Endogenous cell repair of chronic demyelination

Abstract

In multiple sclerosis lesions, remyelination typically fails with repeated or chronic demyelinating episodes and results in neurologic disability. Acute demyelination models in rodents typically exhibit robust spontaneous remyelination that prevents appropriate evaluation of strategies for improving conditions of insufficient remyelination. In the current study, we used a mouse model of chronic demyelination induced by continuous ingestion of 0.2% cuprizone for 12 weeks. This chronic process depleted the oligodendrocyte progenitor population and impaired oligodendrocyte regeneration. Remyelination remained limited after removal of cuprizone from the diet. Fibroblast growth factor 2 (FGF2) expression was persistently increased in the corpus callosum of chronically demyelinated mice as compared with nonlesioned mice. We used FGF2 mice to determine whether removal of endogenous FGF2 promoted remyelination of chronically demyelinated areas. Wild-type and FGF2 mice exhibited similar demyelination during chronic cuprizone treatment. Importantly, in contrast to wild-type mice, the FGF2 mice spontaneously remyelinated completely during the recovery period after chronic demyelination. Increased remyelination in FGF2 mice correlated with enhanced oligodendroglial regeneration. FGF2 genotype did not alter the density of oligodendrocyte progenitor cells or proliferating cells after chronic demyelination. These findings indicate that attenuating FGF2 created a sufficiently permissive lesion environment for endogenous cells to effectively remyelinate viable axons even after chronic demyelination.

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 < 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 < 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone. (B-D) Representative MOG immunostaining of coronal sections through the corpus callosum. (B) Control without cuprizone treatment (age matched to [D]). (C) Continuous cuprizone for 12 weeks. (D) continuous cuprizone for 12 weeks followed by 6 weeks of recovery on normal chow. Midline at left side of each image. CC, corpus callosum; DF, dorsal fornix; Cg, cingulum. Scale bar for (B, C, D) shown in (D) = 250 μm.

FIGURE 2.

Oligodendroglial repopulation of the corpus callosum was compromised after chronic demyelination of C57Bl/6 mice. (A) Quantification of the density of oligodendrocytes in the corpus callosum of C57Bl/6 mice. In situ hybridization for PLP mRNA, which identifies premyelinating and myelinating oligodendrocytes, was quantified using unbiased stereology with at least three sections per mouse and at least four mice per condition. Bar colors indicate no cuprizone (white), acute cuprizone (gray), or chronic cuprizone (black). The oligodendrocyte density after 12 weeks of cuprizone followed by 6 weeks for recovery was still significantly below nonlesioned values (p > 0.05; n = 5 for both conditions). Significant differences, p < 0.05 or less, are noted by an asterisk (*) for comparisons with no cuprizone and with a carrot (^) for comparisons of recovery stages with 12 week cuprizone. (B-D) Representative PLP in situ hybridization of coronal sections through the corpus callosum. (B) Control without cuprizone treatment (age matched to [D]). (C) Continuous cuprizone for 12 weeks. (D) Continuous cuprizone for 12 weeks followed by 6 weeks of recovery on normal chow. Midline is at left side of each image and areas are similar to those shown Figure 1. Scale bar for (B, C, D) shown in (D) = 250 μm.

FIGURE 3.

Chronic demyelination compromised oligodendrocyte progenitor (OP) and cycling cell densities. (A, B) Quantification of the density of OP cells ( [A], identified by in situ hybridization for PDGFαR mRNA) and cycling cells ( [B], detected by Ki-67 immunostaining) in the corpus callosum of C57Bl/6 mice. Bar colors indicated no cuprizone (white), acute cuprizone (gray), or chronic cuprizone (black). At least three sections were quantified per mouse and at least four mice per condition. (A) The density of PDGFαR+ cells was significantly increased during acute demyelination but decreased to nontreated levels during chronic cuprizone administration (p < 0.001 for 5 weeks cuprizone, denoted by asterisk [*], in comparison with all other conditions; p > 0.05 for each comparison of chronic cuprizone with no treatment; no cuprizone, n = 5; 5 weeks cuprizone, n = 5; 12 weeks cuprizone, n = 4; 12 weeks cuprizone + 3 weeks off, n = 6; 12 weeks cuprizone + 6 weeks off, n = 3). B) The density of Ki-67+ cells indicated a significant increase in the population of cycling cells after 12 weeks of cuprizone that declined during the recovery phase (p < 0.01 for 12 weeks cuprizone, denoted by asterisk (*), in comparison with all other conditions; no cuprizone, n = 4; 12 weeks cuprizone, n = 5; 12 weeks cuprizone + 6 weeks off, n = 5). (A, inset) Example of a cell immunolabeled for NG2 (red) and exhibiting a chromatin arrangement (blue, DAPI staining) that was characteristic of metaphase and indicative of active mitosis. Scale bar for inset in (A) = 10 μm.

FIGURE 4.

In chronically demyelinated corpus callosum of C57Bl/6 mice, FGF2 expression continued to be upregulated in lesions. (A, B) Representative coronal sections through the corpus callosum with the midline along the left edge of each image. In situ hybridization for FGF2 mRNA showed many labeled cells in neuronal structures, for example, cortex (above corpus callosum) and hippocampus (below corpus callosum). In the corpus callosum, few cells exhibited detectable FGF2 mRNA in nontreated mice (A) but FGF2 mRNA expression was markedly increased in the corpus callosum of mice fed 0.2% cuprizone for 12 weeks (B). Scale bar for (A, B) shown in (B) = 250 μm.

