Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction.
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 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). 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.
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Anthony R
AR
Calabria
Christian
C
Weidenfeller
Angela R
AR
Jones
Helga E
HE
de Vries
Eric V
EV
Shusta
D000818:Animals; D001812:Blood-Brain Barrier / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D001921:Brain / Q000098:blood supply* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D062507:Claudin-5; D017097:Electric Impedance; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D008833:Microcirculation / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008954:Models, Biological; D062793:Occludin; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology*; D051381:Rats; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
C107466:CLDN5 protein, human; D062507:Claudin-5; D005938:Glucocorticoids; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568987:Ocln protein, rat; D011500:Protein Synthesis Inhibitors; D011691:Puromycin