Extension of the thrombolytic time window with minocycline in experimental stroke

Abstract

Background and purpose: Thrombolysis with tPA is the only FDA-approved therapy for acute ischemic stroke. But its widespread application remains limited by narrow treatment time windows and the related risks of cerebral hemorrhage. In this study, we ask whether minocycline can prevent tPA-associated cerebral hemorrhage and extend the reperfusion window in an experimental stroke model in rats.

Methods: Spontaneously hypertensive rats were subjected to embolic focal ischemia using homologous clots and treated with: saline at 1 hour; early tPA at 1 hour, delayed tPA at 6 hours; minocycline at 4 hours; combined minocycline at 4 hours plus tPA at 6 hours. Infarct volumes and hemorrhagic transformation were quantified at 24 hours. Gelatin zymography was used to measure blood levels of circulating matrix metalloproteinase-9 (MMP-9).

Results: Early 1-hour thrombolysis restored perfusion and reduced infarction. Late 6-hour tPA did not decrease infarction but instead worsened hemorrhagic conversion. Combining minocycline with delayed 6-hour tPA decreased plasma MMP-9 levels, reduced infarction, and ameliorated brain hemorrhage. Blood levels of MMP-9 were also significantly correlated with volumes of infarction and hemorrhage.

Conclusions: Combination therapy with minocycline may extend tPA treatment time windows in ischemic stroke.

Figure 1

Cerebral perfusion during embolic focal ischemia and reperfusion after tPA thrombolysis, as measured with laser Doppler flowmetry (mean+SEM). Cerebral perfusion dropped rapidly below 20% after clot injection. Early tPA therapy at 1 hour almost fully restored perfusion. Delayed tPA therapy at 6 hours did not fully achieve reperfusion. No effects of minocycline on perfusion were detected.

Figure 2

Effects of tPA and minocycline on tissue outcomes. a, Representative images of TTC-stained brain sections are shown for each group. Infarction volume (b) and hemorrhage volume (c) at 24 hour. Data expressed as mean+SEM. tPA at 1 hour reduced infarction. Delayed tPA at 6 hours had no effects on infarction and instead induced cerebral hemorrhage. Combination therapy with 6-hour tPA plus 4-hour minocycline reduced infarction and prevented hemorrhage. *P<0.01.

Figure 3

Effects of tPA plus minocycline treatments on plasma MMP levels, all collected during the acute phase at 1 hour after tPA administration. a, Representative zymogram comparing tPA alone vs tPA plus minocycline treatment groups. tPA alone at 6 hours amplified plasma MMP-9 levels. Combination therapy with tPA plus minocycline prevented this tPA-associated amplification of MMP-9. b, Quantified densitometry of the zymogram data for MMP-9 responses. c, Quantified densitometry of the zymogram data for MMP-2 responses. *P<0.01.

Figure 4

Correlation between plasma MMP-9 levels at 1 hour after tPA administration with volumes of (a) infarction (R²=0.80, P<0.01) and (b) hemorrhage (R²=0.73, P<0.01). Closed circles indicate samples from rats treated with tPA alone at 6 hours. Open circles indicate samples from rats treated with minocycline at 4 hours plus tPA at 6 hours.