Figure legends
Figure 1. Scheme of the in vivo study design. (Experimental 1) Time course of sample preparation. (Experimental 2) Evans blue assay. (Experimental 3) Behavior test. GA (50 mg∙kg-1, i.p) was injected immediately after SCI and then received the same dose of GA at 6 h and 12 h, and then further treated once a day for 7 d.
Figure 2. GA directly binds to the active site of Jmjd3 and inhibits Jmjd3 activity. (A) Stereoview of the intermolecular interactions between the bound GA and the Jmjd3 enzyme residues that line the catalytic pocket shown as dashed lines. (B) The lysates from OGD/reperfusion-injured bEnd.3 cells were immunoprecipitated with the anti-Jmjd3, and immunoblotted with the anti-H3K27me3. GSK-J4 was used as a positive control. Immunoblotting was analyzed quantitatively (n = 5). All data represent mean ± SD. *p < 0.05 vs. Jmjd3 only.
Figure 3. GA inhibits the activation and expression of Jmjd3 after SCI. Rats were administered immediately with GA (50 mg∙kg-1) after SCI and spinal cord tissues were isolated at indicated time after injury. (A) The expression level of Jmjd3 was determined by RT-PCR (at 6 h and 1 d) and (B) Western blot (at 8 h and 1 d) after SCI and each experiment was analyzed quantitatively (n = 5). (C) The activity of Jmjd3 was measured by Western blot for anti-H3K27me3 at 8 h and 1 d after SCI and Western blot was quantified (n = 5). All data represent mean ± SD. *p < 0.05 vs. vehicle. (D) Representative fluorescence microscopic photographs showed that Jmjd3-positive blood vessel endothelial cells are positive for RECA1 at 1 d after injury. Scale bar, 30 μm.
Figure 4. GA inhibits the increase of BSCB permeability by suppressing MMP-9 expression and activation after SCI. Rat was administered with GA after SCI and spinal cord tissues from 6 h and 1 d were processed for RT-PCR (n = 5) and gelatin zymography (n = 5). (A) RT-PCR for Mmp-2 and -9 and densitometric analysis of RT-PCR. (B) Gelatin zymography. (C) Densitometric analysis of zymography. All data represent mean ± SD. *p < 0.05 vs. vehicle. (D) Representative spinal cord showing Evans blue dye permeabilized into moderately injured spinal cord and (E) quantification of the amount of Evans blue at 1 d after injury. Data represent as mean ± SEM. *p < 0.05 vs. vehicle. (F) The expression levels of TJ molecules were determined by Western blot with anti-ZO-1 (at 1 d) and anti-occludin (at 5 d) after injury. Western blot was analyzed quantitatively (n = 5). Data represent mean ± SD. *p < 0.05 vs. vehicle.
Figure 5. GA suppresses Jmjd3-mediated MMP-3 and -9 gene activation and inhibits TJs loss in OGD-induced bEnd.3 cells. (A) ChIP assay was performed in bEnd.3 cells subjected to 1 h of OGD/reperfusion injury using anti-Jmjd3. The occupancy of anti-Jmjd3 at the MMP-3 and MMP-9 promoter region was calculated via quantitative PCR. (B) Western blots for Jmjd3 and H3K27me3 was determined in bEnd.3 cells subjected to OGD/reperfusion injury. (C) Densitometric analysis of Western blot for Jmjd3 (Left) and H3K27me3 (Right) (n = 5). (D) Western blots for ZO-1 and occludin was determined in bEnd.3 cells subjected to OGD/reperfusion injury. (E) Densitometric analysis of Western blots for ZO-1 (Left) and occludin (Right) (n = 5). Data represent mean ± SD. *p< 0.05 vs. +OGD.
