Isolation method of brain microvessels from small frozen human brain tissue for blood-brain barrier protein expression analysis
The blood-brain barrier (BBB), formed by brain capillary endothelial cells, regulates the movement of substances between the blood and the brain through tight junction protein complexes and selective transport systems, thereby maintaining central nervous system (CNS) homeostasis. Disruption or dysfunction of the BBB plays a critical role in the onset and progression of various CNS diseases. For instance, BBB breakdown is observed in Alzheimer’s disease (AD), even before the appearance of dementia symptoms  Additionally, impairment of the basement membrane in brain capillaries has been observed in AD model mice . BBB breakdown is also observed in Parkinson’s disease . Alterations in proteins associated with BBB transport systems are closely linked to CNS pathologies. Dysfunction of glucose transporter 1 (GLUT1/ solute carrier family 2 member 1 [SLC2A1]) and creatine transporter (CRT/solute carrier family 6 member 8 [SLC6A8]) leads to deficiency syndromes characterized by intellectual disability and epilepsy due to reduced brain glucose and creatine levels . Insulin resistance affects the expression of BBB transporters such as multidrug resistance 1 (MDR1)/ATP-binding cassette sub-family B member 1 (ABCB1), GLUT1, and monocarboxylate transporter 1 (MCT1)/solute carrier family 16 member 1 (SLC16A1) in high-fat diet-fed mice, potentially impacting cognitive function . In AD, amyloid-β peptide (Aβ) accumulation in the brain inversely correlates with MDR1 levels . Systemic inflammation promotes leukocyte migration across the BBB via changes in CD antigens, contributing to neuroinflammation . Similarly, lymphocyte interaction with the BBB plays a key role in multiple sclerosis progression . These findings underscore the critical role of BBB integrity and transport system alterations in CNS diseases. Elucidating the molecular mechanisms and pathogenesis underlying BBB disruption and dysfunction in human CNS diseases requires comprehensive analysis of brain microvessels. Quantitative proteomic analysis of isolated human brain microvessels provides critical insights into their physiological functions and pathological alterations . However, obtaining high-purity brain microvessels from human tissue poses significant challenges due to low yields, often necessitating large cortical samples (Table ) . For example, Uchida et al.  successfully isolated adequate brain microvessels from approximately 1–10 g of brain cortex, with other studies reporting similar requirements of 3–10 g. Such substantial tissue demands restrict the feasibility of human brain microvessel studies. Recently, a study demonstrated the isolation of brain microvessels from as little as 100 mg of human brain tissue for proteomic analysis (Table ) . However, this method lacked critical purification steps, such as mesh filtration and glass bead purification, potentially resulting in contamination by large vessels and blood cell components. Therefore, there is a critical need for efficient methods to isolate high-purity brain microvessels from small amounts of frozen human brain tissue, for accurate protein expression analysis. We previously developed an efficient method for isolating brain capillaries from a single frozen mouse brain sample (approximately 0.3 g) using a bead homogenizer . This method offers several advantages, such as a smaller scale and a five-fold increase in brain microvessel recovery compared to previously reported methods in mice . Given the shared principles underlying the isolation of brain microvessels from human and mouse brains , we hypothesized that this method could be adapted to achieve high-purity brain microvessel isolation from small amounts of human brain tissue. In the present study, we aimed to develop an efficient method for isolating microvessels from small amounts of frozen human brain tissue, optimized for protein expression analysis. Additionally, we performed quantitative proteomic analysis to characterize the protein expression profiles in the isolated human brain microvessels, highlighting the advantages and potential applications of our isolation method. 

