Search for marker proteins to assess blood-brain barrier development.

The blood-brain barrier (BBB) separates the central nervous system from the peripheral blood circulation, and performs various functions such as dictating central nervous system (CNS)-specific pharmacokinetics, and maintaining brain homeostasis. While previous studies have clarified the mechanisms underlying cerebrovascular network development, details regarding in vivo BBB maturation remain unknown. In this regard, we previously reported that the development of physical tightness of the BBB and the formation of perivascular glial structures begins on postnatal day 4 and is almost complete by postnatal day 15. Moreover, the difficulty in predicting BBB permeability has hindered the development of CNS drugs, and microphysiological systems (MPSs) that mimic the BBB have been extensively developed to address this issue. Building on this information, in this study, we examined the temporal expression patterns of vascular proteins (CD31, Tie2, CD34, CD146, and agrin), tight junction proteins (ZO-1, claudin-5, and occludin), transporters (P-gp, BCRP, and Glut1), and transferrin receptors (TfRs) during the postnatal period, along with the development of BBB tightness. Based on their temporal expression patterns, these proteins were divided into five groups. We selected representative proteins from groups 1-5, respectively, and examined the temporal expression changes to determine the developmental stage of the BBB. The expression patterns of these proteins can be used to determine the maturation stage of BBB-MPSs.