Fig. 1

Cranial window and mTBI model preparation. A Experimental design timeline. Caspase-1 activation reporter mice were subjected to three closed-head mTBIs over a period of three weeks (each injury occurred one week apart). Corresponding IVIS imaging (in vivo and ex vivo), von Frey, Hargreaves, mouse grimace score (MGS) behavioral assessment, and protein analysis (immunohistochemistry and Western blotting) were performed as shown. B, C The mouse skull was thinned and strengthened by high-strength transparent surgical cyanoacrylate glue (blue area). Sample image shows the thinned-skull window position of the transparent cranial window (C). C, D The thinned cranial hemisphere becomes transparent (right, C) compared to the not-yet-thinned side (left, C). Unilateral closed-head mTBI was induced on an area (black circle B, C, and D) that was approximately 1 mm rostral to the anterior edge of the windows. No apparent tissue damage was observed (C). E Increases in intraocular pressure of the eye ipsilateral to the injury cortex immediately after impact. F, G Brain sections were obtained from four groups of C57BL mice, including sham control (1 day), sham control (3 days), 3 × mTBI (1 day), and 3 × mTBI (3 days). E. Sample confocal images of coronal cortical slices stained for DAPI, GFAP, and IBA1 for each group. G Quantification of GFAP (left) and IBA1 (right) positive cells showed increases in GFAP and IBA1 labeling at 1 day and 3 days after the third injury (n = 3 mice per group; *p < 0.05, **p < 0.01, One-way ANOVA, Tukey’s HSD). Scale bar: 50 μm