Fig 2: Reduction in aquaporin expression and cell damage by HMGB1. (a) Cell viability of chondrogenic C3H10T1/2 cells by HMGB1. (b) Expressions of AQP1, -3 and -4 mRNA by HMGB1. (c) Western blot analyses for AQP1, -3 and -4 protein expression by HMGB1. Densitometric analyses of the western blots are shown on the right. (d) HMGB1 and AQP4 protein expression in young and old aged cartilage tissues (n = 5 per group). Densitometric analyses of the western blots are shown on the right. (e) Morphology of chondrogenic C3H10T1/2 cells by HMGB1 administration. (f) Flow cytometry analysis in HMGB1-treated C3H10T1/2 cells. Forward scatter (FSC) represent the cell size, and side scatter (SSC) data represent the complexity or granularity of the cell.

Fig 3: Glymphatic related protein expression changes. (A) Densitometric ratio of the levels of Podoplanin, LYVE1, and AQP4 with respect to GAPDH as measured in the FCtx, H, CRB and BS 15 days following an explosive-driven double blast exposure, n = 6 per group, * indicates p values < 0.05, *** indicates p values < 0.001 and **** indicates p values < 0.0001, as determined by 2-tailed, unpaired t-tests. Error bars represent standard error of the mean (SEM). (B) Representative western blots # for each antibody used. All gels were run in triplicate and data represents the average of 3 runs per sample. # For full length blots, see Supplementary Figures S5–S7.

Fig 4: AQP4 IF intensity changes in the BS following double blast exposure. AQP4 in control (Ctl.) versus double blast (2×B) rat BS. (A) AQP4-ir positive staining in the BS of a control rat not exposed to blast versus BS of a rat exposed to double blast, taken at 10× magnification. The insets at 40× magnification, defined as superior raphe obscurus-mfl, indicate the area (dorso-medial area) of MFI quantification. (B) Graphs showing MFI of total BS, 50% superior BS midline, and dorso-medial area including the superior portion of the raphe obscurus nuclei and mlf. * indicates p values < 0.05 and ** indicates p values < 0.01 as determined by 2-tailed, unpaired t-tests. Indices are MFI of each ROI over MFI of the total brainstem. IF = Immunofluorescence; BS = brainstem; mlf = medial longitudinal fasciculus; indices = IF intensity ratios between total BS/dorso-medial areas; MFI = mean fluorescent intensity.

Fig 5: Plasma concentration of AQP4 following double blast exposure. Histogram representing the plasma concentration of AQP4 15 days following an explosive-driven double blast exposure, Ctl, n = 6, Blast, n = 4.