In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

The glial cell population was characterized using three-color immunofluorescence which enabled simultaneous visualization of three antigens, each labeled by one of three different fluorochromes (rhodamine, fluorescein, or coumarin). Oligodendrocytes were identified by expression of cell surface antigens recognized by anti-GC (Raft et al., 1978) , Cells containing intracellular filaments immunostained with anti-GFAP were classified as astrocytes (Biguami et al., 1972; Rafter al., 1979) . Type 2 astrocytes were distinguished from type 1 astrocytes by the expression of cell surface antigens recognized by the 04 antibody (Trotter and Schachner, 1989) . O-2A progenitors derived from adult CNS were identified as cells binding 04 in the absence of GC or GFAP immunolabeling (Wolswijk and Noble, 1989) . Thus, any particular cell could be identified as an oligodendrocyte (04+ GC+ GFAP-), a type 2 astrocyt¢ (04+ GFAP+ GC-), an O-2A progenitor (04+ G C -GFAP-), a mixed oligodendrocyte-astrocyte phenotype cell (04+ GC+ GFAP+), or a type 1 astrocyte (GFAP+ 0 4 -GC-). (In this paper, the term "type 2 astrocyte" will be used to denote only the 04+ GFAP+ G C -antigenic phenotype and is not intended to indicate a specific localization or function in vivo.) Glial cells were isolated and cultured in parallel from spinal cords of infected and age-matched control mice at several intervals after viral inoculation. These cultures contained various types of glial cells, of which O-2A lineage cells (oligodendrocytes, O-2A progenitors, type 2 astrocytes, and mixed phenotype cells) represented a small proportion of the total. The number of O-2A lineage cells was counted after 1 d in culture (Fig. 2) . This number was similar for control and experimental tissue at 1 wpi (Fig. 2) when demyelination was not yet detected (see Fig. 3, A and B) . As demyelination and vacuolation progressed (3-5 wpi, see Fig. 3 C) , O-2A lineage cells were much more abundant in cultures of the demyelinated tissue (Fig. 2) . With the onset of remyelination (6-8 wpi, see Fig. 3 D) , the number of O-2A lineage cells derived from lesioned tissue declined (Fig. 2) . It is possible that CNS inflammation and vacuolation may have facilitated dissociation of the demyelinated tissue and thereby improved the yield of growing c¢11s. Yet the cultures from control versus demyelinated CNS differed not only in cell The other cell types in our cultures were type 1 astrocytes, fibroblasts, and microglia. Flat calls expressing GFAP but not 04 antigens were identified as type 1 astrocytes. Type 1 astrocytes seemed to be more prevalent in cultures of lesioned tissue. However, at 1 div such cells were usually found in dense clusters that prohibited accurate quantitation of the initial type 1,astrocyte population (Fig. 4, A and C) . By 3 and 6div, type 1 astrocytes grew out from these clusters and proliferated, as assayed by [3H]thymidine incorporation (Fig. 4, B and D) . Flat cells presumed to be fibroblasts, due to the absence of 04, GC, or GFAP immunostaining, were present and p~liferated in all cultures examined (not shown).