Abstract
Brain microvasculature forms a specialized structure, the blood-brain barrier (BBB), to maintain homeostasis and integrity of the central nervous system (CNS). The BBB dysfunction is emerging as a critical contributor to multiple neurological disorders, including stroke, traumatic brain injury, autoimmune multiple sclerosis, and neurodegenerative diseases. The brain microvasculature exhibits highly cellular and regional heterogeneity to accommodate dynamic changes of microenvironment during homeostasis and diseases. Thus, investigating the underlying mechanisms that contribute to molecular or cellular changes of the BBB is a significant challenge. Here, we describe an optimized protocol to purify microvessels from the mouse cerebral cortex using mechanical homogenization and density-gradient centrifugation, while maintaining the structural integrity and functional activity of the BBB. We show that the isolated microvessel fragments consist of BBB cell populations, including endothelial cells, astrocyte end-feet, pericytes, as well as tight junction proteins that seal endothelial cells. Furthermore, we describe the procedures to generate single-cell suspensions from isolated microvessel fragments. We demonstrate that cells in the single-cell suspensions are highly viable and suitable for single-cell RNA-sequencing analysis. This protocol does not require transgenic mice and cell sorting equipment to isolate fluorescence-labeled endothelial cells. The optimized procedures can be applied to different disease models to generate viable cells for single-cell analysis to uncover transcriptional or epigenetic landscapes of BBB component cells.
Highlights
The brain vasculature is a complex system composed of endothelial cells, pericytes, astrocytes, smooth muscle cells, and extracellular matrix components, forming the unique blood-brain barrier (BBB), which lies at the interface between circulating blood and the neural tissue (Abbott and Friedman, 2012; Obermeier et al, 2013)
Previous literature suggests that the BBB disruption can lead to neuronal injury mediated through peripheral immune cells or harmful agents that enter into the central nervous system (CNS) in multiple neurological disorders, such as Alzheimer’s disease (AD; Sweeney et al, 2018; Nation et al, 2019), ischemic stroke (Kassner and Merali, 2015), multiple sclerosis (Ortiz et al, 2014), and traumatic brain injury (Alluri et al, 2015)
Despite the importance of BBB highlighted in many neurological disorders, the molecular and cellular mechanisms that lead to BBB dysfunction or disruption in these disease conditions remain largely unknown
Summary
The brain vasculature is a complex system composed of endothelial cells, pericytes, astrocytes, smooth muscle cells, and extracellular matrix components, forming the unique blood-brain barrier (BBB), which lies at the interface between circulating blood and the neural tissue (Abbott and Friedman, 2012; Obermeier et al, 2013). The BBB tightly regulates the transport of ions and nutrients necessary for neuronal health and function. Previous literature suggests that the BBB disruption can lead to neuronal injury mediated through peripheral immune cells or harmful agents that enter into the CNS in multiple neurological disorders, such as Alzheimer’s disease (AD; Sweeney et al, 2018; Nation et al, 2019), ischemic stroke (Kassner and Merali, 2015), multiple sclerosis (Ortiz et al, 2014), and traumatic brain injury (Alluri et al, 2015). We describe an optimized protocol that would allow isolating brain microvessels that closely resemble in vivo structures suitable for downstream applications of molecular and single-cell analyses to characterize molecular signatures of BBB component cells
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