Quantifying Brain Injury and Recovery

Evaluation of neuropathology was performed with blinding to treatment group. Histologic sections from the anterior third of the brain, encompassing the frontal lobes, were used to estimate the ipsilateral and contralateral hemisphere volume of structurally normal appearing cerebral cortex and the volume of peri-lesion subcortical white matter. Every 12th serial coronal section was stained with 0.1% Luxol fast blue followed by 0.1% cresyl violet staining after dehydration in a series of increasing alcohol concentrations. Luxol fast blue staining was used to identify white matter. Cresyl violet was used to counterstain tissue sections for lesion identification and neocortex loss. Using Image J software, we traced the area of each hemisphere excluding the contusion injury (cavity plus pale cresyl violet stained tissue) and the area of white matter in each hemisphere. We calculated the volume by summing section area × 600 µm spacing between sections along the entire axial length of the anterior third of the cerebral hemisphere. The percentage of contusion injury volume was calculated 100 (left–right)/left hemisphere volume. The percentage of white matter volume loss was calculated as 100 (left–right)/left white matter volume. For this analysis, 15–20 brain sections were used to calculate the percentage of contusion injury volume and the percentage of white matter volume loss for each brain.