Experimental Designs

The present study was categorized into two parts: in vivo experiments and in vitro experiments.

Initially, the cortical expression of S100A8 was assessed after TBI. For the experimental part, 21 adult, male ICR mice, weighing approximately 28–32 g, were selected and randomly divided into seven groups: Sham group (n = 3/group) and TBI groups (3, 6, 9, 12, 24, and 72 h) (n = 3/group). Sham mice were euthanized at 24 h, and the skin incision was performed only during the surgery. Furthermore, TBI mice were euthanized at the indicated time-point after TBI. Then, after euthanizing mice in each group, cerebral cortex tissues around the lesion site were collected and estimated by Western blot analysis. In addition, six other mice were added into the Sham (n = 3) and TBI (n = 3) groups, which were euthanized at exactly 24 h after the TBI, to collect all the brains for performing the immunofluorescence staining.

Furthermore, as the experiment proceeded, one-time point (24 h) was selected for the experiments, to perform this according to the time-course of the S100A8 expression. Afterward, 36 male mice were randomly divided into six groups: (1) sham group, (2) NS group, (3) TBI group, (4) TBI+TAK-242 group, (5) S100A8 group, and (6) S100A8+TAK-242 group for the Western blot, and 60 male mice were randomly divided into five groups: (1) sham group, (2) TBI group, (3) TBI+TAK-242 group, (4) S100A8 group, and (5) S100A8+TAK-242 group for Nissl staining, enzyme-linked immunosorbent assay (ELISA), and wet/dry method. For mice in the sham group, the skin incision was performed only during the surgery, and these mice were subsequently euthanized accurately at 24 h. Mice in the TBI group were euthanized, as mentioned below. TAK-242 (MCE; Monmouth Junction, NJ, United States) was intraperitoneally administered (0.3 mg/kg) to mice in the TBI+TAK-242 and S100A8+TAK-242 groups at 0.5 h prior to the intracerebroventricular administration of S100A8 or TBI. For mice in the S100A8 and S100A8+TAK-242 groups, S100A8 recombinant protein (3 μg) at a volume of 5 μl was injected into the lateral ventricle of the brain, according to a previous report (Yamada et al., 2011). However, for mice in the NS group, sterile normal saline (5 μl) was injected into the left lateral ventricle. Briefly, the coordinates for the intracerebroventricular injection were 1.8 mm lateral from the midsagittal suture, 0.7 mm posterior to the bregma, and -2.5 mm from the flat skull surface. The intracerebroventricular injection was performed at 30 min after the TAK-242 was intraperitoneally administered to mice.

To perform the immunological assays, the brain samples were collected for the Western blot (n = 6) and ELISA (n = 3). Nissl staining (n = 3, for each group) was also performed. In addition to the above techniques, the brain water content was measured by using the wet/dry method (n = 6), as described below. The neurological deficits (n = 6) were evaluated.

BV-2 cells were cultured in six-well plates and were randomly divided into five groups: (1) sham group, (2) S100A8 group, (3) S100A8+TAK-242 group, (4) LPS group, and (5) LPS+TAK-242 group. Then, cells were collected for Western blot analysis (n = 3), and the cell-free supernatants were collected for ELISA (n = 3).