Delayed minocycline inhibits ischemia-activated matrix metalloproteinases 2 and 9 after experimental stroke

Abstract

Background: Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are increased in the brain after experimental ischemic stroke in rats. These two proteases are involved with the degradation of the basal lamina and loss of stability of the blood brain barrier that occurs after ischemia and that is associated with thrombolytic therapy in ischemic stroke. Minocycline is a lipophilic tetracycline and is neuroprotective in several models of brain injury. Minocycline inhibits inflammation, apoptosis and extracellular matrix degradation. In this study we investigated whether delayed minocycline inhibits brain MMPs activated by ischemia in a model of temporary occlusion in Wistar rats.

Results: Both MMP-2 and MMP-9 were elevated in the ischemic tissue as compared to the contra-lateral hemisphere after 3 hours occlusion and 21 hours survival (p < 0.0001 for MMP-9). Intraperitoneal minocycline at 45 mg/kg concentration twice a day (first dose immediately after the onset of reperfusion) significantly reduced gelatinolytic activity of ischemia-elevated MMP-2 and MMP-9 (p < 0.0003). Treatment also reduced protein concentration of both enzymes (p < 0.038 for MMP-9 and p < 0.018 for MMP-2). In vitro incubation of minocycline in concentrations as low as 0.1 mug/ml with recombinant MMP-2 and MMP-9 impaired enzymatic activity and MMP-9 was more sensitive at lower minocycline concentrations (p < 0.05).

Conclusion: Minocycline inhibits enzymatic activity of gelatin proteases activated by ischemia after experimental stroke and is likely to be selective for MMP-9 at low doses. Minocycline is a potential new therapeutic agent to acute treatment of ischemic stroke.

Figure 1

The ischemic hemisphere has increased MMP-9 (significant increase) and MMP-2 activity as compared to the contra-lateral non ischemic control hemisphere in non-treated animals. Sample sizes: n = 8 for both PBS and minocycline treated groups (p < 0.0001 for MMP-9 increase). Each band represents one different replicate sample. Molecular weights were determined with the use of pre-stained protein standards.

Figure 2

Intra-peritoneal minocycline 45 mg/kg treatment twice a day (first dose immediately after reperfusion), significantly reduced the ischemia-induced increase in MMP-2 and MMP-9 gelatinolytic activities (represented by the active form bands; 67 and 86 kDa respectively). Sample sizes: PBS n = 6; Minocycline n = 8 (p < 0.0003). Each band represents one different replicate sample.

Figure 3

Intra-peritoneal minocycline 45 mg/kg treatment twice a day (first dose immediately after reperfusion), significantly reduced the ischemia-induced increase in MMP-2 and MMP-9 total protein concentrations as compared to PBS treated control animals. Sample sizes: PBS n = 6; Minocycline n = 6 (p < 0.038 for MMP-9 and p < 0.018 for MMP-2). Each band represents one different replicate sample.

Figure 4

Minocycline concentrations ranging within 0.1 and 1000 μg/ml inhibits the activity of 0.05 ng recombinant human MMP-2 and MMP-9 as compared to the control enzymatic activity. At low concentrations, MMP-9 inhibition is greater than that of MMP-2 (p < 0.05).