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

In a previous in vivo study of this demyelinating disease, O-2A lineage cells which had incorporated [3I-l]thymidine during a 2-h terminal pulse were detected in 1-1zm frozen sec- tions (Godfraind et al., 1989) . To further explore the mitotic potential of O-2A lineage cells from adult CNS, we prepared cultures from spinal cords of normal and infected mice after in vivo labeling with [3H]thymidine during a 2-h terminal pulse (Table I) . Cells undergoing active DNA synthesis were identified after 1 div by [3I-I]thymidine autoradiography combined with triple-label immunocytochemistry, as outlined in Fig. 1 A. The proportion of O-2A lineage cells (oligodendrocytes, O-2A progenitors, and type 2 astrocytes) which were thymidine-labeled was extremely low in control cultures prepared from normal mice at 5 wk of age (0.52%) and at 8 wk of age (0 %). In contrast, the proportion of O-2A lineage cells (oligodendrocytes, O-2A progenitors, type 2 astrocytes, and mixed phenotype cells) that were pH]thymidine-labeled in cultures derived from infected tissue was markedly higher both in the early phase of the disease (4.96% at 1 wpi; 5 wk of age) and at the time of widespread demyelination (5.38% at 4 wpi; 8 wk of age). We next examined the proliferative capacity of the cultured cells during an in vitro pulse of [3H]thymidine (Table I ). In this case proliferation in vitro was measured by adding [SH] thymidine to the culture medium at 2 div, fixing the cells 20 h later, and then identifying [3H]thymidine-labeled cells by autoradiography combined with three-fluorochrome immunocytochemistry, as outlined in Fig. 1 B. The percentage of O-2A lineage cells that were labeled in vitro with pH]thymidine was approximately ninefold higher in cultures derived from diseased mice at 1 wpi than in cultures from age-matched control mice, and was increased more than threefold at 4 wpi (Table I) . Oligodendrocytes (Fig. 10) , type 2 astrocytes (Fig. 11) , O-2A progenitors (Fig. 12) , and mixed phenotype cells were labeled with [3H]thymidine after the 20-h terminal pulse. Remarkably, O-2A progenitors were the only cell type for which the increased percentage of [3H]thymidine-labeled cells from demyelinated tissue was highly significant (4 wpi; demyelinated = 38.3 %, control = Figure 8 . A cell with a mixed oligodendrocyteastrocyte phenotype isolated from demyelinated tissue (4 wpi) which was grown in culture for 3 d, fixed, and processed for three-color immunofluorescence. (A) The cell surface membrane is immunostained with an mAb against GC, visualized with fluorescein. (B) Intracellular filaments are immunostained with a polyclonal antiserum recognizing GFAP, visualized with coumarin. This cell was also labeled by 04, visualized with rhodamine (not shown). Bar, 50 ttm. 0%; p = 0.027, as determined by paired two-tailed t test). Approximately 12-14% of each of the other O-2A lineage cell types (oligodendrocytes, type 2 astrocytes, and mixed phenotype cells) were pH]thymidine-labeled in cultures derived from demyelinated tissue. Although the percentage of [3H]thymidine-labeled cells within each of these phenotypes alone was not increased significantly, when combined as a single group these three phenotypes accounted for a significant proliferative response in cultures of demyelinated tissue (4 wpi; demyelinated = 13.5 %, control = 4.3 %; p = 0.029, as determined by paired two-tailed t test). Thus the in vivo proliferative response of O-2A lineage cells isolated from demye