ABSTRACT
There is a growing interest to use in vitro BBB cell assays in early safety assessment of compounds. By modifying a well-validated co-culture model of brain capillary endothelial and glial cells, developed by Dehouck et al. [Dehouck, M.P., Meresse, S., Delorme, P., Fruchart, J.C., Cecchelli, R., 1990. An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro. Journal of Neurochemistry 54 (5), 1798-1801], it has been possible to develop a new in vitro BBB system suitable for high throughput screening (HTS). In addition, this new procedure substantially reduces the use of experimental animals and considerably facilitates the process of obtaining a functional in vitro BBB model. The model is ready to use after only 4 days of culture and then shows the typical expression and localization of tight junction proteins. The function of the P-glycoprotein and the transcriptional expression of other efflux transporters such as MRP 1, 4 and 5 have been demonstrated. In addition, the model produces a good in vitro/in vivo correlation for 10 compounds (R2=0.81). Furthermore, studies were undertaken within the European ACuteTox consortium with the objective to assess BBB toxicity and make risk assessments of potentially toxic compounds according to their predicted ability to reach the CNS compartment. These investigations demonstrated that the results produced in the HTS BBB model were similar to the standard co-culture model.
Subject(s)
Blood-Brain Barrier/drug effects , Drug Evaluation, Preclinical/methods , ATP Binding Cassette Transporter, Subfamily B/biosynthesis , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Blotting, Western , Carrier Proteins/metabolism , Cattle , Cells, Cultured , Chemistry, Pharmaceutical , Data Interpretation, Statistical , Endothelial Cells/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Microscopy, Fluorescence , Permeability/drug effects , Pharmaceutical Preparations/metabolism , RNA/biosynthesis , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Tight Junctions/drug effectsABSTRACT
Increased lymphocyte trafficking across blood-brain barrier (BBB) is a prominent and early event in inflammatory and immune-mediated CNS diseases. The adhesion molecules that control the entry of leukocytes into the brain have not been fully elucidated. Although the role of ICAM-1 and VCAM-1 has been well documented, the expression and role of selectins is still a matter of controversy. In a mouse syngenic in vitro BBB model, highly relevant for examining immunological events, mouse brain capillary endothelial cells (MBCECs) do not express selectins. Treatment of MBCECs with LPS, induced E- and P-selectin expression, whereas TNF-alpha or IFN-gamma treatments did not. Finally, P-selectin but not E-selectin expression was induced in IL-1beta treated MBCECs. Thus, our study suggests that diverse inflammatory stimuli could differentially regulate selectin expression at the BBB.
Subject(s)
Cerebral Cortex/cytology , Cytokines/pharmacology , Endothelial Cells/drug effects , Lipopolysaccharides/pharmacology , Neuroglia/metabolism , Selectins/metabolism , Animals , Animals, Newborn , Antigens, CD/metabolism , Cell Adhesion Molecules/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Flow Cytometry/methods , Fluorescent Antibody Technique/methods , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Inflammation/chemically induced , Inflammation/metabolism , Intercellular Adhesion Molecule-1/metabolism , Mice , Neuroglia/drug effects , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Although cerebral endothelium disturbance is commonly observed in central nervous system (CNS) inflammatory pathologies, neither the cause of this phenomenon nor the effective participation of blood-brain barrier (BBB) in such diseases are well established. Observations were mostly made in vivo using mouse models of chronic inflammation. This paper presents a new mouse in vitro model suitable for the study of underlying mechanistic events touching BBB functions during CNS inflammatory disturbances. This model consists of a coculture with both primary cell types isolated from mice. Mouse brain capillary endothelial cell (MBCEC)s coming from brain capillaries are in culture with their in vivo partners and form differentiated monolayers that retain endothelial markers and numerous phenotypic properties of in vivo cerebral endothelium, such as: (1) peripheral distribution of tight junction proteins (occludin, claudin-5, claudin-3 and JAM-1); (2) high trans-endothelium electrical resistance value; (3) attenuated paracellular flux of sucrose and inulin; (4) P-gp expression; (5) no MECA-32 expression. Furthermore, this endothelium expresses cell adhesion molecules described in vivo and shows intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 upregulation under lipopolysaccharide-treatment. Therefore, this well-differentiated model using autologous cells appears as a suitable support to reconstitute pathological in vitro BBB model.
