Figure 7

Reduced post-SCI hemorrhage and improved BSCB function in Bmal1^−/− mice. Immunostaining analyses were performed on coronal sections of the spinal cord from WT (dpi 1: M:F = 2:5 including 2 KO littermates; dpi 7: M:F = 2:2) and Bmal1^−/− (dpi 1: M:F = 0:4; dpi 7: M:F = 1:2) mice that received moderate T9 SCI. (a) In WT mice, signals for the hemorrhage marker hemoglobin (Hb) and extravasated plasma proteins including fibrin/fibrinogen and IgG are strong in the lesion area including the epicenter as indicated by negative staining for the astrocyte marker GFAP or densely packed Hoechst⁺ nuclei of the fibrotic scar cells (arrows). Partial overlap between fibrinogen and IgG signals is also observed (arrowheads). In Bmal1^−/− mice, signals for Hb, fibrin/fibrinogen, and IgG, but not the microvascular marker PECAM1 or GFAP, are reduced. Representative images of the dpi 1 staining are shown in Supplementary Fig. S7. (b) Quantification of relative signal area for Hb, IgG, fibrin/fibrinogen, PECAM1 and GFAP in the lesion core (1,500 μm region centered on the injury epicenter and also including the injury penumbra) confirmed reduced subacute hemorrhage, and both acute and subacute BSCB disruption with unaffected content of spinal cord microvasculature or reactive astroglia. Data are means ± SD (*p < 0.05, u-test). Similar patterns were observed for another plasma protein, IgM (Supplementary Fig. S8). IgG was detected using an anti-mouse IgG F(ab’)2 fragment to avoid binding to cells that express IgG receptors. Calibration bar is 500 μm.