Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Abstract

Citation: Molino, Y., Jabès, F., Lacassagne, E., Gaudin, N., Khrestchatisky, M. Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport. J. Vis. Exp. (88), e51278, doi:10.3791/51278 (2014). AbstractThe blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm 2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10 -3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Gene expression comparison reveals distinct basal expression of HOX members and differential TNF-induced response between brain- and spinal cord-derived microvascular endothelial cells

“…The production of in vitro BBB and BSCB models is based on our previously described protocol [12]. Briefly, primary cultures of BMECs and SCMECs, prepared from 5- to 6-week-old Wistar rats, were seeded in the luminal compartment of six-well plate polyethylene insert filters (Merck Millipore, Billerica, MA, USA), pre-coated with collagen type IV and fibronectin (BD Biosciences, Franklin Lakes, NJ, USA) to establish the endothelial cell monolayers.…”

“…After 24 h inflammation with recombinant rat TNF-α (5 ng/mL, Peprotech), barrier integrity of the in vitro models was controlled with Lucifer Yellow (LY CH lithium salt, Sigma Aldrich), a small hydrophilic molecule (MW 457 g/mol) retained by the monolayers as previously described [12]. Briefly, quantification of the LY paracellular leakage from the luminal to the abluminal compartment was assessed by fluorimetric analysis (excitation at 430 nm and emission at 535 nm) and expressed in LY permeability, Pe (LY) .…”

“…Although, the structural and functional differences might explain that certain disease states differentially affect BMEC and SCMEC populations, very little is known concerning their intrinsic differences, including their response to inflammation. To gain more insight into such differences, a comparative transcriptomic analysis was performed on RNAs extracted from MEC monolayers of in vitro models of the BBB and BSCB [12] left untreated or treated with TNF-α for 12, 24, and 48 h.…”

Anti-transferrin receptor-modified amphotericin B-loaded PLA&ndash;PEG nanoparticles cure Candidal meningitis and reduce drug toxicity

“…These cells are rich in TfR. 27,28 AMB is an insoluble broad spectrum antifungal drug that does not efficiently traverse the BBB, making it difficult to achieve effective inhibitory concentrations of AMB in the brain. 29 High circulating concentrations of AMB can cause hemolysis or severe side effects such as nephrotoxicity and hepatotoxicity.…”

Role of Microvascular Disruption in Brain Damage from Traumatic Brain Injury

“…Alternatively, astrogliosis can also lead to BBB modulation over time (100). Astrocytes support endothelial cell function at the neurovascular unit and direct control of permeability (115). Astrocytes and neurons help support the basement membrane surrounding the endothelial cells (161).…”