Frozen human brain tissue Frozen human brain tissue (frontal cortex) was obtained from ProteoGenex (Inglewood, CA, USA) of the KAC Corporation (Kyoto, Japan). KAC Corporation has ethical approval for the use of human specimens. The tissues were stored at -80 °C until use. The donor information is summarized in Table . Human brain microvessel isolation Microvessels were isolated from frozen human brains, as previously described in mice . Briefly, thawed human brains were cut into 0.30–0.31 g pieces and homogenized in solution A (101 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 15 mM HEPES, pH 7.4) using a bead homogenizer (Bead Mill 4, Thermo Fisher Scientific) with stainless beads (diameter 3.2 mm, approximately 1.8 g, TOMY SEIKO). Part of the brain homogenate was collected in another tube and used as whole-brain lysate. The brain microvessels were then concentrated by centrifugation (1,000 × g , 10 min, 4 °C) with 16% dextran, and the collected brain microvessels were purified by mesh filtration through a cell strainer (70 μm mesh) with glass beads using solution B (25 mM NaHCO 3 , 10 mM glucose, 1 mM pyruvate, and 5 g/L bovine serum albumin). Isolated brain microvessel fractions were lysed by sonication in a hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl 2 , pH 7.4). The amount of protein in the brain microvessel fraction was measured using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Proteins were used for western blot and proteomic analyses as described below. Western blot analysis Western blot analysis of the isolated brain microvessel fractions and whole-brain lysates was performed as previously described  using primary antibodies (claudin 5 [CLDN5], 35-2500, Thermo Fisher Scientific; GLUT1, ab652, Abcam, Cambridge, UK; MDR1, #13978, Cell Signaling Technology [CST]; β-actin, 8H10D10, CST) and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG HRP, ab6721, Abcam; goat anti-mouse IgG HRP, ab6789, Abcam). Targeted quantitative proteomic analysis Multiplexed multiple reaction monitoring (MRM) analysis was used to determine the protein expression levels of target molecules in the brain microvessel fraction as previously described . The brain microvessel fraction was subjected to trypsin digestion using the phase-transfer surfactant (PTS) method . Stable isotope-labeled peptides were spiked into the digested peptides with internal standards, and the peptide samples were desalted and dissolved in 0.1% trifluoroacetic acid for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Standard samples were prepared as a dilution series of unlabeled standard peptides at relevant injection levels (0.5–100 fmol) with 30 fmol of internal standards. The samples were analyzed using a micro-LC system with a ChromXP C18CL column (Ekisigent, Redwood City, CA, USA) coupled with a QTRAP6500 electrospray ionization triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA). The transitions are listed in Table . Data were analyzed using Skyline version 19.1 (MacCoss Laboratory, University of Washington, WA, USA). The quantification value was determined as the average of the peak ratios of 3–4 transitions. Label-free quantitative proteomic analysis The Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) method was used for label-free quantitative proteomic analysis of both brain microvessel fractions and whole-brain lysates. After trypsin digestion of the samples using the PTS method, proteins were quantified based on specific peptide data detected by LC-MS/MS. The data were acquired using information-dependent acquisition (IDA) and SWATH modes on a TripleTOF6600 (SCIEX) interfaced with the Eksigent NanoLC400 system (SCIEX). Proteins were identified using Protein Pilot 4.5 (SCIEX) with MS/MS data obtained from IDA. Protein expression data were calculated from the precursor peak area data using the MaxLFQ algorithm integrated into DIA-NN . The average protein peak areas of the samples in each group were compared between the two groups. Data analysis of quantitative proteomic analysis using RNA-Seq database Among the proteins identified by quantitative proteomic analysis, we selected cell-type markers for brain endothelial cells, astrocytes, neurons, oligodendrocytes, and microglia. These selections were based on the criterion that the mRNA expression ratio (the ratio of expression levels in the most highly expressed cell type to the second most highly expressed cell type) was greater than 10-fold. The human brain RNA sequencing database used for this analysis has been previously published . Statistics analyses Numerical data are presented as mean ± standard deviation (SD). Pearson’s correlation analysis was employed to determine the correlations between the two groups. The statistical significance of differences was assessed using Student’s t -test using Microsoft Excel version 16.0 (Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). 

Frozen human brain tissue (frontal cortex) was obtained from ProteoGenex (Inglewood, CA, USA) of the KAC Corporation (Kyoto, Japan). KAC Corporation has ethical approval for the use of human specimens. The tissues were stored at -80 °C until use. The donor information is summarized in Table . 

Microvessels were isolated from frozen human brains, as previously described in mice . Briefly, thawed human brains were cut into 0.30–0.31 g pieces and homogenized in solution A (101 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 15 mM HEPES, pH 7.4) using a bead homogenizer (Bead Mill 4, Thermo Fisher Scientific) with stainless beads (diameter 3.2 mm, approximately 1.8 g, TOMY SEIKO). Part of the brain homogenate was collected in another tube and used as whole-brain lysate. The brain microvessels were then concentrated by centrifugation (1,000 × g , 10 min, 4 °C) with 16% dextran, and the collected brain microvessels were purified by mesh filtration through a cell strainer (70 μm mesh) with glass beads using solution B (25 mM NaHCO 3 , 10 mM glucose, 1 mM pyruvate, and 5 g/L bovine serum albumin). Isolated brain microvessel fractions were lysed by sonication in a hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl 2 , pH 7.4). The amount of protein in the brain microvessel fraction was measured using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific). Proteins were used for western blot and proteomic analyses as described below. 

Western blot analysis of the isolated brain microvessel fractions and whole-brain lysates was performed as previously described  using primary antibodies (claudin 5 [CLDN5], 35-2500, Thermo Fisher Scientific; GLUT1, ab652, Abcam, Cambridge, UK; MDR1, #13978, Cell Signaling Technology [CST]; β-actin, 8H10D10, CST) and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG HRP, ab6721, Abcam; goat anti-mouse IgG HRP, ab6789, Abcam). 

