Transient oxygen-glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation

Abstract

Thrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective.

Keywords: BBB; BCEC; DMEM; Dulbecco's modified Eagle's medium; EC; GC; GFAP; H/PI; Hoechst/propidium iodide; LDH; LY; Lucifer yellow; NGS; OGD; P(e); PBS; R; RH; Ringer–HEPES buffer; SEM; TEM; TJ; WGA-HRP; bFGF; basic fibroblast growth factor; blood–brain barrier; brain capillary endothelial cell; endothelial cell; endothelial permeability coefficient; glial cell; glial fibrillary acidic protein; lactate dehydrogenase; normal goat serum; oxygen–glucose deprivation; phosphate buffer saline; recombinant tissue-type plasminogen activator; reoxygenation; rtPA; standard error of the mean; tight junction; transmission electron microscopy; wheat germ agglutinin-horseradish peroxidase.