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The isolation and molecular characterization of cerebral microvessels

Abstract

The study of cerebral microvessels is becoming increasingly important in a wide variety of conditions, such as stroke, sepsis, traumatic brain injury and neurodegenerative diseases. However, the molecular mechanisms underlying cerebral microvascular dysfunction in these conditions are largely unknown. The molecular characterization of cerebral microvessels in experimental disease models has been hindered by the lack of a standardized method to reproducibly isolate intact cerebral microvessels with consistent cellular compositions and without the use of enzymatic digestion, which causes undesirable molecular and metabolic changes. Herein, we describe an optimized protocol for microvessel isolation from mouse brain cortex that yields microvessel fragments with consistent populations of discrete blood–brain barrier (BBB) components (endothelial cells, pericytes and astrocyte end feet) while retaining high RNA integrity and protein post-translational modifications (e.g., phosphorylation). We demonstrate that this protocol allows the quantification of changes in gene expression in a disease model (stroke) and the activation of signaling pathways in mice subjected to drug administration in vivo. We also describe the isolation of genomic DNA (gDNA) and bisulfite treatment for the assessment of DNA methylation, as well as the optimization of chromatin extraction and shearing from cortical microvessels. This optimized protocol and the described applications should improve the understanding of the molecular mechanisms governing cerebral microvascular dysfunction, which may help in the development of novel therapies for stroke and other neurologic conditions.

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Fig. 1: Cortical microvessel isolation protocol overview.
Fig. 2: Characterization of microvessel preparations: purity, structural features (size) and consistency in cellular composition.
Fig. 3: Morphological characterization of microvessel preparations: structural integrity and cell composition.
Fig. 4: Characterization of microvessel fragments: size and α-SMA content.
Fig. 5: Impact of the microvessel isolation method on RNA integrity and sample-to-sample variation.
Fig. 6: Quantification of changes in gene expression in cerebral microvessels after stroke.
Fig. 7: Quantification of phospho-Ser473 Akt levels in cerebral microvessels in wild-type mice after administration of the S1PR2 antagonist JTE-013.
Fig. 8: Detection of DNA methylation in Bdnf, S1pr2 and S1pr1 promoter regions in microvessels and whole brain.
Fig. 9: Optimization of chromatin extraction and shearing from cortical microvessels.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. No datasets were generated or analyzed during the current study.

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Acknowledgements

This work was supported by funds from the American Heart Association (Grant-in-Aid, 12GRNT12050110), the NIH (HL094465) and the Leducq Foundation (14CVD02) to T.S. A.I. was supported by a grant from the Roche Foundation.

Author information

Authors and Affiliations

Authors

Contributions

H.S. and T.S. designed the protocol. T.S., H.S., Y.-K.L. and H.U. modified and updated the protocol to its current state. A.I. conducted the stroke surgeries. H.U. conducted the in vivo pharmacological treatments. Y.-K.L. and H.U. optimized and conducted the molecular assays with cerebral microvessels, as well as the immunofluorescence analysis. H.U., Y.-K.L., H.S. and T.S. wrote the manuscript with contributions from all the authors.

Corresponding author

Correspondence to Teresa Sanchez.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Xavier Declèves, Sven Meuth and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Yanagida, K. et al. Proc. Natl. Acad. Sci. USA 114, 4531–4536 (2017): https://doi.org/10.1073/pnas.1618659114

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Lee, YK., Uchida, H., Smith, H. et al. The isolation and molecular characterization of cerebral microvessels. Nat Protoc 14, 3059–3081 (2019). https://doi.org/10.1038/s41596-019-0212-0

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