Background

The Blood-brain barrier (BBB) is a complex vascular structure that separates and protects the brain from systemic circulating factors and immune cells while allowing selective transport of critical nutrients. This highly specialized vasculature regulates selective transport of metabolites across the barrier. BECs also form a central component of the neurovascular unit (NVU), with close association and crosstalk with various cell types in the brain including neural precursor cells, microglia and the brain-resident immune cells. The BBB not only limits the passage of ions and other molecules such as glucose but also prevents the uncontrolled exchange of toxins, bacteria, viruses and cells between the blood and the brain parenchyma (Abbott et al., 2010). Nutrient-rich, oxygenated blood is pumped into the brain through cerebral arterial BECs (arteries and arterioles), which are protected and supported by smooth muscle cells (SMCs) that cover the endothelium and form a basement membrane layered by astrocytic end-feet of the brain parenchyma (Figure 1). The blood is transferred to highly specialized capillaries, which are comprised of BECs that form unique tight junctions and are wrapped by pericytes (Peric.) within the endothelial basement membrane, which is then covered by astrocytic end-feet. BBB capillaries are the site of controlled transport of fluids and solutes into the CNS. Immuno-surveillance and occasional extravasation of leukocytes (Leuk.) into the CNS parenchyma occurs at the level of postcapillary venous cells (venules and veins) the vascular segments into which blood flows after passing through the capillaries. Postcapillary Venules contain enlarged perivascular space between the endothelial and astrocytic basement membranes where occasional immune cells can reside (Banks, 2016; Engelhardt et al., 2017).

The outlined fluorescent activated cell sorting (FACS) single cell isolation method is based on previously described procedure for BEC isolation (Tam et al., 2012), that have been further developed and modified in context of a study aiming to determine molecular changes occurring in brain endothelial cells during aging and exposure to aged blood (Yousef et al., 2018). A schematic of the FACS procedure for BEC isolation and an example of cell gating are shown in Figure 2. Aging results in the upregulation of chronic inflammatory processes involving endothelial cell activation that contributes to reduced neural precursor cell activity and increased neuroinflammation in the aging brain (Yousef et al., 2018). The technique was successfully applied in a collaborative effort to investigating the effects of an aged systemic milieu on hippocampal neurogenesis and microglia activation (Yousef et al., 2018). The study highlights the role of the vascular cell adhesion molecule 1 (VCAM1) as a negative regulator of adult neurogenesis and inducer of microglial activity. VCAM1 is only expressed in a very small subpopulation of cells under baseline conditions and thus, was not sufficiently immunolabeled using conventional two-step antibody staining to perform FACS analysis. To that end, mice were stimulated systemically with Lipopolysaccharide (LPS) from Salmonella typhimurium to increase VCAM1 expression. The mice were then retro-orbitally injected with fluorescently labeled anti-mouse VCAM1, which resulted in a reliable detection of the VCAM1 positive brain endothelial cell subpopulation. The CD31⁺VCAM1⁺ BECs in LPS-stimulated mice could be used to set positive and negative gates in the flow sorter to quantitatively assess CD31⁺VCAM1⁺ expression in normal young and aged mice, or young mice treated with young or aged plasma and to isolate this rare subpopulation (Yousef et al., 2018; Figure 3). Additionally, VCAM1 is enriched in arterial and venous BECs and can be used to enrich and study these two vessel segmental populations (Vanlandewijck et al., 2018; Yousef et al., 2018).

In the last decade, several protocols for brain endothelial cell isolation have been employed, which tend to use transgenic endothelial cells labeled with fluorescent proteins such as GFP (Daneman et al., 2010; Vanlandewijck et al., 2018). Because these techniques are dependent on transgenic endothelial cell markers, they may not easily be applied to diseased or normal aged mouse models. This protocol allows the isolation of highly pure brain endothelial cell population from any mouse strain or murine animal model using papain-based enzymatic digestion using the commercial neural dissociation kit (MACS Miltenyi Biotec, Miltenyi).


Figure 1. Schematic of the blood-brain barrier. Nutrient-rich, oxygenated blood is pumped into the brain through cerebral arterial BECs (arteries and arterioles), which are protected and supported by smooth muscle cells (SMCs) that cover the endothelium and form a basement membrane layered by astrocytic end-feet of the brain parenchyma. The blood is transferred to highly specialized capillaries, which are comprised of BECs that form unique tight junctions and are wrapped by pericytes (Peric.) within the endothelial basement membrane, which is then covered by astrocytic end-feet. BBB capillaries are the site of controlled transport of fluids and solutes into the CNS. Immuno-surveillance and occasional extravasation of leukocytes (Leuk.) into the CNS parenchyma occurs at the level of postcapillary venous cells (venules and veins) the vascular segments into which blood flows after passing through the capillaries. Postcapillary Venules contain enlarged perivascular space between the endothelial and astrocytic basement membranes where occasional immune cells can reside (Banks, 2016; Engelhardt et al., 2017). (Adopt from Yousef et al. [2018] Figure 2a)


Figure 2. Mouse Brain endothelial cell isolation through flow cytometry. A. Schematic of flow sorting of CD31⁺CD45^- BECs from mouse cortex and hippocampi. Each isolated RNA sample is a pool of BECs from 2 mouse brains. B. FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11b⁺ and CD45⁺ cells were gated to exclude monocytes/macrophages and microglia. CD31⁺Cd11b^-CD45^- cells were defined as the BEC population. (Adopt from Yousef et al. [2018] Supplemental Figure 1a-b)


Figure 3. Identification of VCAM1⁺CD31⁺ BECs through flow cytometry. Vcam1^fl/fl Slco1c1-Cre^ERT2+/- mice (Cre+) received tamoxifen (100 mg/kg; i.p.) once daily for 5 days. After a 3-day resting period, mice were treated with LPS for 16 h and 2 h before perfusion (1 mg/kg, i.p.) and fluorescently labeled anti-VCAM1 mAb (100 µg, r.o.) for 2 h prior to cell isolation and flow cytometry analysis. Gating plots of CD31⁺VCAM1⁺ cells isolated from Gating plots are of tamoxifen-treated, LPS stimulated aged (19-month-old) Slco1c1-Cre^ERT2+/--Vcam1^fl/fl (Cre+) and littermate control mice lacking a copy of the Cre gene (Cre-), injected with fluorescently-tagged DL488 anti-VCAM1 mAb or IgG-DL488 isotype control (r.o.) 2 h before sacrifice.