London, United Kingdom 2013 2 Vascular biology 8:40 - 8:50 Dynamic crosstalk of neutrophil granulocytes and microglia at the ischemic blood-brain interface - role of very late antigen-4. J. Neumann1, M. Riek-Burchardt2, R. König3, M.W. Görtler4, H.J. Heinze5, K.G. Reymann6, M. Gunzer7 Department of Neurology, University of Magdeburg, Magdeburg, GERMANY1,Leib-niz Institute for Neurobiology, Magdeburg, GERMANY2, Leibniz Institute for Neurobiology, Magdeburg, GERMANY3, Department of Neurology, University of Magdeburg, Magdeburg, GERMANY4, Department of Neurology, University of Magdeburg, Magdeburg, GERMANY5, German Centre for Neurodegenerative Diseases (DZNE), Magdeburg, GERMANY6, Institute of Experimental Immunology and Imaging, University of Duisburg-Essen, Essen, GERMANY7 Background: The pathophysiology of ischemic stroke resulting from the occlusion of arterial vessels which leads to irreversible neuronal cell injury is further aggravated by invading pe-ripheral immune cells. Histologically, early inflammatory infiltrates are mainly characterized by neutrophil granulocytes. However, the cellular dynamics of neutrophil entry and the direct con-sequences for the affected brain area have remained unclear. Methods: Dynamic of neutrophil adhesion, infiltration as well as interaction between microglia and neutrophils after focal cerebral ischemia (occlusion of middle cerebral artery) in mice have been visualized by intravital, intracranial two-photon microscopy. Results: We show by intravital two-photon microscopy in mice that neutrophils immediately recognize endothelial activation in stroke-related brain areas and start rolling and firm adhesion on such sites. The following neutrophil invasion is associated with breakdown of the endo-thelial barrier and edema formation around sites of neutrophil exit. Microglia within the brain recognize both, endothelial damage and neutrophil invasion. They physically shield activated endothelia and cooperate to form membrane networks that can trap infiltrating neutrophils. Sys-temic blockade of Very-late-antigen 4 (VLA-4) effectively inhibits endothelial interaction as well as brain entry of neutrophils. Conclusion: Thus, neutrophil invasion of ischemic brain is rapid, massive and strongly com-promises endothelial and microglial physiology in the affected area. Interfering with neutrophil entry via blockade of VLA-4 might thus be a powerful early stroke treatment. Cerebrovasc Dis 2013; 35 (suppl 3)1-854 27 8:30-10:00 Oral Session Room 17 Vascular biology & Translational stroke research Chairs: R. Bordet, France and M. Fatar, Germany 1 Vascular biology 8:30 - 8:40 Inhibition of Rho-kinase prevents oxygen-glucose deprivation-mediated cerebral barrier disruption K. Srivastava1, U. Bayraktutan2 The University of Nottingham, Nottingham, UNITED KINGDOM1,The University of Not-tingham, Nottingham, UNITED KINGDOM2 Background: Ischaemic brain injury characterised by oxygen-glucose deprivation (OGD) is known to induce cerebral barrier damage. Although underlying mechanisms remain largely un-known, RhoA/Rho-kinase pathway seems to be involved. Methods: Human brain microvascular endothelial cells (HBMEC) were exposed to normox-ia (25% O2, 5.5mM D-glucose), OGD (1% O2) alone and followed by reperfusion (OGD+R; 25% O2, 5.5mM D-glucose) in the absence/presence of Y-27632, a specific Rho-kinase inhib-itor. The protein levels of RhoA, Rho-kinase, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5 and beta-catenin were detected by Western blotting. An in vitro model of human blood-brain barrier (BBB) was established by co-culture of HBMEC with human astrocytes. The integrity and functional capacity of the BBB were studied by measure-ments of transendothelial electrical resistance (TEER) and flux of Evan’s blue-labelled albumin (EBA), respectively. Results: OGD+/-R produced dramatic increases in the protein expressions of Rho-kinase, gp91- phox, antioxidant enzyme catalase and in RhoA and pro-oxidant enzyme NADPH oxidase ac-tivities. Exposure of HBMEC to OGD+/-R led to emergence of oxidative stress as ascertained by the increased formation of superoxide anion. Reperfusion of cells elevated claudin-5 and beta-catenin protein levels, a tight junction and an adherens junction protein, respectively. OG-D+/- R impaired cerebral barrier integrity as evidenced by decreases in TEER and concurrent increases in EBA flux. Coexposure of HBMEC to OGD+/-R and Y-27632 effectively reversed all the changes observed in aforementioned protein expressions, enzyme activities and barrier integrity and function while suppressing superoxide anion levels. Conclusions: Attenuation of Rho-kinase activity via Y-27632 prevents the damage inflicted on cerebral barrier integrity and function by OGD+/-R through regulations of oxidative stress and maintenance of proper junctional complex.
Karger_ESC London_2013
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