Our goals are to extend the therapeutic window and to reduce adverse effects associated with thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) of embolic stroke. To accomplish these goals we will treat embolic stroke in the rat with a combination of rtPA and anti-adhesion molecule therapy using an antibody against the intercellular adhesion molecule (ICAM-1). The fundamental outcome measurements of the combination therapy will be volume of cerebral infarction and net hemorrhage. The mechanistic bases for secondary damage associated with delayed thrombolytic therapy will be investigated using quantitative laser scanning confocal microscopy (LSCM), immunohistochemistry, histology and Western blot analysis. We will quantify secondary embolization, thrombosis and vascular occlusion after embolic stroke, with and without combination therapy. Microvascular perfusion deficits will be measured under experimental conditions, as will adhesion molecule expression and localization of proinflammatory cytokines (IL1beta, TNFalpha) and vascular endothelial growth factor (VEGF). Cytokine and VEGF expression may be modulated by rtPA, and contribute to inflammatory responses and vascular disruption. Astrocytic response to treatment conditions and vascular deficits will also be quantified. The major strengths of our proposed studies are: 1) we focus on an immediate and highly relevant clinical problem, the need to expand access of stroke patients to rtPA treatment and to reduce secondary adverse effects from thrombolytic therapy. 2) the model of embolic stroke that we have developed and characterized closely adheres to the pathophysiological events and the etiology of human thromboembolic stroke. 3) we have novel data and new insights into the mechanisms of secondary rtPA induced damage, e.g. the enhancement of the inflammatory response to stroke evoked by rtPA treatment, the correlation of expression of VEGF and activation on astrocytes, and the reduction of perfusion. 4) we have developed, characterized and employed a novel technology, quantitative LSCM to the study of experimental stroke. This technology and the proposed experiments will enhance our understanding of the mechanisms underlying microvascular dysfunction and occlusion after stroke and thrombolytic treatment.
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