title: Characterisation of a mouse cerebral microvascular endothelial cell line (bEnd.3) after oxygen glucose deprivation and reoxygenation.
abstract: Studies have utilised immortalised mouse cerebral endothelial cells (bEnd.3) exposed to oxygen glucose deprivation (OGD) to study blood-brain barrier (BBB) disruption after ischaemia. However, there is a paucity of literature describing the duration of OGD (and reoxygenation [RO]) required to best simulate BBB disruption in vivo. In this study we assessed BBB disruption in bEnd.3 cells after exposure to a range of OGD periods, and also after OGD + RO. Exposure of bEnd.3 monolayers to 4, 6, 16, or 24 hours of OGD resulted in a significant increase in permeability. The hyperpermeability after 16 or 24 hours was associated with decreased expression of tight junction proteins (occludin and claudin-5). Furthermore, there was a decrease in cell viability and increased expression of the pro-apoptotic protein, cleaved caspase-3. Exposure of bEnd.3 monolayers to 1 hour OGD+ 23 hours RO exacerbated hyperpermeability relative to 1 hour OGD, which was associated with decreased expression levels of occludin and ZO-1, but no change in cell viability or caspase-3. 4 hours OGD + 23 hours RO exacerbated hyperpermeability, decreased expression levels of tight junction proteins, decreased cell viability, and increased caspase-3 expression. Thus, bEnd.3 cells exhibit hyperpermeability, a loss of tight junction proteins, and undergo cell death, after exposure to prolonged periods of OGD. Moreover, they exhibit exacerbated hyperpermeability, a loss of tight junction proteins, and increased expression of caspase-3 after OGD + RO. These findings will facilitate the use of this cell line in studies of BBB disruption and for the testing of therapeutics.
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Ku
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.
authors.forename: Mohammadali
authors.initials: M
authors.lastname: Taher
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.
authors.forename: Kai Yee
authors.initials: KY
authors.lastname: Chin
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.
authors.forename: Megan
authors.initials: M
authors.lastname: Grace
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: McIntyre
authors.affiliation: School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.
authors.forename: Alyson A
authors.initials: AA
authors.lastname: Miller
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism; D002199:Capillary Permeability / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000502:physiology; D002560:Cerebrovascular Circulation / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism*; D005947:Glucose / Q000172:deficiency*; D051379:Mice; D055806:Microvessels / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*
chemical_list: D005947:Glucose; D010100:Oxygen
keywords: blood-brain barrier; cell death; cultured cells; ischaemia; permeability; tight junctions