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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.

High levels of serum unconjugated bilirubin (UCB) in newborns are associated with axonal damage and glial reactivity that may contribute to subsequent neurologic injury and encephalopathy (kernicterus). Impairments in myelination and white matter damage were observed at autopsy in kernicteric infants. We have recently reported that UCB reduces oligodendrocyte progenitor cell (OPC) survival in a pure OPC in vitro proliferative culture. Here, we hypothesized that neonatal hyperbilirubinemia may also impair oligodendrocyte (OL) maturation and myelination. We used an experimental model of hyperbilirubinemia that has been shown to mimic the pathophysiological conditions leading to brain dysfunction by unbound (free) UCB. Using primary cultures of OL, we demonstrated that UCB delays cell differentiation by increasing the OPC number and reducing the number of mature OL. This finding was combined with a downregulation of Olig1 mRNA levels and upregulation of Olig2 mRNA levels. Addition of UCB, prior to or during differentiation, impaired OL morphological maturation, extension of processes and cell diameter. Both conditions reduced active guanosine triphosphate (GTP)-bound Rac1 fraction. In myelinating co-cultures of dorsal root ganglia neurons and OL, UCB treatment prior to the onset of myelination decreased oligodendroglial differentiation and the number of myelinating OL, also observed when UCB was added after the onset of myelination. In both circumstances, UCB decreased the number of myelin internodes per OL, as well as the myelin internode length. Our studies demonstrate that increased concentrations of UCB compromise myelinogenesis, thereby elucidating a potential deleterious consequence of elevated UCB.

Measuring the effects of sphingosine-1-phosphate (S1P) receptor modulators on human primary neural cells is of particular interest given the recent application of these central nervous system-accessible agents to the treatment of neurodegenerative diseases, such as multiple sclerosis. Issues to consider in experimental studies include the ability of some of these modulators to bind multiple S1P receptor subtypes simultaneously, the nonspecificity of commercially available S1P receptor antibodies, and activation of multiple intracellular signaling cascades by a given S1P receptor. Here, we discuss how to assay S1P receptor expression and activation using multiple agonists/antagonists, by linking the results of real-time reverse transcriptase polymerase chain reaction with the assessment of intracellular signaling derived from Western blot analyses.