Fig. 1

Blood-spinal cord barrier permeability increases and NETs accumulate in the spinal cords of CFA mice. A. Timepoint of the schematic design. B. Paw withdrawal threshold of mice in each group (n = 6, group: F_{1, 5 =} 173.3, P < 0.0001; time: F_{4, 20 =} 22.22, P < 0.0001; interaction: F_{4, 20} = 21.19, P < 0.0001). C. Paw withdrawal latency of mice in each group (n = 6, group: F_{1, 5} = 2792, P < 0.0001; time: F_{4, 20} = 165.5, P < 0.0001; interaction: F_{4, 20} = 129.9, P < 0.0001). D. Quantification of Evans blue dye in the spinal cord of mice (n = 5, F_{4, 20} = 5.568, P = 0.0035). E–G. Representative blots and quantification of ZO-1 and occludin in the spinal cord of sham (Day 7) and CFA mice (n = 5, ZO-1: F_{4, 20} = 14.74, P < 0.0001; occludin: F_{4, 20} = 16.89, P < 0.0001). H and I. Representative blots and quantification of Ly6G and PAD4 in the spinal cord of sham (Day 7) and CFA mice (n = 5, Ly6G: F_4,20 = 10.96, P < 0.0001; PAD4: F_{4, 20} = 8.688, P = 0.0003). J. Immunofluorescence staining for PAD4 in the spinal cords of sham (Day 7) and CFA (Day 7) mice (n = 3, scale bars: 100 μm). K and L. Representative blots and quantification of MPO, and citH3 in the spinal cord of sham (Day 7) and CFA mice (n = 5, MPO: F_{4, 20} = 7.445, P = 0.0008; citH3: F_{4, 20} = 4.185, P = 0.0127). Ly6G, PAD4, MPO, and citH3 levels in the sham group were set as 1 for quantification. M. Double immunofluorescence staining for citH3 (red) and MPO (green) in the spinal cords of sham (Day 7) and CFA (Day 7) mice. NETs were visualized by colocalization of citH3 and MPO staining (merged images; n = 3, scale bars: 50 or 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the sham group mice