Multiplexed multiple reaction monitoring (MRM) analysis was used to determine the protein expression levels of target molecules in the brain microvessel fraction as previously described . The brain microvessel fraction was subjected to trypsin digestion using the phase-transfer surfactant (PTS) method . Stable isotope-labeled peptides were spiked into the digested peptides with internal standards, and the peptide samples were desalted and dissolved in 0.1% trifluoroacetic acid for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Standard samples were prepared as a dilution series of unlabeled standard peptides at relevant injection levels (0.5–100 fmol) with 30 fmol of internal standards. The samples were analyzed using a micro-LC system with a ChromXP C18CL column (Ekisigent, Redwood City, CA, USA) coupled with a QTRAP6500 electrospray ionization triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA). The transitions are listed in Table . Data were analyzed using Skyline version 19.1 (MacCoss Laboratory, University of Washington, WA, USA). The quantification value was determined as the average of the peak ratios of 3–4 transitions. 

The Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) method was used for label-free quantitative proteomic analysis of both brain microvessel fractions and whole-brain lysates. After trypsin digestion of the samples using the PTS method, proteins were quantified based on specific peptide data detected by LC-MS/MS. The data were acquired using information-dependent acquisition (IDA) and SWATH modes on a TripleTOF6600 (SCIEX) interfaced with the Eksigent NanoLC400 system (SCIEX). Proteins were identified using Protein Pilot 4.5 (SCIEX) with MS/MS data obtained from IDA. Protein expression data were calculated from the precursor peak area data using the MaxLFQ algorithm integrated into DIA-NN . The average protein peak areas of the samples in each group were compared between the two groups. 

Among the proteins identified by quantitative proteomic analysis, we selected cell-type markers for brain endothelial cells, astrocytes, neurons, oligodendrocytes, and microglia. These selections were based on the criterion that the mRNA expression ratio (the ratio of expression levels in the most highly expressed cell type to the second most highly expressed cell type) was greater than 10-fold. The human brain RNA sequencing database used for this analysis has been previously published . 

Numerical data are presented as mean ± standard deviation (SD). Pearson’s correlation analysis was employed to determine the correlations between the two groups. The statistical significance of differences was assessed using Student’s t -test using Microsoft Excel version 16.0 (Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). 

