BBG accumulates in the lesion area following spinal cord injury in rats. (A) A rat injected with BBG (50 mg/kg) exhibits blue coloration of the eye and skin 3 days later. The blue color fades slowly and is not noticeable after 1 week. A vehicle-injected rat is shown for comparison. (B) Quantification of tissue BBG concentrations at the injury site and distant area. BBG accumulates in the lesion, but is detectable in the uninjured tissue in rats that received 50 mg/kg. *, P < 0.05; **, P < 0.01; two-way ANOVA with Tukey-Kramer test; n = 9 rats per group. Error bars indicate SEM.

BBG reduces microglia cell activation, suppresses reactive gliosis, and decreases the number of infiltrating neutrophils in the spinal cord at 4 days after injury. BBG also reduces activation of microglial cells 6 weeks after injury. (A) Longitudinal sections of spinal cord at the traumatic lesion (injury area outlined). Green, GFAP; red, CD68; blue, DAPI. Red arrows indicate CD68⁺ cells most distant from the lesion site. (A, Bottom) The scheme used for quantification of immune response. Immunoreactivity in 4 regions close to the lesion was compared with the intensity of the signal in 4 regions located >10 mm from the border of the injury in the same section. (B) BBG did not change the distribution or the intensity of Iba-1 immunoreactivity (Iba-1, red, n = 5), but significantly reduced the number of activated microglial cells (CD68, green, n = 5). (C and D) BBG also reduces the posttraumatic up-regulation of GFAP (green, n = 5) and infiltrating neutrophils (MPO, red, n = 7), but not the number of infiltrating cytotoxic T-lymphocytes (CD8, green, n = 5–6). (B–D, Bottom) Summary of the quantification of immunolabeling at 4 days or 6 weeks postinjury. The data are plotted as the relative ratio of the immunoreactivity near the injury site compared with distant area for Iba1, GFAP, and MPO, and the number of CD68⁺ or CD8⁺ cells. Blue, DAPI. Scale bars, 50 μm. *, P < 0.05; **, P < 0.01 by one-way ANOVA with a Newman-Keuls posthoc test. Error bars indicate SEM. Bottom graphs quantify % increase (Iba1, GFAP, and MPO) or number of cells (CD68⁺ or CD8⁺) at 6 weeks postinjury.

Schematic diagram of the experimental design. Spinal cord injury in rats was inflicted by a weight-drop impact (10 g weight dropped from a height of 12.5 mm at T11–T12). Vehicle or BBG at 10 or 50 mg/kg was administered intravenously right after injury and once daily on days 2 and 3. Motor function was evaluated in a first group of rats from days 1–42. Tissue injury was evaluated on longitudinal sections of spinal cord prepared at day 42. Acute inflammatory responses were quantified in a second group of rats processed for immunohistochemistry at day 4.

BBG (10 or 50 mg/kg) significantly improves motor function and reduces tissue loss following traumatic spinal cord damage. (A) The graph depicts the BBB scores for locomotor function, which was evaluated immediately after injury and every 2–3 days thereafter. BBG significantly improved hindlimb motor function from day 13 after injury and onwards. Improvement persisted until 6 weeks postinjury. *, P < 0.05; * *, P < 0.01 compared with vehicle; one-way ANOVA with Dunnett's test. n = 11–17 rats per group. Error bars indicate SEM. (B) Longitudinal spinal cord sections stained with Luxol Fast Blue/Cresyl Violet display how the area of tissue injury and atrophy was quantified. The injury area exhibits less vacuolation and cell death when treated with 10 or 50 mg/kg BBG. (C) The bar histogram compares the loss of tissue following traumatic injury (the sum of the volumes of tissue injury and atrophy) in control vehicle or BBG treated animals. BBG at 10 mg/kg reduced the loss of tissue relatively more than 50 mg/kg. *, P < 0.05; * *, P < 0.01; compared to control volume, one-way ANOVA with Tukey-Kramer test. n = 11–17 rats per group. Error bars indicate SEM. (D) Whole perfusion-fixed spinal cords at 6 weeks. BBG (blue color) was deposited in the injury area in rats treated with BBG.