London, United Kingdom 2013 6 Translational stroke research 9:20 - 9:30 Co-application of the neuroprotectant FTY720 and rt-PA in an experimental model of ischemic stroke A. Cai1, J. Pfeilschifter2, H. Steinmetz3, C. Foerch4, W. Pfeilschifter5 Goethe University Hospital, Department of Neurology, Frankfurt am Main, GER-MANY1, Goethe University Hospital, Department of General Pharmacology and Toxicology, Frankfurt am Main, GERMANY2, Goethe University Hospital, Department of Neurology, Frankfurt am Main, GERMANY3, Goethe University Hospital, Department of Neurology, Frankfurt am Main, GERMANY4, Goethe University Hospital, Department of Neurology, Frankfurt am Main, GERMANY5 Background: Thrombolysis with rt-PA is the standard treatment for ischemic stroke patients. It harbors an increased risk of intracerebral hemorrhage due to a degradation of the blood-brain barrier by proteolytic enzymes, e. g. matrix metalloproteinases. FTY720 (Fingolimod), a sphin-gosine 1-phosphate receptor agonist which provides neuroprotection in stroke has also been shown to protect endothelial barriers in various other studies. The aim of this study is to illus-trate whether FTY720 has a positive impact on the blood-brain barrier disruption triggered by thrombolysis. Methods: We performed 3 h MCAO in 4 groups of 10 weeks old C57Bl/6 mice (n = 18/group). Prior to reperfusion, the groups were treated with vehicle (0.9 % NaCl) or FTY720 (1mg/kg) i.p., either alone or in combination with rt-PA (10 mg/kg) i. v. After 23 hours neurological im-pairment was tested and 200 μL of Evans Blue were injected i.v. and allowed to circulate for 1 hour before sacrifice. We measured MMP-9 activity by gelatin zymography and Evans Blue extravasation by photometry. Results: FTY720 in combination with rt-PA did not show a beneficial effect in terms of surviv-al and preservation of the blood-brain barrier. It did not reduce MMP-9 activation induced by cerebral ischemia and thrombolysis. In contrast, we saw a tendency, however non-significant despite a large sample size of 18 mice/group, towards a greater extent of mortality and blood-brain barrier disruption in mice who received both rt-PA and FTY720. Conclusion: Our results do not support a specific protective effect of FTY720 on the blood-brain barrier in cerebral ischemia and thrombolysis. The combination of FTY720 with rt-PA may even increase the risk of thrombolysis-induced blood-brain barrier disruption and second-ary intracerebral hemorrhage. This caveat should be considered if the neuroprotectant FTY720 or ist analogues are advanced towards clinical use in acute ischemic stroke. Cerebrovasc Dis 2013; 35 (suppl 3)1-854 29 5 Vascular biology 9:10 - 9:20 NADPH Oxidase is a key mediator of blood-brain barrier breakdown after an ischaemic stroke K. Vashisht1, U. Bayraktutan2 University of Nottingham, School of Clincial Sciences, Division of Stroke, Nottingham, UNITED KINGDOM1,University of Nottingham, School of Clincial Sciences, Division of Stroke, Nottingham, UNITED KINGDOM2 Background Enhanced NADPH oxidase activity and generation of superoxide anion (O2.-) may be a key mechanism in cerebral ischaemia/reperfusion injury. O2.- may compromise blood-brain barrier (BBB) integrity by regulating plasminogen activators (PAs) and downstream matrix metallo-proteinases (MMPs). Methods Human brain microvascular endothelial cells (HBMEC) were exposed to 4 or 20 hours of oxy-gen- glucose deprivation (OGD) alone or followed by 20 hours of reperfusion with and without inhibitors for NADPH oxidase and urokinase-type plasminogen activator, apocynin and ami-loride. HMBEC were also transfected with p22phox cDNA (a NADPH oxidase subunit) and then exposed to amiloride or an anti-uPA receptor antibody. O2.- levels and NADPH oxidase activity were measured using cytochrome C reduction and lucigenin-enhanced chemilumines-cence assays. MMP activity was measured by gelatin-zymography. Protein levels were assessed by Western blotting and PA activities were measured using an ELISA-based assay. BBB integ-rity was assessed on a HBMEC-human astrocyte co-culture model by transendothelial electrical resistance and flux of Evan’s blue albumin and sodium fluorescein. Results BBB permeability was disrupted by OGD alone which was further exacerbated by reperfusion. O2.- levels and NADPH oxidase activity were increased after 4 hours of OGD alone and fol-lowing reperfusion. Both OGD and reperfusion resulted in an increase in PA levels and activi-ties, whereas MMP-2 activity was only increased after reperfusion. Transfection with p22phox cDNA increased O2.- levels, NADPH oxidase activity and PA levels. Treatment with apocynin normalised the increases in BBB permeability and enzyme activities observed. Amiloride exert-ed similar effects. Conclusion Reperfusion injury appears to be linked to an increase in NADPH oxidase activity and ensuing increases in PA and MMP-2 activities. Inhibition of NADPH oxidase plays a therapeutic role in decreasing O2.- levels, MMP-2 activity and BBB damage.
Karger_ESC London_2013
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