Abstract
Background Protein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, the isolation of brain microvessels from human brain tissue, particularly in small amounts, poses significant challenges. This study presents a method for isolating brain microvessels from a minimal amount of frozen human brain tissue, using techniques from an established mouse brain capillary isolation method.Methods The human brain microvessel fraction was obtained from approximately 0.3 g of frozen human brain (frontal cortex) using a bead homogenizer for homogenization and a combination of cell strainer and glass beads for purification. Protein expression in human microvessel fractions and whole-brain lysates was analyzed by western blot and proteomic analysis.Results Microscopic images showed successful isolation of brain microvessels from a frozen human brain. Protein assays indicated that sufficient protein was extracted from the microvessel fraction for detailed expression analysis. Western blot analysis showed enrichment of BBB-selective proteins (MDR1/ABCB1, GLUT1/SLC2A1, and CLDN5) in the brain microvessel fraction compared to whole-brain lysates. In addition, multiple reaction monitoring quantification of six BBB-selective proteins (MDR1, BCRP/ABCG2, GLUT1, MCT1/SLC16A1, transferrin receptor, and CLDN5) revealed expression levels consistent with those observed in larger human brain samples. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomics further revealed significant enrichment of human microvascular endothelial cells within the isolated fraction, mirroring the findings in mouse models.Conclusions The developed method successfully isolated brain microvessels from a small volume of frozen human brain tissue, facilitating the study of BBB proteome changes due to aging or pathological conditions. This technique provides valuable insights into BBB dysfunction in central nervous system disorders and may improve drug delivery strategies in the brain.