In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 ± 13 W · cm 2 ; HC 218 ± 66 W · cm 2 ) than those treated by the glucocorticoid alone (CORT 57 ± 5 W · cm 2 ;HC 70 ± 2 W · cm 2 ). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations. The cerebral microvasculature separates the brain interior from the bloodstream and has been termed the blood-brain barrier (BBB) as a result of its impermeable properties. The BBB assists in maintaining brain homeostasis and protects the brain against harmful blood-borne substances. A single layer of brain microvascular endothelial cells (BMEC) is responsible for the limited solute transfer between blood and brain, and these specialized endothelial cells (EC) display distinctive attributes when compared with peripheral endothelium. Low BMEC permeability results from continuous tight junctions between adjoining ECs (Reese and Karnovsky 1967), low levels of pinocytosis and a general lack of fenestrae (Brightman and Reese 1969;Joo 1971). Because of the impermeable phenotype, the BBB plays major roles in disease pathology and hinders drug delivery efforts. Because of the inherent difficulties in performing molecular level studies of disease pathology in vivo, and the fact that prediction of BBB drug permeability prior to animal studies would be highly advantageous, a representative in vitro model would be of high utility. Unfortunately, when

The blood-brain barrier (BBB) is composed of uniquely differentiated brain microvascular endothelial cells (BMEC). Often, it is of interest to replicate these attributes in the form of an in vitro model, and such models are widely used in the research community. However, the BMEC used to create in vitro BBB models de-differentiate in culture and lose many specialized characteristics. These changes are poorly understood at a molecular level, and little is known regarding the consequences of removing BMEC from their local in vivo microenvironment. To address these issues, suppression subtractive hybridization (SSH) was used to identify 25 gene transcripts that were differentially expressed between in vivo and in vitro BMEC. Genes affected included those involved in angiogenesis, transport and neurogenesis, and real-time quantitative polymerase chain reaction (qPCR) verified transcripts were primarily and significantly downregulated. Since this quantitative gene panel represented those BMEC characteristics lost upon culture, we used it to assess how culture manipulation, specifically BMEC purification and barrier induction by hydrocortisone, influenced the quality of in vitro models. Puromycin purification of BMEC elicited minimal differences compared with untreated BMEC, as assessed by qPCR. In contrast, qPCR-based gene panel analysis after induction with hydrocortisone indicated a modest shift of 10 of the 23 genes toward a more 'in vivo-like' gene expression profile, which correlated with improved barrier phenotype. Genomic analysis of BMEC de-differentiation in culture has thus yielded a functionally diverse set of genes useful for comparing the in vitro and in vivo BBB.

No abstract