Isolation of brain microvessels from a small amount of frozen human brain Using our established brain microvessel isolation method, which utilizes a bead homogenizer in the homogenization step and a combination of a cell strainer and glass beads in the purification step, we successfully isolated brain microvessels from approximately 0.3 g of the frontal cortex from four male human donors (aged 35–42 years) (Fig. A). Microscopic images showed enrichment of brain microvessels in this fraction (Fig. B). To estimate the diameter of the isolated brain microvessels, we examined magnified images from Fig. B and found that they measured approximately 5–10 μm in diameter (Figure ). The average protein content of the brain microvessel fraction, as determined by BCA assay, was 28.2 µg of protein (Fig. C), and the protein content per gram of brain tissue was 93.0 µg (Fig. D). Western blot analysis revealed the presence of a single band derived from MDR1, GLUT1, and CLDN5, which are selectively expressed in brain microvascular endothelial cells, in all isolated brain microvessel fractions, but not in whole-brain lysates (Fig. A). The bands of MDR1 detected in the western blot analysis were broad, likely because MDR1 is a membrane protein with multiple N-linked glycosylation sites—a phenomenon we also observed in our previous mouse study using the same antibody . Among the four brain microvessel samples, Sample No. 1 exhibited the highest band intensity for MDR1, GLUT1, and claudin-5 in western blot analysis (Fig. A). The band intensities of β-actin were comparable across the four brain microvessel samples, and the quantified band intensities of MDR1, GLUT1, and CLDN5 normalized to β-actin were similar (Fig. B). These results demonstrate that human brain microvessels were successfully enriched in the isolated fractions. To evaluate the efficiency of our isolation method in enriching brain microvessels in the isolated fractions, we conducted a comparative analysis of the absolute expression levels of representative BBB transport-related proteins measured by MRM against previously reported . Quantitative proteomics using the MRM method revealed the expression of MDR1, GLUT1, and CLDN5, as well as additional protiens such as BCRP/ABCG2, MCT1/SLC16A1, and transferrin receptor (TFRC) in the isolated brain microvessel fraction (Fig. A). These quantitative values were compared with those of previous studies using the brain microvessel fraction obtained from 5.23 to 5.57 g of the human frontal brain  (Fig. B). The fold-change of the proteins relative to the reported values ranged from 0.759 to 2.58, with a mean of 1.60, indicating that the expression levels of almost all proteins were within a 2-fold range of the previous values (Fig. B). These results suggest that, compared to earlier reports, the brain microvasculature was effectively recovered from approximately 0.3 g of frozen human frontal cortex for proteomic analysis. Enrichment of brain cell-selective proteins in the human-isolated brain microvessels To investigate which types of brain cells were enriched in the isolated brain microvessel fraction compared with the whole-brain lysate, SWATH-MS-based label-free quantitative proteomic analyses were performed. A Venn chart showed that 18.3% (649 proteins) and 28.1% (1131 proteins) of the proteins were quantified in the brain microvessel fraction and whole brain lysate, respectively (Fig. A, Table , and ). At least 70% of the proteins (2899 proteins) were quantified in both the brain microvessel fraction and the whole-brain lysate (Fig. A and Table ). The volcano plot showed that 70.4% of the proteins (2042 proteins) were significantly different between the isolated brain microvessel fraction and whole-brain lysate (Fig. B). Reproducibility among replicates ( n = 4) of the brain microvessel fractions was evaluated using the coefficient of variance (%CV) of 3548 quantified proteins. The average %CV was 32.6% and the 75th percentile was 41.9% (Figure A). The reproducibility of the enrichment process was assessed using the %CV of the brain microvessel fraction-to-whole brain lysate enrichment ratio. The average %CV of the enrichment ratio was 39.3% and the 75th percentile was 49.0% (Figure B). These values were larger than those reported previously in our proteomic analysis of the mouse brain microvessel fraction and whole brain lysate ; however, this may be due to the inclusion of individual differences in the human brain itself. In addition, capillary isolation in mice uses the whole mouse brain, while only a small portion of the brain is used in humans. These differences also explain the large %CV in human samples. Among the 2899 proteins quantified in both the brain microvessel fraction and whole-brain lysate, we selected 105 proteins that were particularly expressed in each brain cell type (20 endothelial cells, 24 astrocytes, 41 neurons, 11 microglia, and 9 oligodendrocyte proteins) using our criteria based on the RNA-seq database  (Fig. C). The levels of proteins selectively expressed in the brain endothelial cells were significantly higher in the isolated brain microvessel fraction than in the whole brain lysate (median: 79.6). One-way ANOVA showed that the ratio of proteins in brain endothelial cells was markedly higher than that in the