FIGURE 5.

Spontaneous remyelination of the corpus callosum after chronic demyelination in FGF2 null mice but not in FGF2 wild-type mice. (A, B) Quantification (like in Figure 1) of corpus callosum myelination estimated by immunostaining for myelin oligodendrocyte glycoprotein (MOG) in cuprizone-treated FGF2^−/− mice (A) and FGF2^+/+ mice (B). The corpus callosum showed persistent demyelination with 0.2% cuprizone ingestion throughout 9 weeks and 12 weeks. After 12 weeks of cuprizone treatment, on return to normal chow, remyelination in FGF2^−/− mice significantly improved (p < 0.001; 12 weeks cuprizone, n = 4; 12 weeks cuprizone 6 weeks off, n = 4) and recovered to nonlesioned levels (p > 0.05; no cuprizone, n = 5; 12 weeks cuprizone 6 weeks off). In contrast, values in FGF2^+/+ mice did not increase after chronic demyelination (p > 0.05; 12 weeks cuprizone, n = 4 vs. 12 weeks cuprizone 6 weeks off, n = 3) and remained significantly below nonlesioned levels (p < 0.001; no cuprizone, n = 5 vs. 12 weeks cup). Significant differences, p < 0.05 or less, are noted by an asterisk (*) for comparison with no cuprizone and with a carrot (^) for comparisons of recovery stages with 12 weeks cuprizone. Comparison of the FGF2^+/+ values with the matching set of conditions in FGF2^−/− mice demonstrated a significant effect of genotype (p < 0.0001). (C-E) Representative coronal sections of MOG immunostaining. Midline is at left side of each image and areas are similar to those shown Figure 1. (C)FGF2^−/−control mouse without cuprizone treatment (age matched to [D]). (D)FGF2^−/− mouse after 12 weeks of cuprizone followed by 6 weeks on normal chow. (E)FGF2^+/+ mouse after 12 weeks of cuprizone followed by 6 weeks on normal chow. Scale bar for (C-E) shown in (E) = 250 μm.

FIGURE 6.

Oligodendroglial repopulation of the corpus callosum after chronic demyelination was dramatically improved in FGF2^−/− mice as compared with FGF2^+/+ mice. (A) Quantification of the density of oligodendrocytes in the corpus callosum of FGF2 mice. In situ hybridization for PLP mRNA, which identified premyelinating and myelinating oligodendrocytes, was quantified using unbiased stereology with at least three sections per mouse and at least four mice per condition. The oligodendrocyte density after 12 weeks of continuous cuprizone was significantly below nonlesioned values (noted by asterisks [*]; p < 0.01 for FGF2^+/+; p < 0.05 for FGF2^−/− mice; n = 4 for each condition). After 12 weeks of cuprizone and a subsequent 3- and 6-week period for recovery on normal chow, oligodendrocyte densities in FGF2^−/− mice were no longer significantly different as compared with nonlesion values (p > 0.05 at 3 weeks and at 6 weeks; n = 4 for each condition). Comparison of the FGF2^+/+ values with the matching conditions in FGF2^−/− mice demonstrated a significant effect of genotype (p = 0.0196). (B, C) Representative PLP in situ hybridization of coronal sections through the corpus callosum of an FGF2^+/+ mouse (B) and an FGF2^−/− mouse (C). Midline is at the left side of each image and areas are similar to those shown Figure 1. In both micrographs (B, C), sections shown are from mice that were fed cuprizone continuously for 12 weeks followed by 6 weeks on normal chow. Scale bar for (B, C) shown in (C) = 250 μm.

FIGURE 7.

Oligodendrocyte progenitor (OP) and cycling cell populations in corpus callosum after chronic demyelination were not altered by FGF2 genotype. (A, B) Quantification of the density of OP cells ([A], identified by in situ hybridization for PDGFαR) and cycling cells ([B], identified by Ki-67 immunostaining) in corpus callosum white matter of FGF2^+/+ mice and FGF2^−/− mice. (A) Compared with nontreated mice, the density of OP cells was significantly higher after chronic (12 weeks) cuprizone regardless of FGF2 genotype (noted by asterisks [*]; FGF2^+/+ mice, p < 0.001; FGF2^−/− mice, p < 0.001; n = 4 for each condition). (B) The density of cycling cells was also similarly increased significantly after 12 weeks of cuprizone, relative to nontreated mice, for both FGF2 genotypes (noted by asterisks [*]; FGF2^+/+ mice, p < 0.05; FGF2^−/− mice, p < 0.05; n = 5 for each no cuprizone genotype, n = 4 for each 12 week cuprizone genotype). Comparison of all three FGF2^−/− conditions with those from FGF2^+/+ mice demonstrated no significant effect of genotype for PDGFαR+ cells (p = 0.2746) or Ki-67+ cells (p = 0.8651). (C) Representative example of Ki-67 immunostaining of coronal sections through the corpus callosum of an FGF2^−/− mouse after 12 weeks of chronic cuprizone. Midline is at the left side of each image and areas are similar to those shown Figure 1. The corpus callosum was approximately within the borders demarcated between the dashed lines. Scale bar in (C) = 250 μm.