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

In the present in vitro study, we have characterized the growth and differentiation of glial cells isolated from the CNS of mice during the course of demyelination and remy- Figure 9 . The relative abundance of type 2 astrocytes and oligodendrocytes in spinal cord cultures from virus-inoculated animals. The number of type 2 astrocytes and oligodendrocytes was determined as described in Fig. 5 . In defined media alone, the proportion of type 2 astrocytes increases during demyelination (3-5 wpi). Addition of bFGF (10 ng/nd) to the defined media between 1 and 3 div exaggerates this shift toward the type 2 astrocyte phenotype by decreasing the relative number of cells expressing GC. Treatment with IGF-I (100 ng/ml) induces a larger proportion of cells to express the oligodendrocyte phenotype. Exogenous PDGF (10 ng/ml) did not alter the ratio of type 2 astxo~tes to oligodendrocytes. To minimize variability between experiments, the cultures were prepared in parallel for each timepoint and as one completely matched set from mice inoculated at the same time with the demyelinating virus. Each value represents the ratio from cell counts in an entire 35-mm dish. A total of 1,793 cells were counted in the set of cultures. To estimate the variability that might be expected between nonmatched experiments, the ratio of type 2 astrocytes to oligodendrocytes was compared in three similar experiments of cultures grown without exogenous growth factors. The asterisks denote values which fall outside of a 95 % confidence interval of the expected variability between experiments for cultures grown without exogenous growth factors. Fig. 1, A and B) . Each value is the percentage (+SEM) of total O-2A lineage cells (oligodendrocytes, type 2 astrocytes, O-2A progenitors, and mixed phenotype cells combined) which were [~Hlthymidine-labeled (greater than 10 autoradiographic silver grains localized over each nucleus) and represents the average of 2-3 dishes. Significance values were determined using the two-tailed paired t test. Studies of neonatal CNS tissue have shown that the growth and differentiation of O-2A lineage cells can be influenced in vitro by several polypeptide growth factors, which are synthesized in the brain (for review, see Raft, 1989; Dubois-Dalcq and Armstrong, 1990) . PDGF stimulates the proliferation of O-2A progenitor cells, which prevents premature differentiation in vitro Raff et al., 1988) . Figure 11 . A type 2 astroeyte cultured from demyelinated tissue (4 wpi) and processed as described in Fig. 10 is identified by the presence of surface staining with 04 (A; rhodamine optics), intracellular expression of GFAP (B; coumarin optics) and the absence of labeling with GC antibody (C; fluoreseein optics). The cluster of silver grains over the nucleus (D, arrow) indicates that this cell incorporated [3H]thymidine during the 20-h in vitro pulse. (E) Phase contrast showing the cell processes. Bar, 50 #m.