Isolation and Preparation of Cells from Focal Remyelinating Central Nervous System Lesions for RNA Sequencing.

Abstract

Remyelination is the regenerative process whereby myelin sheaths are restored around axons following nervous system injury, allowing reinstatement of electrical impulse conduction, trophic/metabolic support, and axon health. Failure of remyelination in progressive multiple sclerosis is considered to contribute to axon loss, a correlate of clinical decline. Lack of approved pro-regenerative therapies for MS highlights the need to understand the cellular and molecular mechanisms underpinning successful remyelination. One approach is to conduct nonbiased gene expression analyses of cell types which regulate remyelination, such as microglia and monocyte-derived macrophages. Recent technological advances address the challenges of RNA sequencing of small tissue samples, thus allowing relatively small numbers of cells to be isolated from discrete lesions for analysis. Here, we present methods for FACS-based isolation of cells from focal remyelinating lesions of the adult mouse brain and subsequent RNA extraction for sequencing, using isolation of microglia/macrophages as an example.