Figure 3
Total and endogenously active MMP-9 are measurable with the FRET peptide-based immunocapture assay in multiple biological samples. Brain homogenates and plasma obtained from rats that underwent MCAO and sacrificed 48 h later were used in the new FRET peptide assay. (A) Total and active MMP-9 activity was measured in 500 μg total protein from ipsilateral and contralateral cerebral cortex. GM6001 (10 μM) was added to total MMP-9 ipsilateral samples. Total ipsilateral, active ipsilateral, and total ipsilateral + GM6001 compared by ANOVA with Newman-Keuls post-hoc test. * Statistically significant decreases compared to ipsilateral, p < 0.001, n = 7–8. # Statistically significant decrease compared to total ipsilateral, p < 0.01, n = 4–8. (B) Total MMP-9 activity recorded from 50 μL plasma from sham and MCAO rats. GM6001 (10 μM) added to MCAO plasma samples significantly reduced MMP-9 activity. Compared by ANOVA with Newman-Keuls post-hoc test. ** Statistically significant difference compared to MCAO, p < 0.001, n = 5. (C) Total MMP-9 activity from MCAO rat plasma samples (50 μL) and MCAO with the gelatinase inhibitor SB-3CT (0.5 μM and 5.0 μM). MMP-9 activity was significantly reduced with addition of SB-3CT in a dose-dependent manner. Compared by ANOVA with Newman-Keuls post-hoc test. *** Significant decrease compared to MCAO, p < 0.0001, n = 5. (D) Total MMP-9 activity in 10, 50, and 100 μL HT-1080 conditioned media (n = 4–6). (E) Zymogram, (F) densitometric analysis, and (G) FRET assay of total MMP-9 activity in lysates from RBE4 cells treated with IL-1β (10 ng/mL) compared to controls. Treatment with IL-1β significantly increased total MMP-9 levels in RBE4 cells as seen with zymography (Student’s t-test, * p < 0.05, n = 3) and the FRET assay (Student’s t-test, * p < 0.05, n = 3 independent experiments).