Breakdown of the blood-brain barrier (BBB) is a key step associated with ischemic stroke and its increased permeability causes extravasation of plasma proteins and circulating leukocytes. Polymorphonuclear neutrophil (PMN) proteases may participate in BBB breakdown. We investigated the role of PMNs in ischemic conditions by testing their effects on a model of BBB in vitro, under oxygen-glucose deprivation (OGD) to mimic ischemia, supplemented or not with high-density lipoproteins (HDLs) to assess their potential protective effects. Human cerebral endothelial cells cultured on transwells were incubated for 4 hours under OGD conditions with or without PMNs and supplemented or not with HDLs or alpha-1 antitrypsin (AAT, an elastase inhibitor). The integrity of the BBB was then assessed and the effect of HDLs on PMN-induced proteolysis of extracellular matrix proteins was evaluated. The release of myeloperoxidase and matrix metalloproteinase 9 (MMP-9) by PMNs was quantified. Polymorphonuclear neutrophils significantly increased BBB permeability under OGD conditions via proteolysis of extracellular matrix proteins. This was associated with PMN degranulation. Addition of HDLs or AAT limited the proteolysis and associated increased permeability by inhibiting PMN activation. Our results suggest a deleterious, elastase-mediated role of activated PMNs under OGD conditions leading to BBB disruption that could be inhibited by HDLs.