other cells ( P < 0.0001, Tukey’s test). The protein abundance of representative transporters, receptors expressed in the BBB, and endothelial cell-specific proteins (endothelial cell-selective adhesion molecule [ESAM], platelet endothelial cell adhesion molecule 1 [PECAM1], and von Willebrand factor [VWF]) were examined in brain microvessel fractions and whole-brain lysates (Fig. D). The protein abundance of MDR1, BCRP, and GLUT1 in the brain microvessel fractions were higher by 112-, 117-, and 122-folds, respectively, than those in the whole brain lysate. Similarly, the receptor protein TFRC and the tight junction protein CLDN5 demonstrated increased protein abundance in the brain microvessel fractions, with fold changes of 18.1 and 60.6, respectively. The significant increase in CLDN5 protein abundance in the brain microvessel fractions indicates an enrichment of tight junction proteins, suggesting that the structural integrity of the brain microvessels is well preserved. In addition, the endothelial cell-specific proteins ESAM, PECAM1, and VWF also showed significant increases in the brain microvessel fractions, with 52.3-, 133-, and 180-fold changes, respectively. Proteins that were selectively expressed in microglia and oligodendrocytes were also higher in the isolated brain microvessel fraction than in the whole brain lysate (median: 2.97 and 1.56, respectively); however, these medians were at least 26.8-fold lower than those in brain endothelial cells (Fig. C). Proteins selectively expressed in astrocytes were less abundant in the isolated brain microvessel fraction compared to the whole brain lysate (median: 0.737) (Fig. C). Similarly, proteins selectively expressed in neurons showed lower abundance in the isolated brain microvessel fraction (median: 0.209) (Fig. C). These results suggest minimal contamination from other cell types in the brain microvessel fraction, highlighting the succesful enrichment of brain microvessels. Pericytes are attached to brain microvascular endothelial cells but are not included in the RNA-seq database . We selected two pericyte marker proteins (chondroitin sulfate proteoglycan 4 [CSPG4] and vitronectin [VTN]), based on the mouse RNA-seq database . The ratio of the brain microvessel fraction to the whole-brain lysate was higher in the isolated brain microvessel fraction (4.71- and 7.22-folds, respectively) (Fig. E). This is likely due to the fact that pericytes are partially recovered attached to microvessels during isolation of brain microvessels. To investigate the extent of enrichment of microvessels and microvascular endothelial cells in the human brain microvessel fraction, we performed a comparative analysis of protein enrichment between human and mouse brain microvessel fractions. Among the identified proteins in both human and mouse samples, 1598 proteins were enriched in the brain microvessel fraction relative to the whole brain lysate (Fig. ). Correlation analysis revealed a strong correlation coefficient ( r = 0.865), indicating a significant similarity in the enrichment patterns of these proteins between humans and mice (Fig. A). Furthermore, when focusing on 420 proteins selected as endothelial cell-specific proteins based on the RNA-seq database, the correlation analysis showed a robust correlation coefficient ( r = 0.889) (Fig. B). These results suggest a high degree of similarity in the protein enrichment profiles of human and mouse brain microvessel fractions. Taken together, our findings indicate that brain microvascular endothelial cells are significantly enriched in the human microvessel fraction. Protein expression of ABC and SLC transporters in the brain microvessel fraction To investigate the proteins involved in the transport of drugs and endogenous substances across the BBB, we selected ATP-binding cassette (ABC) and solute carrier (SLC) transporters expressed in brain microvascular endothelial cells based on the following criteria: (1) exclusive or highly selective identification in the brain microvessel fraction (fold change > 3) and (2) highest fragments per kilobase of exon per million reads mapped (FPKM) value in endothelial cells compared to other brain cells, as determined using the RNA-seq database . A total of three ABC transporters and 14 SLC transporters were identified (Table ). ABCA9, SLC6A12, SLC12A7, SLC19A1, and SLC38A5 were quantified exclusively in the brain microvascular fraction. Additionally, the fold changes in BCRP, MDR1, and GLUT1—transporters known to be expressed in brain microvascular endothelial cells—were at least 3-fold higher compared to other transporters. These findings suggest that these five transporters are likely specifically expressed in brain vascular endothelial cells. We examined the similarities in characteristics between the human and mouse brain microvascular fractions (Fig. ). However, it is also possible to elucidate species differences by analyzing the specificity of protein expression in both fractions. Therefore, among the major ABC and SLC transporters involved in BBB transport, we selected endothelial cell-selective molecules that were uniquely identified in human or mouse brain microvascular fractions (Table ). As a result, seven molecules were identified as human-specific transporters, and four as mouse-specific. 

