Neutral lipid reshaping in Blood Brain Barrier is induced in modelled ischemia-reperfusion.

Aims: Changes in lipids metabolism under ischemia-reperfusion (I/R) injury is implicated in oxidative stress and inflammatory response that lead to blood brain barrier (BBB) break-down. An increase in cell membranes cholesterol outcomes in the amyloid-β (Aβ) production during OGD/ogR [1]. The present study is aimed to monitor lipids fluctuations and peroxidation as well signaling pathway linked to those, in order to understand their contribute in BBB dysfunctions. Materials: Rat brain endothelial (RBE4) cells shows typical endothelial morphology retaining many characteristics; 5%CO2: 95%N2 gaseous mixtures (Sapio); hypoxia chamber (Billups- Rothenberg). Methods: RBE4 cells are subjected to oxygen and glucose deprivation (OGD). After medium replacement with a glucose-free balanced salt solution, cells are incubated for 3 hours in a gaseous mixture saturated chamber. The restoration solution (glucose and FBS) is administrated to cells (ogR) for one hour or 24 hours. Lipids analyses are performed by means high performance liquid chromatography (HPLC). Results: After OGD followed by 1 or 24 h ogR, the enhancement of inflammatory (COX-2) and oxidative stress (MDA) markers is detected. Phospholipids levels do not change, while neutral lipids metabolism is significant altered. Triglycerides (TG) levels increase accumulating in lipid droplets (LD), which move from nuclear to plasma membrane following a specific direction of actin filaments during ogR (1-24h). In parallel, cholesterol ester (CE), gathered in LD, decrease about 40% at ogR1h, returning to the control level at ogR24h. Activation of autophagy pathway is revealed by the increase of LC3-II/LC3-I ratio and the decrease in p62 levels. Finally, immunofluorescence experiments, show an increase in LD and LC3 co-localization during ogR. Discussion: I/R injury seems to induce a reshaping in triglycerides and in CE, sustaining the lipid droplets biogenesis up to an excessive lipid storage. During ogR the autophagy pathway is significant activated to counteract the cell engulfment (lipids and protein), as showed by LD and LC3 co-localization. The movement of LD to plasma membrane, suggest that conventional autophagy might be flanked by exocytosis pathways. Moreover, the increase of CE hydrolysis might play a role in Aβ stabilization along to a diminished Aβ lysosomal degradation [2]. Conclusions: I/R injury leads to the reshaping in neutral lipids that alters cellular membrane and cellular pathways, including Aβ stabilization and accumulation. Nevertheless, unconventional lipid exocytosis (TG and CE) might represent a type of cell communication to counteract the harmful effects of ischemic insult [3]