule for both group and each time point. At 3‐month post injury, the FA values in the right internal capsule are significantly lower compared with the left one in the mTBI group (mTBI, n = 6; sham, n = 6; avg ± SEM; Student's t‐test, *p < 0.05).

FIGURE 7

Behavioral assessment of mTBI and sham pigs. The results from the open field test revealed that mTBI pigs traveled a significantly greater distance than sham pigs. (A) The normalized distance traveled by the pigs over a 10‐min period in the weeks following the injury. (B) Displays the group mean distance walked. (C) The normalized number of escape attempts in the weeks following the injury. (D) Group means for the number of escape attempts show a significant increase in mTBI pigs. (E) The novel object recognition test recorded the number of interactions with a novel object for each pig, normalized to the pre‐injury baseline. (F) Shows a substantial decrease in group means for mTBI pigs (n = 6 mTBI, n = 6 sham control, average ± SEM, **p < 0.005, ***p < 0.0005).

FIGURE 8

Correlation heatmap of behavioral test outcomes with imaging data. The heatmap illustrates the Pearson correlation analysis between behavioral test and other imaging features like FA and volume from brain regions corpus callosum, left and right hemispheres of internal capsule and hippocampus at 1‐ and 3‐month post injury using R‐squared (R ²), which indicates the proportion of variance in one variable explained by others. The color gradient represents the strength of the R ², where darker shades indicate stronger correlations and lighter shades represent weaker or no correlation. Significant p‐values (p < 0.05) are highlighted with an asterisk (*) to indicate meaningful correlation that meet the threshold for statistical significance. The x‐axis lists the target features, while the y‐axis lists the contributing features.

FIGURE 9

SVM model with polynomial kernel. (A) The contour plot (as shown in our results) illustrate how different feature combinations influence classification. The red and blue regions represent the model's decision areas for mTBI and control groups, respectively. The contour lines show areas where the model is uncertain or where class boundaries lie. The circles are the individual pigs classified into mTBI (red) or control (blue). There were four misclassified cases where some blue points fall into the red region (or vice versa). (B) The confusion matrix helps assess the accuracy and error types of the classifier, showing the balance between correct and incorrect predictions for both classes.

FIGURE 10

Histological analysis. Luxol fast blue histology. Left: Pig anatomical atlas overlayed on the LFB histology. An image of 4‐μm thick brain slice stained for LFB showing the area of the RCC that was analyzed to evaluate myelin in the pig's brain. Right: A 40× magnification of the area shows myelin architecture with high resolution.