In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

Using 1-#m frozen sections and triple immunofluorescence, we have found cells with the O-2A adult progenitor antigenic phenotype in normal and virus infected mice. These cells were increased in number during viral infection and, since they incorporated tritiated thymidine, were actively Figure 8 . Example of a cell with a mixed phenotype. A CNP+ 04+ GFAP+ cell in the spinal cord of an infected mouse, 2 WPI. Such cells were found infrequently in spinal cord of infected mice but never in controls. They were most abundant at 2 WPI and were never labeled by [3H]thymidine injected shortly before killing. Bar, 10 #m. dividing. While not definitive, the evidence at hand suggests that these O4+ GFAP-C N P -cells, seen in situ for the first time, might give rise to oligodendrocytes formed in these adult animals during recovery from viral infection. Similarly, induction of oligodendrocyte proliferation and remyeli-nation after chronic demyelination in guinea pigs and multiple sclerosis has been described (Raine et al., 1988) . In addition to the 04 + only O-2A adult progenitor cell, we found increased numbers of 04+ GFAP+ cells in infected mice. Many of these cells also incorporated tritiated thymidine, as was seen previously in developing optic nerve (Miller et al., 1985) . Type 2 astrocytes are normally formed during developmental myelination, where they extend processes to the nodes of Ranvier (ffrench-Constant and Raft, 1986) ; the 04+ GFAP+ cells which proliferate in the infected animals might play the same role during remyelination. In addition, we found occasional cells with the mixed phenotype 04+ GFAP+ CNP+. The existence of this intermediate form raises the possibility that transdifferentiation of type 2 (04+ GFAP+) astrocytes might also contribute to the generation of oligodendrocytes. Interestingly, the expression of GFAP in immature oligodendroglia has been described before in the developing human spinal cord (Choi and Kim, 1984) and coexistence of the oligodendroeyte marker carbonic anhydrase and GFAP have been reported in radial Figure 9 . Dorsal funiculus of infected spinal cord, 7 WPI. a; b', c'are close up of the upper region of a, b, and c. CNP immunofluorescence shows nearly normal intensity and is distributed as rings around and within myelin sheaths reformed during remyelination (a and a'). 04 immunofluorescence is still greater than normal though somewhat reduced as compared to lesions at earlier stages; however, several large 04+ GFAP+ cells are scattered in the remyelinated area (white arrowheads in b and c). Such cells were never found in control specimens. Bars: (a-c) 50 tzm; (a'-c') 20 ~m. fibers of developing spinal cord (Hirano and Goldman, 1988) . Similarly cultures of spinal cord from our remyelinating animals yield a substantial number of cells expressing both oligodendrocyte and astrocyte markers (Armstrong, R., V. Friedrich, K. V. Holmes, and M. Dubois-Dalcq, manuscript in preparation).