A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature

Katrine Dahl Bjørnholm; Francesca Del Gaudio; Hao Li; Weihan Li; Elisa Vazquez-Liebanas; Maarja Andaloussi Mäe; Urban Lendahl; Christer Betsholtz; Per Nilsson; Helena Karlström; Michael Vanlandewijck

doi:10.1016/j.crmeth.2023.100431

A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature

Cell Rep Methods. 2023 Mar 27;3(3):100431. doi: 10.1016/j.crmeth.2023.100431.

Affiliations

¹ Department of Neurobiology, Care Sciences, and Society, Division of Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, Solna, Sweden.
² Department of Cell and Molecular Biology, Karolinska Institutet, Solna, Sweden.
³ Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
⁴ Department of Medicine, Huddinge, Karolinska Institutet, Solna, Sweden.

Abstract

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture.

Keywords: blood-brain barrier; capillary; endothelial cell; microvascular isolation; neurovascular unit; pericyte; single-cell RNA sequencing; vasculature.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Brain / blood supply
Cardiovascular System*
Cells, Cultured
Mice
Proteomics*