Abstract 18873: Protection of CD39 Transgenic Mice From Cerebral Ischemic Injury

Abstract

Cerebral tissue damage after an ischemic event can be exacerbated by inflammation and thrombosis. Elevated levels of extracellular ATP and ADP are associated with cellular injury and promote inflammation and thrombosis. Ectonucleoside triphosphate diphosphohydrolase 1 (CD39), an enzyme expressed on the plasmalemma of leukocytes and endothelial cells, suppresses platelet activation and leukocyte infiltration by phosphohydrolyzing ATP and ADP. To investigate the effects of increased CD39 in an in vivo model of cerebral ischemia, we developed a transgenic mouse expressing human CD39 (hCD39). A floxed stop sequence was inserted prior to the transcriptional start site of hCD39, generating a mouse in which the expression of hCD39 can be controlled in a tissue-specific manner with specific Cre recombinase mice. Here, we generated mice that express hCD39 globally, using an EIIA-Cre mouse. Bone marrow analysis showed similar endogenous CD39 mRNA between transgenic (TG) and wild type (WT) mice while expression of the human transgene was >3,600-fold greater than the mouse gene (p<0.0001, n=3). Cerebral ischemia was induced using a photothrombotic model of middle cerebral artery (MCA) occlusion, which is clinically relevant to ischemic stroke. After 48 hours, mice underwent brain MRI and infarct volumes were quantified. TG hCD39 mice had 37.8% smaller infarct volumes (p<0.05, n=5). A second cohort of mice was assessed for neurological deficits - TG mice showed lower deficits compared to WT (1.0 vs 1.5 based on an established 5-point scale p<0.02, n=8). Western blot analysis of non-ischemic hemispheres showed that TG mice had significantly more CD39 expression than WT mice (5.4-fold, n=3, p<0.0001) Leukocytes from ischemic and contralateral hemispheres were purified for flow cytometric analysis. While contralateral hemispheres had the same numbers of macrophages and neutrophils (CD45+/ F4/80+ and CD45+/Ly6g+ respectively), ischemic hemispheres from TG mice had 64% fewer infiltrating macrophages and 68% fewer neutrophils (p<0.001, n=8). This is the first report of transgenic overexpression of CD39 in mice imparting a protective phenotype following stroke, with reduced leukocyte infiltration, smaller infarct volumes and decreased neurological deficit.