Characterization of Oligodendroglial Populations in Mouse Demyelinating Disease Using Flow Cytometry: Clues for MS Pathogenesis

Abstract

Characterizing and enumerating cells of the oligodendrocyte lineage (OLCs) is crucial for understanding demyelination and therapeutic benefit in models of demyelinating disease in the central nervous system. Here we describe a novel method for the rapid, unbiased analysis of mouse OLCs using flow cytometry. The assay was optimized to maximize viable yield of OLCs and maintain OLC antigen integrity. Panels of antibodies were assembled for simultaneous analysis of seven antigens on individual cells allowing for characterization of oligodendroglial cells throughout the lineage. We verified the utility of the assay with cultured OLCs and through a time course of developmental myelination. Next we employed the assay to characterize OLC populations in two well-characterized models of demyelination: cuprizone-induced demyelination and experimental autoimmune encephalomyelitis (EAE). In EAE we observed a dramatic loss of mature oligodendrocytes coincident with a dramatic expansion of oligodendrocyte progenitors cells (OPCs) at the onset of disease suggesting an attempt of the host to repair myelin. This expanded OPC pool was maintained through remission and relapse suggesting an arrest in differentiation in the face of the chronic autoimmune T cell-mediated inflammatory response. These robust, reproducible changes in OLCs through disease provide a rapid quantitative global analysis of myelin-producing cells in the adult mouse brain and important information regarding effects of disease on oligodendroglial proliferation/differentiation which is useful for defining the pathogenesis and therapy of MS.

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

“…2) while maintaining the efficient detection of numerous cell markers (O4,NeuN,CD45,CD11b,CD3,CD4,CD8). Similarly to our results, Robinson et al (2014) obtained the highest cell yield using Accutase, a commercially available mix of proteolytic and collagenolytic enzymes, and other collagenase-based commercial solutions (e.g. Liberase), compared with undigested tissues, or trypsin or papain digested tissues.…”

“…1). One group has also reported that the linear Percoll separation is superior to the Percoll gradient to recover cells of the oligodendrocyte lineage from mouse CNS (Robinson et al, 2014). We found that amongst several digestion conditions (papain + DNase I, trypsin + DNase I, collagenase D + DNase I, and none), the collagenase D + DNase I step allowed the best cell number recovery (Fig.…”

“…The low viscosity and low osmolarity of this non-toxic solution has favored its use to isolate cells (Dunkley et al, 2008;Pertoft et al, 1978). Recently, one group has characterized cells of the oligodendrocyte lineage from rodent adult spinal cord samples using flow cytometry (Robinson et al, 2014). Numerous groups routinely investigate the profile of CNS infiltrating immune cells in animal models of neurological diseases such as multiple sclerosis.…”

Concurrent cell type–specific isolation and profiling of mouse brains in inflammation and Alzheimer’s disease

“…Utilizing analogous antibodies and reporter lines and varied strategies of dissociation, neurons (6)(7)(8)(9)(10), astrocytes (6,7,(10)(11)(12)(13), microglia (6,7,12,(14)(15)(16)(17)(18)(19)(20), endothelia (21,22), and oligodendrocytes (6,7,23,24) have been collected separately. In addition, multiple cell types have been isolated and profiled concurrently by using a combinatorial approach of reporter lines and/or multiple antibodies (1,5,6).…”

Cyclin-dependent kinase inhibitor flavopiridol promotes remyelination in a cuprizone induced demyelination model

“…[3][4][5]8 Moreover, certain antioxidants such as melatonin, 9 quetiapine, 10 and resveratrol, 11 showed protective effects on the CPZ-induced oligodendrocyte loss and myelin breakdown in recent animal studies. Interestingly, oligodendrocyte loss and myelin breakdown were accompanied by increase in the number of OPCs labeled as NG2 C (chondroitin sulfate proteoglycan) and PDGFRa C (platelet-derived growth factor a) cells in CPZexposed mice, [12][13][14] suggesting the existence of an aberrant cell cycle regulation following CPZ administration.…”