Optimized Enrichment of Murine Blood-Brain Barrier Vessels with a Critical Focus on Network Hierarchy in Post-Collection Analysis
Authors: Abdelazim Hanaa , Barnes Audra , Stupin James , Hasson Ranah , Muñoz-Ballester Carmen , Young Kenneth L. , Robel Stefanie , Smyth James W. , Lamouille Samy , Chappell John C. 
Published: September 2024

SUMMARYCerebrovascular networks contain a unique region of interconnected capillaries known as the blood-brain barrier (BBB). Positioned between upstream arteries and downstream veins, these microvessels have unique structural features, such as the absence of vascular smooth muscle cells (vSMCs) and a relatively thin basement membrane, to facilitate highly efficient yet selective exchange between the circulation and the brain interstitium. This vital role in neurological health and function has garnered significant attention from the scientific community and inspired methodology for enriching BBB capillaries. Extensive characterization of the isolates from such protocols is essential for framing the results of follow-on experiments and analyses, providing the most accurate interpretation and assignment of BBB properties. Seeking to aid in these efforts, here we visually screened output samples using fluorescent labels and found considerable reduction of non-vascular cells following density gradient centrifugation (DGC) and subsequent filtration. Comparatively, this protocol enriched brain capillaries, though larger diameter vessels associated with vSMCs could not be fully excluded. Protein analysis further underscored the enrichment of vascular markers following DGC, with filtration preserving BBB-associated markers and reducing – though not fully removing – arterial/venous contributions. Transcriptional profiling followed similar trends of DGC plus filtration generating isolates with less non-vascular and non- capillary material included. Considering vascular network hierarchy inspired a more comprehensive assessment of the material yielded from brain microvasculature isolation protocols. This approach is important for providing an accurate representation of the cerebrovascular segments being used for data collection and assigning BBB properties specifically to capillaries relative to other regions of the brain vasculature.HIGHLIGHTS–We optimized a protocol for the enrichment of murine capillaries using density gradient centrifugation and follow-on filtration.–We offer an approach to analyzing post-collection cerebrovascular fragments and cells with respect to vascular network hierarchy.–Assessing arterial and venous markers alongside those associated with the BBB provides a more comprehensive view of material collected.–Enhanced insight into isolate composition is critical for a more accurate view of BBB biology relative to larger diameter cerebrovasculature.MOTIVATIONThe recent surge in studies investigating the cerebrovasculature, and the blood-brain barrier (BBB) in particular, has inspired a broad range of approaches to target and observe these specialized blood vessels within murine models. To capture transcriptional and molecular changes during a specific intervention or disease model, techniques have been developed to isolate brain capillary networks and collect their cellular constituents for downstream analysis. Here, we sought to highlight the benefits and cautions of isolating and enriching microvessels from murine brain tissue. Specifically, through rigorous assessment of the output material following application of specific protocols, we presented the benefits of specific approaches to reducing the inclusion of non-vascular cells and non-capillary vessel segments, verified by analysis of vascular-related proteins and transcripts. We also emphasized the levels of larger- caliber vessels (i.e. arteries/arterioles and veins/venules) that are collected alongside cerebral capillaries with each method. Distinguishing these vascular regions with greater precision is critical for attributing specific characteristics exclusively to the BBB where metabolic, ion, and waste exchange occurs. While the addition of larger vessels to molecular / transcriptional analyses or follow-on experiments may not be substantial for a given protocol, it is essential to gauge and report their level of inclusion, as their contributions may be inadvertently assigned to the BBB. Therefore, we present this optimized brain microvessel isolation protocol and associated evaluation methods to underscore the need for increased rigor in characterizing vascular regions that are collected and analyzed within a given study.Graphical AbstractConstructed using resources from BioRender.com.