Microvessel isolation protocol for RNA visualization and profiling

Abstract

Abstract Disruptions in pericyte and endothelial cell communication can compromise the integrity of the blood-brain barrier (BBB), leading to neurovascular dysfunction and the development of neurological disorders. However, the evaluation of microvessel RNAs has been limited to tissue homogenates, with spatial visualization only available for protein targets. The aim of the present study is the development of an innovative microvessel isolation technique that is RNA-friendly for the purpose of coupling with in situ hybridization RNAscope analysis. RNA-friendly microvessel isolation combined with RNAscope analysis enables the visualization of cell-specific RNA within the spatial and histological context of the BBB. Using this approach, we have gained valuable insights into the structural and functional differences associated with the microvessels of 5xFAD mice, a mouse model of Alzheimer’s disease (AD). RNAscope analysis revealed a decrease in pericytes from microvessels isolated from 5xFAD mice in comparison to wild-type mice. Additionally, the microvessels of 5xFAD mice exhibited an increase in TYRO protein tyrosine kinase binding protein (TYROBP) mRNA expression. These findings significantly advance our understanding of neurovascular interactions and hold great promise for guiding the development of targeted therapeutic interventions. This innovative approach enables visualization of cell RNA while preserving the spatial and histological context of the BBB, shedding light on the mechanisms underlying neurovascular unit communication.

Keywords

• Blood-brain barrier
• Microvessels
• Pericytes
• Alzheimer’s disease
• RNAscope
• Endothelial cells