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

ABSTRACT: Studying disease related changes in the brain vasculature is pivotal due to its crucial role in supplying oxygen and nutrients, and removing waste. To study the vascular cells in detail, improved techniques for isolation of the vascular components are warranted. In this study, 22,515 cells were isolated using CD31 coupled magnetic beads and analyzed using single cell RNA sequencing. Their transcriptomes separated into 23 clusters corresponding to all known vascular and perivascular cell types. Western blot analysis using markers for these cell types confirmed their presence in the isolate and suggests that the protocol is suitable also for proteomic analysis. Finally, we adapted the protocol to accommodate primary culture. In conclusion, this method can successfully isolate murine brain microvasculature independent of cell sorting, alleviating the need for reporter mouse lines. The protocol is suitable for several testing modalities, including single-cell analyses, WB, and primary cell culture.

ORGANISM(S): Mus musculus

PROVIDER: GSE214326 | GEO | 2022/10/01

REPOSITORIES: GEO

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Project description:Functional decline and structural alterations of the brain microvasculature are associated with cognitive impairment and development of dementias such as Alzheimer’s disease during aging. Although mechanisms of leading to microvascular changes during aging are not clear, the loss of mitochondria and reduced efficiency of remaining mitochondria appear to play a major role. While pharmacological agents which target mitochondria such as SS-31 have been shown to be effective in beneficial during aging and diseases, the full extent on mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested the hypothesis that attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option in the aging brain. We used male, aged (>18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31 or vehicle saline. Baseline cerebral blood flow (CBF) was determined using laser speckle imaging during the two-week treatment period. Then, isolated cortical microvessels (MVs), composed of end arterioles, capillaries, and venules, were used for Orbitrap Eclipse Tribrid mass spectrometry. Baseline CBF was similar between the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle groups. Specifically, we identified 6,266 proteins in our data set from which 12% were mitochondrial or mitochondria associated. 107 of these proteins were significantly differentially expressed between the treatment and vehicle groups, and were associated with the oxidative phosphorylation, metabolism, antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 also affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the mechanisms of rejuvenating actions of SS-31 in the aging phenotype.

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

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