Experimental design and statistical rationale

The experimental design incorporated four biological replicates for each group (total five groups: sham control, 3 and 24 h, and 7 and 30 DPI after blast), across two independent TMT 10-plex tagging experiments to examine brain cortex. In order to perform robust quantitative analysis between the two independent TMT 10-plex tagging experiments, we pooled two biological replicates from the sham group together as an index for two TMT tagging cross-batch analysis. All experimental groups in the studies were handled in a randomized, double-blinded manner. For quantification purposes, all proteomics data were normalized across all proteins within each 10-plex batch to eliminate the batch effect. Welch's test and t-distribution were performed with R function “t.test” for proteomics data, and other statistical comparisons were made by student's t test for two groups and by one way analysis of variance (ANOVA) test followed by Tukey test for multiple comparisons. GraphPad Prism (version 6 for Windows; GraphPad Software) were used to calculate the p values. The relative protein abundance ratios (fold changes) between blast and sham groups were calculated. As previously described, the changes in protein levels were considered significant if fold change was >1.0 (upregulated) or <1.0 (downregulated), and the p value was <0.05 in two independent experiments.²⁰ The threshold of this open-field LIB was determined based on peak overpressure (46.6 kPa) as compared with literature reports (majority >100 kPa),^6,21 and our previous pathological observations demonstrating the absence of macroscopic damage or necrosis in the presence of nanoscale ultrastructural injuries.⁵