Abstract
BackgroundThe blood–brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis.MethodsTranswell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments.ResultsIn contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations.ConclusionsThe co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.
Highlights
The blood–brain barrier (BBB) is altered in several diseases of the central nervous system
This loss of function can be measured by increased entry of permeability markers into the central nervous system (CNS). This can be determined non-invasively by measuring the reduction of the transendothelial electrical resistance (TEER). Glucose transporters such as SLC2A1 (Glut-1) are upregulated, by which Brain capillary endothelial cell (BCEC) try to take up the remaining glucose for stabilizing the energy balance
Human brain endothelial hCMEC/D3 cells express several tight junction and multidrug‐resistant ATP binding cassette (ABC) transporter proteins First the relative expression of claudins and ABC transporters in hCMEC/D3 cells was investigated under standard cultivation in EBM-2 medium with 5% Fetal Calf Serum (FCS) for 5 days followed by a serum reduction step in EBM-2 medium with 0.25% FCS for additional 24 h from day 5 to day 6
Summary
The blood–brain barrier (BBB) is altered in several diseases of the central nervous system. The breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression This functional damage is one key event which is attempted to be mimicked in in vitro models. In the case of cerebral ischemia, the BBB is damaged within a few hours, whereby the TJ lose their integrity and some ABC transporters are regulated to protect the cells [5, 17, 28, 41] This loss of function can be measured by increased entry of permeability markers into the CNS. One major objective of this study was to establish a human in vitro BBB model of cerebral ischemia that achieves a functional damage of approximately 35–60% TEER decline in less than 6 h. Cell line C6 was chosen, since their usability for inducing BBB breakdown has already been proven in a mouse ischemia model [31, 32]
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