Mouse syngenic in vitro blood–brain barrier model: a new tool to examine inflammatory events in cerebral endothelium

Abstract

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 bloodbrain 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) Keywords: BBB; in vitro model; coculture; tight junction; P-glycoprotein; cell adhesion moleculeThe maintenance of brain interstitial fluid homeostasis is established by the presence of the bloodbrain barrier (BBB) which can be considered as the main interface between blood and brain parenchymal cells. This endothelium can be distinguished from the other vascular beds by the presence of continuous tight junctions and the absence of fenestration or channel. Both of these characteristics reduce the unspecific transport of molecules across the BBB. The blood/brain exchanges involve specific carrier-mediated transport systems that facilitate the uptake of nutrients. 1,2 BBB damage commonly associated to inflammatory events has been reported in several central nervous system (CNS) infections and neurodegenerative diseases. [3][4][5] Although BBB permeability disturbances seem to be critical steps as they lead to brain damage, neither the cause of these phenomena nor the effective participation of BBB in such diseases are established.The cerebral vascular wall was formally considered as a barrier that isolates brain from immunecontrol. However, the presence of immune cells in the brain parenchyma in healthy animals, and their increased cerebral residence in pathogenic conditions, demonstrate that the recruitment of inflammatory cells into the CNS, through the BBB, takes place. 6,7 In fact, the presence of cell adhesion molecules (CAMs) on the BBB suggests a direct and selective interaction between blood cells and the cerebral endothelium. These molecules, first described in peripheral capillaries, may mediate leukocyte transmigration across the BBB in a multistep process. 8,9 BBB damages and leukocyte infiltration in brain parenchyma were mostly examined using in vivo mouse models of chronic inflammation, which are

Identification of an essential endogenous regulator of blood–brain barrier integrity, and its pathological and therapeutic implications

“…Capillaries were extracted from the cortex of 4-mo-old WT or AnxA1-null mice according to published protocols (43). Isolated vessels were put in culture for further analysis (e.g., paracellular permeability) or fixed in 2% (wt/vol) PFA for 15 min, washed twice in 0.05 M PBS solution, and immunostained as described earlier for cell line-based analyses.…”

“…Four-month-old male C57BL/6 WT or ANXA1 −/− mice (42) were bred in house, with all ANXA1 −/− mice being genotyped by PCR before use. BBB permeability was assessed in vivo by administration of Evans blue dye as described previously (43). In some experiments, hrANXA1 was administered i.v.…”

Comparison of Immortalized bEnd5 and Primary Mouse Brain Microvascular Endothelial Cells as in vitro Blood–Brain Barrier Models for the Study of T Cell Extravasation

“…Primary mouse brain microvascular endothelial cells were isolated from gender-matched 4-to 6-week old C57BL/6 mice (Harlan Laboratories, Horst, The Netherlands), cultured in DMEM, 20% fetal calf serum, 1 mmol/L sodium pyruvate, 1% minimal essential medium nonessential amino acids, 50 mg/mL gentamycin, and 1 ng/mL basic fibroblast growth factor (Coisne et al, 2005;Lyck et al, 2009). For standardization of culture conditions between bEnd5 and pMBMECs, we used identical DMEM, and supplements for both types of brain endothelial cells were applicable.…”

“…Moreover, both pMBMECs and bEnd5 have previously been characterized for cytokine-induced upregulation of P-selectin, VCAM-1, and ICAM-1, all three of which are known to be involved in T cell interaction with the brain endothelium upon proinflammatory stimuli (Coisne et al, 2005(Coisne et al, , 2006Reiss et al, 1998;Rohnelt et al, 1997). However, although the expression of the tight junction protein occludin has been described for both in vitro BBB models (Coisne et al, 2005;Yang et al, 2007), we recently found a dramatically reduced occludin mRNA expression level in bEnd5 as compared with pMBMECs (Lyck et al, 2009).…”

“…However, to date, few mouse BBB in vitro models using primary brain endothelial cells have been established. Coisne et al (2005) recently described an in vitro model using primary mouse brain microvascular endothelial cells (pMBMECs) in coculture with primary mouse glial cells. Primary mouse brain microvascular endothelial cells form differentiated endothelial monolayers that retain numerous phenotypic properties of the CNS microvasculature in vitro, such as complex tight junctions and barrier formation (Coisne et al, 2005(Coisne et al, , 2006Lyck et al, 2009).…”