An aged circulatory environment can activate microglia, reduce neural precursor cell activity, and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of Vascular Cell Adhesion Molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, the shed, soluble form of VCAM1 is prominently increased in plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and young mouse hippocampi. Systemic anti-VCAM1 antibody or genetic ablation of VCAM1 in BECs counteracts the detrimental effects of aged plasma on young brains and reverses aging aspects including microglial reactivity and cognitive deficits in old mouse brains. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier (BBB) as a possible target to treat age-related neurodegeneration.

The blood-brain barrier (BBB) proper is composed of endothelial cells (ECs) of the cerebral microvasculature, which are interconnected by tight junctions (TJs) that in turn form a physical barrier restricting paracellular flux. Tight control of vascular permeability is essential for the homeostasis and functionality of the central nervous system (CNS). In vitro BBB models have been in use for decades and have been of great benefit in the process of investigating and understanding the cellular and molecular mechanisms underlying BBB establishment. BBB integrity changes can be addressed in vitro by determining cell monolayer permeability (Pe) to different solutes and measuring trans-endothelial electrical resistance (TEER).This chapter describes procedures that can be utilized for both freshly isolated mouse brain microvascular ECs (MBMECs) and murine or human brain EC lines (bEnd5 or hCMEC/D3), cultivated either as a single monolayer or in cocultivation with primary mouse astrocytes (ACs). It starts with detailed information on how to perform transwell cell culture, including coating of inserts and seeding of the ECs and ACs. Moreover, it encompasses instructions for electrical assessment of the in vitro BBB using the more recent cellZscope(®) device, which was traditionally performed with chopstick electrodes of voltohmmeter type (EVOM). From continuous impedance measurements, the cellZscope(®) device provides TEER (paracellular resistance) and cell membrane capacitance (Ccl-transcellular resistance), two independent measures of monolayer integrity. Additionally, this chapter provides guidance through subsequent experiments such as permeability analysis (Pe, flux), expression analysis (qRT-PCR and Western blotting), and localization analysis of BBB junction proteins (immunocytochemistry) using the same inserts subjected earlier to impedance analysis.As numerous diseases are associated with BBB breakdown, researchers aim to continuously improve and refine in vitro BBB models to mimic in vivo conditions as closely as possible. This chapter summarizes protocols with the intention to provide a collection of BBB in vitro assays that generate reproducible results not only with primary brain ECs but also with EC lines to open up the field for a broader spectrum of researchers who intend to investigate the BBB in vitro particularly aiming at therapeutic aspects.

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