Figure 5

Expression of MMPs in ischemic EC in Timp-3 KO and WT mice at 72 h. A. Immunohistochemistry for MMPs in ischemic external capsule (EC) at 72 h reperfusion in Timp-3 KO and WT mice. The Timp-3 knockout had increased MMP-2 (red) immunoreactivity in ischemia, which co-localized with GFAP-expressing astrocytes (yellow). MMP-9 and MMP-3 were co-localized with astrocytes in the WT. Arrowheads show MMP-9 and -3 in astrocytes around vessels. Insert shows co-localization of MMP-3 and GFAP-positive astrocyte. Scale bar = 100 μm. B. Immunostaining shows MMP-3 expression in CC1-positive OLs in ischemic WT EC. C. Immunostaining shows MMP-3 expression in Iba-1 positive microglia/macrophages. Scale bar = 50 μm. D. Western blot of MMP-3 at 72 h reperfusion in Timp-3 KO and WT mouse brains. Western blots of MMP-3 standard and mouse brain tissues show the proform of MMP-3 at 57 kDa and an active form at 45 kDa. A 60 kDa glycosylated form of MMP-3 is also present in all brain samples. Brilliant blue R staining on the same blots was performed as control for protein loading and transfer. Quantification of pro and active MMP-3 forms together showed a significant increase in MMP-3 (57 kDa and 45 kDa) in ischemic WT compared with ischemic KO, sham KO and WT and nonischemic KO and WT (p < 0.05, n = 6 for each group). No significant difference was found between ischemic and nonischemic hemispheres in the KO. E. Fluorogenic substrate assay for MMP-3 activity shows significant increase in MMP-3 relative activity in both ischemic and nonischemic WT compared with sham WT (p < 0.05, n = 6 for each group).