Combination of tissue-plasminogen activator with erythropoietin induces blood–brain barrier permeability, extracellular matrix disaggregation and DNA fragmentation after focal cerebral ischemia in mice

Abstract

Background and Purpose— After 1 clinical study in which recombinant erythropoietin (EPO) protected against ischemic stroke and improved clinical outcome, the German multicenter EPO trial recently reported increased mortality in stroke patients receiving EPO after tissue-plasminogen activator (t-PA)-induced thrombolysis. The reasons for the adverse effects of EPO in t-PA–treated patients are unknown. Methods— Mice were submitted to 90 minutes of middle cerebral artery occlusion. Immediately after reperfusion, animals were treated with normal saline or t-PA (10 mg/kg). Animals subsequently received injections of normal saline or EPO that were administered after reperfusion and 12 hours later (2500 IU/kg each). Ischemic injury and brain edema were analyzed at 24 hours after reperfusion by cresyl violet staining and terminal transferase biotinylated-dUTP nick end labeling. Blood–brain barrier integrity was assessed by histochemistry for extravasated serum IgG. Matrix metalloproteinase activity was evaluated by gelatinase zymography. Results— EPO did not influence ischemic infarct size but reduced brain swelling. This effect was abolished by t-PA, which exacerbated serum IgG extravasation in ischemic tissue. Gelatinase zymographies revealed that EPO promoted matrix metalloproteinase-9 activity that was markedly elevated by t-PA. Add-on treatment with t-PA increased the density of DNA-fragmented cells in ischemic tissue of EPO-treated, but not vehicle-treated, mice. Conclusions— Our data demonstrate a hitherto unknown interaction of t-PA with EPO at the blood–brain interface, ie, promotion of vascular permeability and extracellular matrix breakdown, which may account for the unfavorable actions of EPO in t-PA–treated patients. After t-PA–induced thrombolysis, EPO may not be suitable as stroke treatment.