Figure 6. GA suppresses Jmjd3-mediated MMP-3 and -9 gene activation and inhibits TJs loss in OGD-induced bEnd.3 cells. (A) Schematic drawings showing infiltrated neutrophils and macrophages positive cells in both the rostral and caudal to the lesion area. MPO-positive neutrophils (green) at 1 d and ED-1-positive macrophages (red) at 5 d in the injured spinal cord tissues. (B) Representative photographs showing MPO-positive neutrophils and ED-1-positive macrophages in the dorsal column of injured spinal tissues at 1500 and 2000 μm rostral to lesion epicenter. Scale bar, 50 μm. (C) Relative fluorescent intensity of MPO- and ED-1-positive cells (n = 5). (D) Western blot (upper) and densitometric analysis (bottom) of ED-1 at 5 d after injury. To determine the expression of cytokines and chemokines, RT-PCR and Western blot were performed at indicated time points after injury (n =5). (E) RT-PCR and (F) densitometric analysis of cytokines (Il-1β and Tnf-α (at 2 h), Il-6 , Cox-2 andiNos (at 6 h) after injury). (G) RT-PCR and (H) densitometric analysis of chemokines (MCP-1 , MIP-1α , MIP-1β andMIP-2α (at 2 h), Gro-α (at 6 h) after injury and (I) Western blot and densitometirc analysis of inflammatory mediators (iNOS and COX-2 at 1 d after injury). All data represent mean ± SD. *p< 0.05 vs. vehicle.
Figure 7. GA inhibits apoptotic cell death of motor neurons and oligodendrocytes after SCI. (A) Representative Cresyl violet staining showing ventral horn of spinal cord at 3 mm rostral from lesion site at 1 d. Scale bar, 50 μm. (B) The spatial pattern of the number of VMN. (C) Representative TUNEL staining in the GM of the spinal cord at 2 mm rostral from lesion site at 1 d and (D) in the WM at 7 mm rostral from lesion site at 5 d. Bottom panels show high-power views (n = 5). Scale bars, 20 μm. (E) Quantitative analysis of TUNEL-positive cells. (F) Immunostaining of cleaved (active) caspase-3 in the WM at 5 d after injury (n = 5). Scale bar, 20 μm. (G) Immunohistochemical analysis of cleaved caspase-3 and CC1. Double labeling shows that oligodendrocytes in the WM were positive for cleaved caspse-3 after SCI (arrow). Scale bar, 30 μm. (H), Quantitative analysis of cleaved caspase-3-positive cells. (I) Western blot and (J) densitometirc analysis of cleaved caspase-3 at 1 d and 5 d after injury (n = 5). All data represent mean ± SD. *p < 0.05 vs. vehicle.
Figure 8. GA improves functional recovery after SCI. (A) BBB scores of vehicle and GA-treated groups after injury. (B) Grid walk test of vehicle and GA-treated groups at 35 d after injury. (C) Inclined plane test of vehicle- and GA-treated groups after injury. (D) Representative footprints obtained from each group at 35 d after SCI. All data are presented as mean ± SEM (n = 10). *p < 0.05 vs. vehicle.
Figure 9. GA alleviates axon and myelin loss and decreases lesion volume after SCI. (A) Representative photographs of NF200-positive axons in spinal cords. Sections were selected 2 mm rostral to the lesion site. Note that GA treatment decreased the extent of axon loss after injury. Scale bars, 20 μm. (B) Quantitative analysis of NF200-positive axons in ventral (left) and dorsolateral (right) funiculi showed that the density of spared axons in the GA-treated group was significantly higher than that in the vehicle. NF200-positive axons were counted as described under Materials and Methods. (C) Representative photographs of 5-HT-positive axons in ventral horn areas in sections 3 mm caudal to the lesion site. Scale bars, 30 μm. (D) Transverse cryosections (lateral funiculus) selected from 2mm rostral to the lesion site were processed for Luxol fast blue staining. Note that the extent of myelin loss was attenuated by GA treatment compared to the vehicle control. Scale bar, 100 μm. (E) Representative spinal cord tissues (1.2 mm from the dorsal surface) showing cavitation in the lesion site and quantitative analysis at 35 d after injury. Scale bar, 1 mm. All data are presented as mean ± SD (n = 5). *p < 0.05 vs. vehicle.