Using our established brain microvessel isolation method, which utilizes a bead homogenizer in the homogenization step and a combination of a cell strainer and glass beads in the purification step, we successfully isolated brain microvessels from approximately 0.3 g of the frontal cortex from four male human donors (aged 35–42 years) (Fig. A). Microscopic images showed enrichment of brain microvessels in this fraction (Fig. B). To estimate the diameter of the isolated brain microvessels, we examined magnified images from Fig. B and found that they measured approximately 5–10 μm in diameter (Figure ). The average protein content of the brain microvessel fraction, as determined by BCA assay, was 28.2 µg of protein (Fig. C), and the protein content per gram of brain tissue was 93.0 µg (Fig. D). Western blot analysis revealed the presence of a single band derived from MDR1, GLUT1, and CLDN5, which are selectively expressed in brain microvascular endothelial cells, in all isolated brain microvessel fractions, but not in whole-brain lysates (Fig. A). The bands of MDR1 detected in the western blot analysis were broad, likely because MDR1 is a membrane protein with multiple N-linked glycosylation sites—a phenomenon we also observed in our previous mouse study using the same antibody . Among the four brain microvessel samples, Sample No. 1 exhibited the highest band intensity for MDR1, GLUT1, and claudin-5 in western blot analysis (Fig. A). The band intensities of β-actin were comparable across the four brain microvessel samples, and the quantified band intensities of MDR1, GLUT1, and CLDN5 normalized to β-actin were similar (Fig. B). These results demonstrate that human brain microvessels were successfully enriched in the isolated fractions. To evaluate the efficiency of our isolation method in enriching brain microvessels in the isolated fractions, we conducted a comparative analysis of the absolute expression levels of representative BBB transport-related proteins measured by MRM against previously reported . Quantitative proteomics using the MRM method revealed the expression of MDR1, GLUT1, and CLDN5, as well as additional protiens such as BCRP/ABCG2, MCT1/SLC16A1, and transferrin receptor (TFRC) in the isolated brain microvessel fraction (Fig. A). These quantitative values were compared with those of previous studies using the brain microvessel fraction obtained from 5.23 to 5.57 g of the human frontal brain  (Fig. B). The fold-change of the proteins relative to the reported values ranged from 0.759 to 2.58, with a mean of 1.60, indicating that the expression levels of almost all proteins were within a 2-fold range of the previous values (Fig. B). These results suggest that, compared to earlier reports, the brain microvasculature was effectively recovered from approximately 0.3 g of frozen human frontal cortex for proteomic analysis. 

