Objective quantification of microglial alignment was performed on Iba1-stained tissue from the S1BF region using a fast Fourier transform (FFT) method. (A) Representative images where radial feather mask was applied to eliminate edge effects. (B) Photomicrographs were then subjected to FFT using NIH Image J software; a ‘starburst’ effect can be seen in the 7d post-FPI image (oriented along the axis defined by the red and green arrows) which corresponds to the microglial alignment. (C) A ring analysis was applied to the FFT image and radial sum intensity value for each angle was calculated around the circumference from the center, as plotted in the histogram. For illustrative purposes, the orange horizontal line represents the root mean square of the radial sum intensities; the red and green vertical lines represent the angle of interest and opposite angle of interest, respectively; the purple and cyan vertical lines represent the orthogonal and opposite orthogonal angles, respectively. (D) Analysis was conducted for uninjured and brain-injured rats at various time points after diffuse brain injury (n = 3/group). At 1, 2, 7, and 28 days post-injury, the ratio from orthogonal was significantly higher than the uninjured sham values (*P <0.05, one-way ANOVA) and the ratio from orthogonal was significantly higher at 7 days post-injury compared to 1 day post-injury (†, P <0.05, one-way ANOVA). Mean ±SEM.