Low Dose TPA Plus Annexin A2 Combination Improves Safety Profile When Treated At A Delayed Time In Rat Embolic Focal Stroke

Background and Purpose:We previously reported that tissue type plasminogen activator (tPA) in combination with recombinant annexin A2 (rA2) reduced acute brain damage and improved neurological outcomes after focal ischemia within 3-hour time window. The purpose of the present study was to ask whether the tPA plus rA2 combination therapy is more efficacious and/or safer when the treatment time window is delayed at 4 hours after stroke.Methods:We compared the effects of intravenous conventional tPA alone (10mg/kg) versus a combination of low-dose tPA (5mg/kg) plus 10 mg/kg rA2 in a focal embolic cerebral ischemia of rats. Totally 152 rats were used. All rats were treated at 4 hours after embolization. Experimental assessments included acute brain tissue damage at 1 day (60 rats), iron deposition at 1 week (24 rats), and neurological outcomes for up to 28 days after stroke (68 rats). Surrogate biomarkers for neurovascular remodeling in peri-infarct area were analyzed by immunohistochemistry at 28 days after stroke.Results:At 24 hours after stroke, there was no difference between saline and conventional tPA alone groups, but brain infarct volume of combination of low-dose tPA plus rA2 showed 9.2% reduction compared to saline,7.4% reduction compared to tPA, although the reductions did not reach statistical significance. However the combination significantly reduced (22.2% reduction) the conventional tPA-elevated intracerebral hemorrhagic (ICH) transformation. At 7 days after stroke, the combination significantly attenuated the conventional tPA alone-elevated iron deposition at peri-lesion area (68.2% reduction).7-day mortality rates in both of the combination (25%,2/8) and conventional tPA alone (37.5%, 3/8) groups were slightly lower than the saline control (50%,4/8). At 28 days after stroke, no significant difference in brain infarction and mortality between groups were detected, but the combination significantly improved neurological function in adhesive tap-removal test, which was accompanied by a significantly higher microvessel density at peri-infarct areas compared to conventional tPA alone group.Conclusions:Compared to conventional high-dose tPA alone, when treated at delayed 4-hour time point after stroke, the combination of low-dose tPA plus rA2 therapy provides a safer profile by lowering risk of ICH transformation and improving long term neurological outcomes after stroke.