Minocycline and tissue-type plasminogen activator for stroke: assessment of interaction potential

Abstract

Background and purpose: New treatment strategies for acute ischemic stroke must be evaluated in the context of effective reperfusion. Minocycline is a neuroprotective agent that inhibits proteolytic enzymes and therefore could potentially both inactivate the clot lysis effect and decrease the damaging effects of tissue-type plasminogen activator (t-PA). This study aimed to determine the effect of minocycline on t-PA clot lysis and t-PA-induced hemorrhage formation after ischemia.

Methods: Fibrinolytic and amidolytic activities of t-PA were investigated in vitro over a range of clinically relevant minocycline concentrations. A suture occlusion model of 3-hour temporary cerebral ischemia in rats treated with t-PA and 2 different minocycline regimens was used. Blood-brain barrier basal lamina components, matrix metalloproteinases (MMPs), hemorrhage formation, infarct size, edema, and behavior outcome were assessed.

Results: Minocycline did not affect t-PA fibrinolysis. However, minocycline treatment at 3 mg/kg IV decreased total protein expression of both MMP-2 (P=0.0034) and MMP-9 (P=0.001 for 92 kDa and P=0.0084 for 87 kDa). It also decreased the incidence of hemorrhage (P=0.019), improved neurologic outcome (P=0.0001 for Bederson score and P=0.0391 for paw grasp test), and appeared to decrease mortality. MMP inhibition was associated with decreased degradation in collagen IV and laminin-alpha1 (P=0.0001).

Conclusions: Combination treatment with minocycline is beneficial in t-PA-treated animals and does not compromise clot lysis. These results also suggest that neurovascular protection by minocycline after stroke may involve direct protection of the blood-brain barrier during thrombolysis with t-PA.

Figure 1

Impact of minocycline on fibrinolytic activity of tPA. The lysis of blood clots was initiated by 2nM tPA at 37°C. The amount of fibrinolysis was determined by measuring the release of soluble ¹²⁵I-fibrin degradation products at various time intervals (35, 75 and 120 minutes). The fibrinolytic activity of tPA was not affected by any concentration tested of minocycline (1 through 30 ug/ ml). The means ± SEM (n=4) are shown.

Figure 2

(A) Brain MMP-2 and MMP-9 activities after stroke as measured by gelatin zymography. MMP-2 and MMP-9 were elevated by tPA compared to stroked saline controls. Minocycline decreased MMP-9 activity to below control levels (NS). (B) Brain MMP-2 (72 kDa) and MMP-9 (87 kDa and 92kDa) protein content 24 hours after stroke as determined by immunoblotting. IV Minocycline group had significantly lower MMP-2 protein, MMP-9 92 kDa and MMP-9 87 kDa proteins compared to tPA animals (p=0.0034; p=0.001and p=0.0084, respectively). Vertical bars indicate SEM.

Figure 2

(A) Brain MMP-2 and MMP-9 activities after stroke as measured by gelatin zymography. MMP-2 and MMP-9 were elevated by tPA compared to stroked saline controls. Minocycline decreased MMP-9 activity to below control levels (NS). (B) Brain MMP-2 (72 kDa) and MMP-9 (87 kDa and 92kDa) protein content 24 hours after stroke as determined by immunoblotting. IV Minocycline group had significantly lower MMP-2 protein, MMP-9 92 kDa and MMP-9 87 kDa proteins compared to tPA animals (p=0.0034; p=0.001and p=0.0084, respectively). Vertical bars indicate SEM.

Figure 2

(A) Brain MMP-2 and MMP-9 activities after stroke as measured by gelatin zymography. MMP-2 and MMP-9 were elevated by tPA compared to stroked saline controls. Minocycline decreased MMP-9 activity to below control levels (NS). (B) Brain MMP-2 (72 kDa) and MMP-9 (87 kDa and 92kDa) protein content 24 hours after stroke as determined by immunoblotting. IV Minocycline group had significantly lower MMP-2 protein, MMP-9 92 kDa and MMP-9 87 kDa proteins compared to tPA animals (p=0.0034; p=0.001and p=0.0084, respectively). Vertical bars indicate SEM.

Figure 3

(A). Hemorrhagic transformation was increased (p = 0.0190) in animals treated with tPA 10 mg/ kg. Adjuvant treatment with minocycline 3 mg/ kg intravenously prevented this increase. (B) Bleeding in an animal in the tPA treated group (10 mg/ kg). Hematomas were seen in the animals that died. (C) Minocycline treatment (3 mg/ kg intravenously) did not significantly decrease edema. Vertical bars indicate SEM.

Figure 4

(A) Effect of treatment on collagen type IV protein content in the brain. Stroke significantly decreased baseline value (p=0.0011). Treatment with tPA 10 mg/ kg further decreased collagen IV. Treatment with 3 mg/ kg minocycline intravenously diminished collagen degradation. (B) Effect of treatment on laminin-α1 protein content in the brain. Stroke significantly decreased laminin and treatment with 3 mg/ kg minocycline strongly prevented laminin degradation (p = 0.0001). Vertical bars indicate SEM.

Figure 4

(A) Effect of treatment on collagen type IV protein content in the brain. Stroke significantly decreased baseline value (p=0.0011). Treatment with tPA 10 mg/ kg further decreased collagen IV. Treatment with 3 mg/ kg minocycline intravenously diminished collagen degradation. (B) Effect of treatment on laminin-α1 protein content in the brain. Stroke significantly decreased laminin and treatment with 3 mg/ kg minocycline strongly prevented laminin degradation (p = 0.0001). Vertical bars indicate SEM.

Figure 5

(A) Infarct size after correction for edema. tPA significantly increased infarct size (p=0.0035). Adjuvant treatment with 3 mg/ kg of minocycline intravenously decreased infarct size. Vertical bars indicate SEM. (B) Illustration with a representative brain of each of the treatment groups.

Figure 6

Effect treatment in behavioral outcome. (A) Improvement in the Bederson scale of animals treated with IV minocycline compared to tPA only treated animals (p=0.0001). (B) tPA + Mino IV treatment group performed better in the paw grasp task (p=0.0391) (B). Vertical bars in all graphs represent SEM.