To investigate which types of brain cells were enriched in the isolated brain microvessel fraction compared with the whole-brain lysate, SWATH-MS-based label-free quantitative proteomic analyses were performed. A Venn chart showed that 18.3% (649 proteins) and 28.1% (1131 proteins) of the proteins were quantified in the brain microvessel fraction and whole brain lysate, respectively (Fig. A, Table , and ). At least 70% of the proteins (2899 proteins) were quantified in both the brain microvessel fraction and the whole-brain lysate (Fig. A and Table ). The volcano plot showed that 70.4% of the proteins (2042 proteins) were significantly different between the isolated brain microvessel fraction and whole-brain lysate (Fig. B). Reproducibility among replicates ( n = 4) of the brain microvessel fractions was evaluated using the coefficient of variance (%CV) of 3548 quantified proteins. The average %CV was 32.6% and the 75th percentile was 41.9% (Figure A). The reproducibility of the enrichment process was assessed using the %CV of the brain microvessel fraction-to-whole brain lysate enrichment ratio. The average %CV of the enrichment ratio was 39.3% and the 75th percentile was 49.0% (Figure B). These values were larger than those reported previously in our proteomic analysis of the mouse brain microvessel fraction and whole brain lysate ; however, this may be due to the inclusion of individual differences in the human brain itself. In addition, capillary isolation in mice uses the whole mouse brain, while only a small portion of the brain is used in humans. These differences also explain the large %CV in human samples. Among the 2899 proteins quantified in both the brain microvessel fraction and whole-brain lysate, we selected 105 proteins that were particularly expressed in each brain cell type (20 endothelial cells, 24 astrocytes, 41 neurons, 11 microglia, and 9 oligodendrocyte proteins) using our criteria based on the RNA-seq database  (Fig. C). The levels of proteins selectively expressed in the brain endothelial cells were significantly higher in the isolated brain microvessel fraction than in the whole brain lysate (median: 79.6). One-way ANOVA showed that the ratio of proteins in brain endothelial cells was markedly higher than that in the other cells ( P < 0.0001, Tukey’s test). The protein abundance of representative transporters, receptors expressed in the BBB, and endothelial cell-specific proteins (endothelial cell-selective adhesion molecule [ESAM], platelet endothelial cell adhesion molecule 1 [PECAM1], and von Willebrand factor [VWF]) were examined in brain microvessel fractions and whole-brain lysates (Fig. D). The protein abundance of MDR1, BCRP, and GLUT1 in the brain microvessel fractions were higher by 112-, 117-, and 122-folds, respectively, than those in the whole brain lysate. Similarly, the receptor protein TFRC and the tight junction protein CLDN5 demonstrated increased protein abundance in the brain microvessel fractions, with fold changes of 18.1 and 60.6, respectively. The significant increase in CLDN5 protein abundance in the brain microvessel fractions indicates an enrichment of tight junction proteins, suggesting that the structural integrity of the brain microvessels is well preserved. In addition, the endothelial cell-specific proteins ESAM, PECAM1, and VWF also showed significant increases in the brain microvessel fractions, with 52.3-, 133-, and 180-fold changes, respectively. Proteins that were selectively expressed in microglia and oligodendrocytes were also higher in the isolated brain microvessel fraction than in the whole brain lysate (median: 2.97 and 1.56, respectively); however, these medians were at least 26.8-fold lower than those in brain endothelial cells (Fig. C). Proteins selectively expressed in astrocytes were less abundant in the isolated brain microvessel fraction compared to the whole brain lysate (median: 0.737) (Fig. C). Similarly, proteins selectively expressed in neurons showed lower abundance in the isolated brain microvessel fraction (median: 0.209) (Fig. C). These results suggest minimal contamination from other cell types in the brain microvessel fraction, highlighting the succesful enrichment of brain microvessels. Pericytes are attached to brain microvascular endothelial cells but are not included in the RNA-seq database . We selected two pericyte marker proteins (chondroitin sulfate proteoglycan 4 [CSPG4] and vitronectin [VTN]), based on the mouse RNA-seq database . The ratio of the brain microvessel fraction to the whole-brain lysate was higher in the isolated brain microvessel fraction (4.71- and 7.22-folds, respectively) (Fig. E). This is likely due to the fact that pericytes are partially recovered attached to microvessels during isolation of brain microvessels. To investigate the extent of enrichment of microvessels and microvascular endothelial cells in the human brain microvessel fraction, we performed a comparative analysis of protein enrichment between human and mouse brain microvessel fractions. Among the identified proteins in both human and mouse samples, 1598 proteins were enriched in the brain microvessel fraction relative to the whole brain lysate (Fig. ). Correlation analysis revealed a strong correlation coefficient ( r = 0.865), indicating a significant similarity in the enrichment patterns of these proteins between humans and mice (Fig. A). Furthermore, when focusing on 420 proteins selected as endothelial cell-specific proteins based on the RNA-seq database, the correlation analysis showed a robust correlation coefficient ( r = 0.889) (Fig. B). These results suggest a high degree of similarity in the protein enrichment profiles of human and mouse brain microvessel fractions. Taken together, our findings indicate that brain microvascular endothelial cells are significantly enriched in the human microvessel fraction. 

To investigate the proteins involved in the transport of drugs and endogenous substances across the BBB, we selected ATP-binding cassette (ABC) and solute carrier (SLC) transporters expressed in brain microvascular endothelial cells based on the following criteria: (1) exclusive or highly selective identification in the brain microvessel fraction (fold change > 3) and (2) highest fragments per kilobase of exon per million reads mapped (FPKM) value in endothelial cells compared to other brain cells, as determined using the RNA-seq database . A total of three ABC transporters and 14 SLC transporters were identified (Table ). ABCA9, SLC6A12, SLC12A7, SLC19A1, and SLC38A5 were quantified exclusively in the brain microvascular fraction. Additionally, the fold changes in BCRP, MDR1, and GLUT1—transporters known to be expressed in brain microvascular endothelial cells—were at least 3-fold higher compared to other transporters. These findings suggest that these five transporters are likely specifically expressed in brain vascular endothelial cells. We examined the similarities in characteristics between the human and mouse brain microvascular fractions (Fig. ). However, it is also possible to elucidate species differences by analyzing the specificity of protein expression in both fractions. Therefore, among the major ABC and SLC transporters involved in BBB transport, we selected endothelial cell-selective molecules that were uniquely identified in human or mouse brain microvascular fractions (Table ). As a result, seven molecules were identified as human-specific transporters, and four as mouse-specific. 

