Chronic Cuprizone Demyelination Provided a Relevant Model of Insufficient Remyelination to Characterize the Repair Capacity of Endogenous Cells C57Bl/6 mice were used to establish parameters for analyzing, and later manipulating, the capacity of endogenous FIGURE 1. Spontaneous remyelination of the corpus callosum was compromised after chronic demyelination of C57Bl/6 mice. (A) Corpus callosum myelination was estimated by immunofluorescence for myelin oligodendrocyte glycoprotein (MOG). Pixel intensity values were normalized between tissue sections by thresholding to exclude values below the immunoreactivity in the dorsal fornix, which was not demyelinated by cuprizone (see DF in [C]). The percent area of the corpus callosum with MOG immunoreactivity above the threshold level was then used as an estimate of myelinated area. Bar colors indicate treatment conditions: no cuprizone (white), acute cuprizone (up to 6 weeks, gray), or chronic cuprizone (9 weeks and above, black). At least three sections were quantified per mouse and at least four mice per condition. After acute cuprizone, myelination returned to near nonlesioned values (p > 0.05; no cuprizone, n = 4; 6 weeks cuprizone 6 weeks off, n = 5). Recovery to nonlesioned levels did not occur after chronic cuprizone (p G 0.001; 12 weeks cuprizone 6 weeks off; n = 6). Values for 12-week cuprizone three off were not significantly different than either 12-week cuprizone or 12-week cuprizone 6 off. Significant differences, p G 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone. oligodendrocyte lineage cells to repopulate and remyelinate chronically demyelinated lesions. We used the cuprizone model to take advantage of the ability to stop active demyelination simply by returning the mice to a normal diet. As characterized in C57Bl/6 mice, continuous cuprizone ingestion results in persistent demyelination with a variable degree of partial remyelination, indicating a regenerative potential of endogenous cells even while mice are still fed cuprizone (24) . However, after 12 weeks of continuous 0.2% cuprizone ingestion in C57Bl/6 mice, spontaneous remyelination remained limited through the 6-week recovery period (Fig. 1 ). This poor remyelination after chronic demyelination contrasted with the almost complete remyelination seen within the same recovery period after acute demyelination (i.e. 6 weeks on cuprizone followed by 6 weeks off cuprizone; Fig. 1 ).