The present method developed by us, which utilizes a bead homogenizer for homogenization and a combination of cell strainers and glass beads for purification, effectively isolates brain microvessels from small amounts of human brain tissue. Specifically, we successfully isolated brain microvessels from as little as 0.3 g of human brain tissue—at least three times less than the amount required in previous studies . This represents a significant improvement in the utilization efficiency. Moreover, the enrichment of key BBB proteins, such as MDR1, GLUT1, and CLDN5, in the isolated microvessel fractions, alongside minimal presence of neuronal proteins, highlights the efficacy and purity of our isolation process. These findings are consistent with those observed in mouse brain capillary fractions isolated using our previously described method . In addition, we found that the enrichment of proteins in the human brain microvessel fraction was similar to that in the mouse brain microvessel fraction. Taken together, these results demonstrate the success of our method in efficiently isolating brain microvessels from small amounts of frozen human brain tissue. Our methodology represents a significant advancement in the homogenization process, a critical step for the efficient isolation of brain microvessels. Previous work by Uchida et al.  emphasized the reliance on extensive manual homogenization, requiring 10 and 20 strokes with a Potter homogenizer for mouse and human brains, respectively, which often results in inconsistencies and variability in microvessel recovery. In contrast, our approach employs a bead homogenizer, which streamlines and standardizes the homogenization of brain tissue with high reproducibility. As demonstrated in our previous studies, the bead homogenizer achieves superior uniformity compared to traditional manual techniques . This innovation not only decreases the variability associated with manual processing but also ensures more consistent recovery of brain microvessels. Furthermore, this bead homogenization approach enables the miniaturization of the brain microvessel purification process by incorporating mesh filtration with the glass bead method. As a result, we successfully isolated highly pure brain microvessels suitable for protein expression analysis from small amounts of frozen human brain tissue. No significant differences were observed in the yield of brain microvessels or in the enrichment of brain microvessel marker proteins detected by western blot analysis, irrespective of the cause of death (Figs. and , Table ). This findings suggest that the cause of death may not substantially impact the efficiency of isolation or the purity of the brain microvessel preparations. However, due to limited sample size in this study, it remains unclear whether the cause of death might influence the molecular characteristics of brain microvessels. Further investigations using a larger number of samples employing this method are needed to clarify this point and to determine any potential subtle effects. Moreover, Brain Sample #1 exhibited a higher yield of brain microvessels and greater enrichment of brain microvessel marker proteins in western blot analysis compared to the other samples. One possible explanation is individual variation; for instance, Sample #1 may have had a higher microvessel density due to genetic factors, physiological conditions, or lifestyle differences. Another contributing factor could be anatomical heterogeneity within the cortex. Previous reports about the density of human brain microvessels have reported that the numerical density and length density of brain microvessels in subcortical gray matter are about 6-fold and 2-fold higher, respectively, than those in subcortical white matter . Since the human brain samples in this study were procured from a vendor, the exact subregions of the frontal cortex from which they were obtained could not be determined. Therefore, variations in the proportion of gray and white matter within the specimens may have influenced the yield and purity of the isolated brain microvessels. Future studies should consider controlling these variables by sourcing samples from well-defined brain regions and by collecting detailed donor information to better understand the factors influencing brain microvessel isolation and characteristics. In our proteomic analysis, we observed that the levels of astrocyte marker proteins in the human brain microvessel fraction did not exceed those in the whole brain lysate. This disparity may be attributed to the detachment of astrocyte endfeet from the brain microvascular endothelial cells during the homogenization process. The extent of this detachment may vary between humans and mice, which could explain the differences in astrocyte protein enrichment. Additionally, regarding the localization of aquaporin-4 (AQP4), an astrocyte marker protein in human and mouse brain microvessels, a previous study  has shown that less AQP4 is localized to the astrocyte endfeet in human brains compared to mouse brains. In our previous study , we observed a 7.87-fold enrichment of AQP4 in the mouse brain microvessel fraction compared to the whole brain lysate, whereas in humans, we found only a 0.917-fold enrichment (Table ). During the brain homogenization process, astrocyte endfeet are thought to be cleaved and co-isolated with brain microvessels. Since humans have less AQP4 localized to the endfeet, no enrichment of AQP4 was observed in the human brain microvessel fraction. Therefore, variations in astrocyte endfeet adhesion and protein localization are likely contributing factors to the observed discrepancies in astrocyte-derived protein enrichment in brain microvessel fractions between humans and mice. In our previous study on isolated mouse brain microvessels, we observed significant reductions in microglia and oligodendrocyte marker proteins in the microvessel fraction compared to the whole brain lysate . This finding contrasts with our current results in humans. Previous studies have shown that the number of microglia per brain volume in the prefrontal cortex is significantly lower in humans compared to mice . These species differences may affect the efficiency of cell disruption during brain homogenization, ultimately influencing the removal efficiency during the brain microvessel purification step. Due to these differences, microglia and oligodendrocytes might not be sufficiently removed in human brain samples, leading to their enrichment in the brain microvessel fraction compared to the whole brain lysate. It has also been reported that in human postmortem brains, microglia became activated with increased process outgrowth within 2–4 h postmortem . Therefore, differences of microglial morphology between mice and humans could also affect the efficiency of microglial removal during brain microvessels isolation process. The consistency of protein enrichment in brain microvessel fractions in both humans and mice was confirmed by the strong correlation between their datasets, which enhances our understanding of their similarities and allows for valuable cross-species comparisons. However, our comparative proteomic analysis of human and mouse brain microvascular fractions revealed species-specific differences in the expression of several ABC and SLC transporters. Among these, transporters such as Abcc4 (Mrp4) and Slc22a8 (organic anion transporter 3 [Oat3]) have been reported to show species-specific differences in their expression at the BBB in both humans and mice, similar to the results of this study . Notably, our study identified SLC5A6 (sodium-dependent multivitamin transporter [SMVT]) as a species-specific gene that is expressed in humans, but not in mice. SMVT is a Na + -dependent multivitamin transporter that is critical for the transport of pantothenic acid and biotin across the human BBB . Additionally, mutations in SMVT have been associated with microcephaly , underscoring its importance in nutrient transport across the human BBB. These findings highlight the complex interactions of transporters within the human and mouse BBB and reveal species-specific differences that may have significant implications for drug delivery and therapeutic strategies targeting the brain. Further investigation of the functional differences associated with SMVT between the two species will enhance our understanding of BBB biology and may lead to more effective approaches for the treatment of CNS diseases. Building upon our developed method, several studies have demonstrated its versatility in various applications. For instance, RNA was extracted from brain microvessels isolated from fresh mouse brains, and was subjected to RNA-seq analysis , leveraging our previously reported method for frozen mouse brains . Additionally, by modifying our method, RNA was extracted from brain microvessels isolated from frozen mouse brains, and RT-qPCR was conducted . These results suggest that RNA analysis using this method is feasible with frozen human brains as well. Furthermore, our method has been adapted for measuring blood-brain barrier (BBB) activity. In one study, mitochondrial function was evaluated in brain microvessels isolated from fresh mouse brains using this technique . However, this study was conducted using brain microvessels isolated from fresh mouse brains and functional assays have yet to be reported for frozen brain samples. While activity analyses such as transporter functions and other assays may also be possible using our method, to our knowledge, there are no reports of such experiments using frozen brains. Addressing this gap represents a future challenge for further research. Elucidating the mechanisms of disease and developing targeted therapies depends on understanding changes in protein expression at the human BBB during disease states. Our study represents a significant methodological breakthrough in neurovascular research, as we have successfully isolated brain microvessels from small amounts of human brain tissue. Aging is associated with changes such as a decrease in brain microvessel density and increased microvessel fragility. Further investigation is needed to determine how these changes may affect the recovery and analysis of brain microvessels using our developed method. In AD model mice, we successfully isolated brain capillaries from mouse brain, but protein assay suggested that the recovery of brain capillaries from 12-month-old AD model mice was lower than that from 2-month-old AD model mice . Therefore, we believe that our developed method is applicable to elderly human brain samples, including those from patients with neurodegenerative diseases, but the recovery of brain microvessels may be low due to aging. This technique allows for in-depth analysis of protein expression changes in the BBB in various CNS diseases, providing valuable insights into their pathophysiology. It is crucial to understand the complex role of the BBB in neurodegenerative diseases for aiding the development of CNS-targeted therapeutics. Our refined isolation method provides the basis for comprehensive studies on the involvement of the human BBB in neurological diseases and its response to pathological changes, thereby enhancing drug delivery research. Moreover, our approach offers the potential for cross-species comparative studies, providing essential data for translational neuroscience by highlighting the differences and similarities between human and mouse BBBs. 

The current study successfully established an innovative approach for isolating brain microvessels from small quantities (approximately 0.3 g) of human frozen brain tissue. This method allows for extensive protein-level analyses of the BBB modifications in various CNS diseases through proteomics. By overcoming the limitations imposed by insufficient brain weight, this advanced technique has enabled us to gain a deeper understanding of the protein expression changes that occur within the BBB across a range of CNS diseases. 

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 

